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Keywords:

  • ectomycorrhiza;
  • genets;
  • Laccaria amethystina;
  • Xerocomus chrysenteron;
  • Xerocomus pruinatus;
  • population structure;
  • RAPD;
  • rDNA

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Supplementary Material
  8. Acknowledgements
  9. References
  •  The knowledge of temporal and spatial structure of populations of ectomycorrhizal fungi, together with the origin and maintenance of their genetic variation, is critical to understanding how populations of these fungi establish, evolve and disappear at different stages of development of forest ecosystems.
  •  Identification and spatial delimitation of genets in populations of the ectomycorrhizal basidiomycetes, Laccaria amethystina, Xerocomus chrysenteron and X. pruinatus were inferred from the polymorphism of two codominant genetic loci, the nuclear rDNA internal transcribed spacers (ITS) and intergenic spacers (IGS), and anonymous dominant RAPD markers from basidiocarps collected in a mixed mature forest in the fungal reserve of La Chanéaz (Switzerland).
  •  The L. amethystina population showed numerous small, short lifespan genets; most closely spaced basidiocarps were genetically unique. Our results confirmed that sexual spore propagation is important in the life history of L. amethystina in undisturbed mature forests. By contrast, we found a single genet for each of the boletoid species colonizing a nearby plot indicating that clonal growth dominated.
  •  In La Chanéaz forest, the intrinsic biological features of the investigated species appear to play a higher role in colonization strategy than the features of local habitat.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Supplementary Material
  8. Acknowledgements
  9. References

Within forest soils, that host a large and diverse community of microbes that compete and interact with each other and with plant roots, ectomycorrhizal fungi are almost ubiquitous (Taylor et al., 2000). The mutualistic ectomycorrhizal symbiosis allows trees to grow efficiently in suboptimal environments in boreal, temperate and some subtropical forest ecosystems (Smith & Read, 1997).

In a forest ecosystem, roots are exposed to an assemblage of several different species of ectomycorrhizal fungi (Taylor et al., 2000; Dahlberg, 2001). Each species exists as a population of many individuals between which there is almost invariably some genetic variation (Debaud et al., 1995). The fact that the host plants are exposed to mycobiont populations which are genetically diverse is a crucial consideration in forest ecology. Individual symbionts vary in their ability to colonize different genotypes of host-plant, their capacity to utilize mineral and organic soil nutrients, and in their adaptation to abiotic factors, such as soil pH and drought. Each of these abiotic and biotic factors might affect the course of a beneficial symbiosis and the establishment and subsequent dissemination of an individual fungal symbiont. Questions about the temporal and spatial structures of communities and populations of ectomycorrhizal fungi, together with the origin and maintenance of their genetic variation, are therefore critical when trying to understand how populations of these symbiotic fungi evolve and disappear at different stages of development of forest ecosystems (Egger, 1994; Dahlberg & Stenlid, 1995; Dahlberg, 2001). By implication, such a knowledge can help in the understanding of biogeochimical cycles in forest ecosystems (Taylor et al., 2000).

When and how genetic individuals of a given species (also referred to as genets) arrive at a site and how interactions between them evolve with maturation of fungal and host-plant populations are key questions in the assessment of dynamics in populations of ectomycorrhizal fungi. To understand the dispersal, establishment, and evolution of ectomycorrhizal populations, it is necessary to analyse these populations over several seasons and compare them at different successional stages of forest ecosystems at different habitats. The source and maintenance of genetic diversity within these ectomycorrhizal populations have been questioned in previous studies (Dahlberg & Stenlid, 1990, 1994; De La Bastide et al., 1994; Gryta et al., 1997, 2000; Bonello et al., 1998; Selosse et al., 1998, 1999; Gherbi et al., 1999; Sawyer et al., 1999; Zhou et al., 1999, 2001; Guidot et al., 2001; Redecker et al., 2001). From these investigations, conclusions about the relative role of vegetative spreads vs spore establishment of ectomycorrhizal basidiomycete can be drawn (Egger, 1994; Dahlberg, 2001).

In open forest communities, genet recruitment from spore germination occurs massively during a short period early in the development of the population, leading to the development of numerous small genetically different individuals. Many early colonizers, such as Hebeloma cylindrosporum and Laccaria spp., appear to have relatively small nonpersistent genets (De La Bastide et al., 1994; Gryta et al., 1997, 2000; Selosse et al., 1998). For some Suillus species, it is believed that no further genet recruitment from spores occurs after the initial colonizing phase and vegetative growth through hyphal networks appears to dominate in closed fungal communities (i.e. older forests). These communities are claimed to be resistant to genet establishment from spores, unless small disturbances in favourable sites create ‘windows of opportunity’ for local new genet recruitment (Dahlberg & Stenlid, 1995). Once thought to be dominated by a few, large (20–40 m) and long lasting genets, undisturbed closed forests can host populations of ectomycorrhizal basidiomycetes having high levels of genetic diversity characterized by small, short life-span genets (Gherbi et al., 1999; Redecker et al., 2001). Some ectomycorrhizal fungal populations found in undisturbed mature forest settings could therefore propagate by intense fruiting and spore germination. Whether population structure is related to microsite heterogeneity (Zhou et al., 1999, 2001; Guidot et al., 2001) or the biological/genetic features of the species is unknown. A way to validate the above mentioned models on the processes driving the evolution of genetic structure in populations of ectomycorrhizal fungi is to determine the genetic structure of a given species in different habitats differing by their ecological characteristics (Redecker et al., 2001).

Laccaria amethystina is found in both recently established plantations and mature forests. We have investigated the genetic structure of L. amethystina populations in a 150-yr-old-monospecific beech (Fagus sylvatica) forest in the Vosges range in north-eastern France (Gherbi et al., 1999). Surprisingly, a high density (> 5000 genets per ha) of small size, short life-span genets was found indicating an important role for sexual reproduction in this mature forest ecosystem. To assess whether this feature can be influenced by the habitat, we used the random amplified polymorphic DNA (RAPD) technique to estimate DNA variation within a population of L. amethystina in the mixed forest of La Chanéaz (Swiss Mittelland). Xerocomus chrysenteron and X. pruinatus, two boletoid species were also analysed for comparative analysis.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Supplementary Material
  8. Acknowledgements
  9. References

Study site

The study was conducted in mixed (conifers and deciduous trees) stands in the mycological reserve of La Chanéaz in Swiss Mittelland (Fribourg canton) (46°48′ N 7°00′ E). The 75 ha-forest is located at an altitude of 575 m above sea level (asl) on a plateau made up of fresh water molasse, and the soil is mainly an acidic moder. It is a typical mixed forest, with deciduous and coniferous trees species, such as Fagus sylvatica, Picea abies, Pseudotsuga menziesii, Pinus sylvestris, and Abies alba. The mean annual temperature is 9.4° and the mean annual precipitation is 900–1000 mm. The reserve was set up in 1979 by the Swiss Federal Institute for Forest, Snow and Landscape Research and mushroom harvesting is not allowed except for scientific investigations. Many studies on fungal ecology and protection were carried out on the site (Egli et al., 1990). The climatic data, ectomycorrhizal communities and fungal species richness of the fungal reserve were described by Egli & Ayer (1997) and Straatsma et al. (2001).

Sampling plots

Within the study site, two sampling areas were established. Plot 59 (10 × 5 m) was used for sampling Laccaria amethystina Cooke basidiocarps, whereas those of Xerocomus chrysenteron (Bull.) Quél. and X. pruinatus (Fr. and Hök) Quél. were collected in the nearby plot 47 (10 × 10 m). Each of these areas was chosen to have mature trees at their edges. Plot 59 comprised two 140-yr-old Pinus sylvestris and two 40-yr-old Fagus sylvatica and several scattered young ecto- and endomycorrhizal trees (F. sylvatica, Corylus avellana, Acer pseudoplatanus, Sorbus aucuparia, and Prunus padus) (Fig. 1). Trees present on plot 47 comprised four 140-yr-old Picea abies and three understorey 40-yr-old Fagus sylvatica (Fig. 2). Plot 47 is surrounded by monospecific closed beech stands (F. Ayer, pers. comm.). Investigated plots were surrounded by 2 m high fences to avoid all inconvenient influences by mushroom pickers and large forest animals. The plot areas were gridded using pegs planted every meter and location of collected basidiocarps were recorded with a precision of a few centimetres (< 5 cm). Only fresh specimens without any signs of decay were used.

image

Figure 1. Schematic depiction of relative positions of Laccaria amethystina basidiocarps and genets plotted on the reference mapping of plot 59 (5 × 10 m) (La Chanéaz forest). Standing trees are represented by grey closed circles, diameters to scale. Pi, Pinus sylvestris; F, Fagus sylvatica; C, Corylus avellana; A, Acer pseudoplatanus; S, Sorbus aucuparia; P, Prunus padus. Each blue (1997), red (1998) or green (1999) circle identifies a basidiocarp. Basidiocarps belonging to the same genet are represented by closed circles and are indicated with bold letters. The shaded zones encompass adjacent basidiocarps belonging to the same genets. Dotted lines connect different patches (ramets) of the same genet. Basidiocarps from neighbour-joining grouping A are indicated by an asterisk; they showed a 39–59% similarity of their RAPD patterns.

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image

Figure 2. Schematic depiction of relative positions of basidiocarps and genets of Xerocomus chrysenteron and X. pruinatus in plot 47 (10 × 10 m) (La Chanéaz forest). Standing trees (P, Picea abies; F, Fagus sylvatica) growing in the plot are represented by grey closed circles, diameters to scale. Basidiocarps of the single X. chrysenteron genet are represented by blue (1997), red (1998) or green (1999) open squares. Basidiocarps of the single X. pruinatus genet are represented by closed circles (dark blue, 1997; purple, 1998; dark green, 1999). The shaded zones encompass basidiocarps belonging to X. pruinatus large genet.

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Survey of basidiocarps

In 1997–99, the fruiting period of L. amethystina was from early October to early November, whereas fruiting of X. chrysenteron and X. pruinatus was from mid-August to mid-November. The Xerocomus species were morphologically identified according to Binder & Fischer (1997) and their identification was confirmed by sequencing their nuclear ribosomal internal transcribed spacer (ITS) and part of the large 25S rDNA subunit (GenBank accession # AF402139 and AF402140). The sites were visited up to twice a week to ensure an exhaustive sampling. In 1997, 1998, and 1999, we sampled a total of 280 L. amethystina basidiocarps (Table 1). The collection of X. chrysenteron totalized 146 basidiocarps (Table 1). Xerocomus pruinatus basidiocarps were rare and only 10 were collected (Table 1). After recording the coordinates of collected basidiocarps on a sampling grid, they were placed in a labelled plastic bag, transported to the laboratory and stored at −20° within 2 h.

Table 1.  Characteristics of the populations of Laccaria amethystina, Xerocomus chrysenteron, and X. pruinatus of the fungal reserve La Chanéaz
 Samplings
 199719981999Total
  • a

    As a result of PCR failures, some of the basidiocarps produced no or poor results.

Laccaria amethystina
Basidiocarps sampled59116105280
IGS1 and RAPD analyses449075209a
Genets detected366952157
Basidiocarps per genet1.221.301.441.33
Proportion of distinguishable genets0.820.770.690.75
Genets per ha per year720013 80010 40010 467
Xerocomus chrysenteron
Basidiocarps sampled395651146
IGS1 and RAPD analyses395651146
Genets detected1111
Basidiocarps per genet39565148
Proportion of distinguishable genets0.020.010.020.02
Genets per ha per year10101010
Xerocomus pruinatus
Basidiocarps sampled72110
IGS1 and RAPD analyses72110
Genets detected1111
Basidiocarps per genet7213.3
Proportion of distinguishable genets0.140.510.3
Genets per ha per year10101010

Molecular methods

Total DNA was extracted directly from basidiocarp tissues using the hexadecyltrimethylammonium bromide (CTAB)/proteinase K protocol as described by Henrion et al. (1994). PCR amplifications of the nuclear rDNA ITS and intergenic spacer 1 (IGS1) were carried out as described in Henrion et al. (1992, 1994), except that a BIOMETRA Uno-thermocycler system was used. RAPD was performed using primers 174 and 157 at high annealing temperature (50°C) according to Selosse et al. (1998). Amplification products were separated by electrophoresis using 1.4% (w/v) Sigma Wide Range agarose/0.8% agarose gel electrophoresis for 5 h (120 V h−1) in a 1X TBE buffer system. Gels were stained with ethidium bromide and photographed with a digital camera.

The amplified nuclear ribosomal DNA ITS and IGS were sequenced using the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems Inc., Norwalk, USA), following the manufacturer’s instructions. Reactions were then electrophoresed on either an ABI 373XL (at the Institut d’Ecologie de l’Université de Lausanne) or an ABI Genotyper 310 (at INRA-Nancy) automated sequencer (Applied Biosystems Inc.). Gels were tracked using the ABI Prism Sequencing programme and raw sequence data were edited using the Sequence Analysis (Applied Biosystems Inc.) or Sequencher (Gene Codes Corporation, Ann Arbor, MI, USA) programmes.

Data and statistical analyses

The different L. amethystina basidiocarps were initially analysed by ITS and IGS1 typing and genets were distinguished by their RAPD patterns. We selected primers yielding both reproducible and polymorphic variations with well defined and darkly stained bands. RAPD were performed twice and only the bands that reproduced consistently were scored for each individual basidiocarp. Similar or identical samples were checked against each other by comparing their electrophoregrams side by side. Basidiocarps having identical ITS, IGS1 and RAPD banding patterns were regarded as belonging to the same genet and were mapped according to the distribution of collected basidiocarps. The area between adjacent basidiocarps sharing identical DNA patterns was assumed to represent the genet surface and the basidiocarps at the edge of this area were used to draw the borders of genets.

RAPD was scored as presence (1) or absence (0) of a band and a matrix of RAPD phenotypes was assembled. This rectangular matrix of individual × RAPD marker containing the band scoring information was transformed to resemblance matrices using four distance and similarity coefficients: simple matching, Sorensen and Jaccard similarity coefficients, and Euclidean distance for binary data (SIMIL in the R package 4.0d3; Casgrain & Legendre, 2001) with similar results. A dendrogram was constructed by unweighted pairgroup method with arithmetic mean (UPGMA) from the similarity matrices as a graphic representation of the relationships between samples using the R package (Casgrain & Legendre, 2001). The binary data matrix was also used for UPGMA and neighbour-joining (NJ) analyses in PAUP*4.08b (PPC/Altivec) (Swofford, 1999). The effect of spatial separation on genetic structure was tested by a regular Mantel test (Mantel & Valand, 1970) on the resemblance (RAPD pattern similarity) matrix and a Euclidean matrix of metric distance between individual basidiocarps (MANTEL module in the R Package). The significance of the correlation between the two matrices was tested by 999 random permutations of the similarity matrix to generate a null distribution of correlation coefficients (z-values). A significant result was inferred if ≥ 95% of the randomly generated statistics were greater than the observed value. Tests for goodness of fit to Hardy–Weinberg equilibrium were computed in POPGENE (Yeh et al., 1997).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Supplementary Material
  8. Acknowledgements
  9. References

Genetic structure and spatial distribution of Laccaria amethystina

Most of the basidiocarps were clustered in the westward part of the plot (Fig. 1). Genetic variability of the L. amethystina population was initially assessed by investigating the size polymorphism of the rDNA ITS and IGS1, regions showing intraspecific variability within Laccaria spp. (Henrion et al., 1992; Selosse et al., 1996, 1998; Gherbi et al., 1999). No size polymorphism was found in the ITS (data not shown). By contrast, three different IGS1 patterns, differring by the number and size of the amplification products, were identified from among the 209 basidiocarps analysed. IGS1-type AA, presenting a single 700 bp DNA fragment, was detected in 113 basidiocarps (54%). Type aa, having a single DNA band at 760 bp, was found in 24 basidiocarps (12%). The remaining 72 analysed basidiocarps (34%) displayed a heterotype Aa with two bands at 700 and 760 bp. Sequencing of the two IGS1 types revealed a low divergence (0.8%) for most of the sequence, but a 47-bp-indel similar to the one observed in L. amethystina in the beech forest of Aubure (Gherbi et al., 1999). The IGS1 types were treated as codominant biallelic markers in a test for Hardy–Weinberg equilibrium expectation in the 209 isolates. Isolates which possessed a single band (700- or 760-bp) were treated as homozygotes (AA, aa) and isolates with two bands (700- and 760-bp) were treated as heterozygotes (Aa). A slight heterozygote deficit was observed in the IGS1 locus (expected and observed heterozygotes: 0.41 and 0.35, respectively; expected and observed homozygotes: 0.58 and 0.64, respectively). A Chi-square test returned the genotype distribution in the IGS1 locus to be significantly different from the genotype distribution under Hardy–Weinberg equilibrium (4.4%; P < 0.05). The heterozygote deficit in the IGS1 locus suggests that inbreeding may occur, although this process should be limited by mating types. However, the significant level of heterozygosity indicates that outcrossing, heterothallic reproductive mode was taking place in this population.

Further molecular typing was carried out using RAPD to ascertain the DNA diversity amongst collected L. amethystina basidiocarps. The two chosen primers yielded 59 scorable markers that ranged in size from 180 to 1200 bp. Up to 157 unique RAPD banding patterns were identified indicating the existence of a large DNA variation in the analysed population. Most of the DNA bands were only detected in a few basidiocarps. Only two DNA fragments, generated by primer 157, were found in all DNA samples. The mean ‘proportion of distinguishable genets’ (i.e. total number of unique RAPD patterns divided by the sample number) from this sample of the population of La Chanéaz forest was 0.75 (Table 1). We found a similar high value (0.61) in L. amethystina populations surveyed in a beech forest in the Vosges mountains (north-eastern France) (Gherbi et al., 1999).

Basidiocarps belonging to the same genet were not necessarily collected on the same day. The persistence of basidiocarps belonging to a discrete genet varied from 5 to 22 d. Only six genets (< 4%) were detected for more than 1 yr (Fig. 1). Genets I, Q, U, and X were detected in both 1997 and 1998, whereas a single genet (i.e. R) was found in 1998 and 1999 samplings. The greatest distance separating two basidiocarps belonging to the same genet and collected in two consecutive years ranged from 0.3 (genet I) to 2.2 m (genet Q & U). Basidiocarps of the largest genet, so-called S, were sampled in 1997 and 1999 (Fig. 1).

The limits of L. amethystina intermingling individuals in plot 59 were often blurred (Fig. 1). The average number of basidiocarps per genet was 1.33 (Table 1). Even in areas of high sampling density (e.g. coordinates B1, C1 and C2), most genets were found only once. The largest genet (S) comprised 13 basidiocarps, all having the IGS1-type AA, and its two main patches covered an area of c. 0.75 m2 (coordinates D2/D4). The average size (largest detectable distance across a genet) of the 26 genets comprising more than one basidiocarp was 1.1 m, ranging from a few cm (genet J, coordinates C1) to 5.40 m (genet B, coordinates A1 to A7). Similarity between genets was assessed by UPGMA (not shown) and NJ (Fig. 3) analyses of RAPD patterns. These statistical analyses produced the same tree topology. Genets comprising at least three basidiocarps formed easily distinguable clusters (labelled shaded boxes in Fig. 3). However, the dendrogram illustrate well the limited number of individuals sharing similar RAPD phenotypes. As an example, the similarity of RAPD patterns clustering at the top of the NJ tree (the so-called grouping A including individuals C714, C807, B731, B817, B824, C802 and C810) ranged from 0.39 to 0.59. These statistical analyses confirmed the lack of a close genetic relationship between most basidiocarps and genets, although breeding occurred as suggested by the significant proportion of heterozygotes at the IGS1 locus (see above). Some of the most divergent RAPD patterns originated from adjacent basidiocarps (see coordinates B1 and C1). The Mantel’s test showed no significant correlation between the spatial distance matrix and the similarity matrix of RAPD patterns (= −0.04, P= 0.13) suggesting that spatial distance between basidiocarps plays a marginal role in structuring this population.

image

Figure 3. Dendrogram constructed by neighbour-joining analysis showing the RAPD relationships among Laccaria amethystina basidiocarps collected in plot 59 (5 × 10 m) of La Chanéaz forest. Analysis was conducted using the distance search algorithm of PAUP 4.0b8. Scale represents 0.01 changes. The letters indicate the IGS1 type (A, type AA; B, type aa; and C, type Aa), the following number stands for the sampling year (7, 1997; 8, 1998; 9, 1999) and the remaining numbers indicate the sample number. Genets comprising more than three basidiocarps are indicated by shaded boxes. Grouping A which includes individuals showing a 39–59% similarity of their RAPD pattern is shown as a shaded box at the tree top. The shaded box at the tree top indicates the grouping A referred to in Figure 1.

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Spatial distribution and DNA typing of Xerocomus chrysenteron and X. pruinatus

Basidiocarps of X. chrysenteron, harvested from 1997 to 1999, were scattered over plot 47 (Fig. 2), as it was recorded since 1992 (F. Ayer, pers. comm.). By contrast, basidiocarps of X. pruinatus were restricted to a surface of 40-m2, less than half of the plot (coordinates H7 to A10). Amplification of the ITS from X. chrysenteron and X. pruinatus generated a single 740 bp-band (data not shown). By contrast, the size of the amplified IGS1 showed a great difference between the two species. X. chrysenteron had a single IGS1 product of 500 bp, whereas X. pruinatus had two bands of 800 and 900 bp.

RAPD typing and spatial distribution of genets

RAPD produced identical patterns, totalling 13 reliable markers, for all X. chrysenteron basidiocarps collected over the 3 yr survey. This genetic homogeneity suggests the presence of a single long-lasting individual in the plot covering a surface as large as 100 m2 (Fig. 3). An attempt to define the edges of this individual was made during the autumn of 2000 by collecting five basidiocarps outside plot 47: four in the northward adjacent plot (up to 50 m), and one 60 meters south-eastward. An additional 10 basidiocarps were collected inside the plot (data not shown). The same IGS and RAPD patterns were found for all basidiocarps. The maximum distance recorded between two basidiocarps from this single genet was, thus, c. 110 m.

All X. pruinatus basidiocarps collected inside plot 47 over the 3 yr belonged to the same genet. The surface covered by this individual was at least 40 m2 and it was restricted to the eastern part of the plot (Fig. 2). Five basidiocarps, collected outside the plot in the autumn of 2000, belonged to four different genets (data not shown) suggesting that this large individual did not extend outside plot 47.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Supplementary Material
  8. Acknowledgements
  9. References

Genet size in ectomycorrhizal fungi can reveal features of colonization strategy and growth. Fruiting body and ectomycorrhiza surveys have shown that the size of the genets from ectomycorrhizal fungi may vary with the forest age. It is widely believed that the presence of many small genets indicates multiple spore establishment events and large genets reflects predominant vegetative spread (Dahlberg & Stenlid, 1990, 1995; Dahlberg, 2001). Estimated densities of populations range from 30 to 5200 genets ha−1 depending on the species and forest age (Gryta et al., 1997; Gherbi et al., 1999; Dahlberg, 2001; Redecker et al., 2001). Populations of Suillus bovinus and S. variegatus in old conifer forests consist mostly of discrete, large (up to 27 m) and old (> 150 yr) genets which do not seem to intermingle (Dahlberg & Stenlid, 1994, 1995; Dahlberg, 2001). One of the largest genet of ectomycorrhizal fungi to date (300 m2) has been found in Suillus pungens in a Bishop pine forest in coastal California (Bonello et al., 1998). It was thus thought that vegetative growth and dissemination over long periods dominate over spore colonization in established forests typified by canopy closure, lack of young trees, and organic N-rich litter (Dahlberg & Stenlid, 1995). However, recent studies (Gherbi et al., 1999; Redecker et al., 2001) have demonstrated that undisturbed mature forests may host fungal populations characterized by numerous, short life-span genets relying mainly on spore dissemination for local colonization. Initial genet recruitment from spores followed by vegetative spread (large, old genets) and repeated recruitment from spores (small, short life-span genets) may be considered as two extremes of a continuum rather than as two clearly distinct recruitment patterns in ectomycorrhizal basidiomycetes.

As spatial structuration of populations of ectomycorrhizal fungi differs between taxa (Dahlberg, 2001), we have analysed populations of three ectomycorrhizal basidiomycetes, Laccaria amethystina, Xerocomus chrysenteron, and X. pruinatus in the undisturbed mixed forest of La Chanéaz. The investigated stands comprised 40-yr-old and 140-yr-old conifer and hardwood species, together with a few young, mainly endomycorrhizal, trees. Molecular typing of sampled basidiocarps revealed two constrasting genetic structures for these species. Both X. chrysenteron and X. pruinatus populations consisted of a discrete, large genet. Assuming a mean growth rate of 0.5 m per year for the underground mycelium of ectomycorrhizal fungi (Dahlberg & Stenlid, 1994; Bonello et al., 1998; Selosse et al., 1998), the founder of the 110 m-large X. chrysenteron clone probably associated with the older Picea abies when they established 140-yr-ago. The closely related species X. pruinatus also formed large clones (40 m2) in the same plot. X. chrysenteron collected in the beech forest of Aubure (Vosges) (Gherbi et al., 1999) also pertained to a single large genet (> 100 m2) (H. Gherbi & F. Martin, unpublished). These results were consistent with the findings of Dahlberg & Stenlid (1994, 1995) showing that, although they produce a considerable number of spores, many boletoids extend mostly by vegetative growth and the genet establishment from spore germination progressively decreased in the ageing population. Their capacity to develop extensive hyphal cords probably enables the large genets to visit the roots of more trees and to pool more total C than species which are more common, but produce smaller genets (Bonello et al., 1998).

By contrast, the L. amethystina population was made of numerous discrete genets (up to 13800 ha−1). Among the analysed genets, only 4% of the identified genets fruited the following years suggesting a considerably large mortality of genets. The genet size varied from the mere diameter of the basidiocarp (i.e. c. 2–4 cm) to 5.40 m in length. The present results are in agreement with a previous survey carried out in a different habitat, a 150-yr-old monospecific beech forest situated at an altitude of 1000 m asl (Gherbi et al., 1999). In the latter study conducted in the Vosges mountains (north-eastern France), L. amethystina populations exhibited up to 5200 short life-span, small-size genets per ha. The dramatic renewal of genets suggested a recolonization of the site by new genets each year and/or erratic fruiting of ‘cryptic’ below ground genets (Gherbi et al., 1999). The numerous L. amethystina genets are likely to have arisen from repetitive establishment from basidiospores, as suggested by their small size. Large amounts of feeder roots with high turnover rates (Vogt et al., 1986) give constant opportunities for ectomycorrhizal fungi to become established (Kranabetter & Wylie, 1998). The growth pattern of L. amethystina suggests that these new host root tips are colonized each season, the spreading of the new genets probably following the elongation of roots. In situations with strong intraspecific competition we could expect a reduced clonal diversity because fewer fitter genets would predominate. Yet, a high DNA diversity was maintained at both sites analysed (i.e. Aubure and La Chanéaz). Obviously, none of the genet produced sufficient clonal offspring to contribute to its predominance.

Colonization by numerous small-sized genets, with high fruiting rates, is reminiscent of young natural populations of ectomycorrhizal fungi (e.g. Suillus bovinus (Dahlberg & Stenlid, 1990); Laccaria bicolor (De la Bastide et al., 1994; Selosse et al., 1998, 1999) and Hebeloma cylindrosporum (Gryta et al., 1997; Guidot et al., 2001)). This population structure was postulated to arise in recently established or disturbed stands (Dahlberg & Stenlid, 1995). Recent investigations have however, shown that this ‘early stage’ population structure is also found for ectomycorrhizal basidiomycetes in closed undisturbed forest settings (Gherbi et al., 1999; Zhou et al., 1999, 2001; Redecker et al., 2001).

The population structure may depend on: the dispersal of the spores; the extent of growth of the monokaryotic mycelium (that germinated from the basidiospores of the previous seasons) and the opportunities for mating between compatible monokaryotic mycelia that thus occur; and the persistence of the below-ground mycelia. The spatial distribution of H. cylindrosporum (Guidot et al., 2001) and Suillus grevillei (Zhou et al., 2001) genets in the soil showed that the development of basidiocarps was tightly correlated with that of the extramatrical and ectomycorrhizal mycelia of the same genet. Disappearance of basidiocarps of these species at a given place was associated with the disappearance of the corresponding subterranean mycelia within 1 yr. If this behaviour is also true for L. amethystina, basidiocarp analysis appears to reflect both the genetic diversity and the spatial structure of its subterranean populations.

Microsite heterogeneity which promotes the coexistence of genets through diversifying selection has been hypothesized to account for the mechanisms underlying the maintenance of a high level of genotypic diversity in fungal species (Frankland, 1998). Because Xerocomus species have been able to maintain a clonal structure for decades, this hypothesis alone is insufficient to explain the high genetic diversity of genets in aged forest ecosystems. The fact that the investigated stands are included in a fungal reserve protected by a fence precludes any major perturbation related to large animal and human activities.

The low similarity between RAPD patterns (Fig. 2) and the low correlation between spatial distance separating basidiocarps and the RAPD similarity matrix (Mantel’s test) indicate that L. amethystina genets found on plot 59 did not arise from a common (or a few) colonization event. These results suggest that establishment of novel genets and colonization of host root tips are largely a stochastic process, with much spore dispersal occurring on a scale greater or equal to that of this study (50 m2). The results of this study are somewhat at odds with the allelic analysis on S. grevillei (Zhou et al., 2001) which has shown that the spread of alleles within and between populations might be by repeats of short-distance spore dispersal.

In conclusion, the prominent role of the sexual spore reproduction over the perennial spread of the vegetative mycelium in the soil for ectomycorrhizal species, such as L. amethystina, is not restricted to pioneer situations, but is found in stable established forest ecosystems. These species are able to establish novel genets from spores repeatedly over several decades without apparent perturbation of the sites and/or host plant genetic structure. This suggests a rapid alteration of the subterranean parts of L. amethystina genets and a constant colonization of new feeder roots. Knowing the spatial structure of the studied populations through the fruiting body location will facilitate the characterization of their genetic structure at the level of ectomycorrhizal roots and extramatrical hyphae using codominant genetic loci (Guidot et al., 2001). So far, investigations on populations of ectomycorrhizal fungi have mainly been descriptive or correlative and no experimental study has yet been performed to manipulate these populations. Identification of causal processes controlling the dynamics and structure of ectomycorrhizal populations will advance our understanding of forest ecosystems.

Supplementary Material

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Supplementary Material
  8. Acknowledgements
  9. References

A map showing the location of study sites, photographs of the ITS and RAPD gels, DNA sequences, distance matrices and UPGMA and NJ trees are available at the following URL: http://mycor.nancy.inra.fr/Aubure/Aubure.html

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Supplementary Material
  8. Acknowledgements
  9. References

This study was part of Anne-Marie Fiore-Donno’s Ph.D. project. It was partially funded by a scholarship from the University of Lausanne and the Special Found of the Swiss Federal Office for Education and Science for Switzerland-France Ph.D. joint programmes. We thank Prof. Heinz Clémençon (University of Lausanne) for his constant support; Simon Egli and François Ayer (WSL, Birmensdorf) for allowing us to establish field sites in the fungal reserve of La Chanéaz for these studies; Christine Delaruelle (INRA-Nancy) for her helpful assistance in molecular methods; François Ayer for his kind assistance and advices in fruiting body surveys; Jesus Diez for stimulating discussions; two anonymous referees for their constructive comments; and Beatrice Senn-Irlet (University of Bern) for suggesting this investigation and her support. Funding was provided by the Bureau des Ressources Génétiques and INRA to FM.

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  6. Discussion
  7. Supplementary Material
  8. Acknowledgements
  9. References
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