Summary• Zn tolerance was investigated in populations of four ectomycorrhizal fungi: Suillus luteus , Suillus bovinus , Rhizopogon luteolus and Paxillus involutus. The fungi were collected in pioneer pine forests at 14 different locations, situated along a Zn pollution gradient. Genetic adaptation to Zn toxicity was previously presumed in a population of S. luteus .• Mycelial biomass production was assessed for 235 isolates exposed to increasing Zn 2+ stress. EC 50 concentrations were determined.• Adaptive Zn tolerance was found in the three species from the Suilloid clade and not in P. involutus . The Suilloid fungi collected within 5 km from a Zn smelter were highly Zn-tolerant, in contrast to isolates collected at least 15 km away from a pollution source. Mixed populations with tolerant and sensitive S. luteus isolates were found in a transition zone, between 5 and 15 km from the Zn smelters.• The severe Zn pollution in the surroundings of the Zn smelters has clearly triggered the evolution of an increased Zn tolerance in the pioneer Suilloid fungi. With increasing distances from the Zn smelters, the frequency of Zn-tolerant genotypes decreases.

Summary• The effects of environmental heavy metal pollution on the genetic structure of pioneer populations of the ectomycorrhizal basidiomycete Suillus luteus were assessed.• Sporocarps were collected from nine different locations and characterized by amplified fragment length polymorphism (AFLP) markers. Six of the sampling sites were contaminated with heavy metals and were dominated by tolerant individuals. Considerable genetic diversity was found within geographic subpopulations, but no reduction of the genetic diversity of populations inhabiting contaminated soils was observed. Neither did significant clustering of subpopulations inhabiting contaminated soils occur. Overall, the genetic differentiation between subpopulations was low, but Bayesian inference indicated the presence of two genetically differentiated clusters of individuals, which may correspond to different intercompatibility groups in S. luteus .• Heavy metal contamination seems to have a limited influence on the genetic structure of populations of S. luteus . Loss of diversity may have been prevented by sexual reproduction and rapid evolution of the tolerance trait or initial genetic bottlenecks may have been reduced by admixture and recurrent migration from surrounding populations colonizing noncontaminated soils.

Complementary DNA (cDNA)-amplified fragmentlength polymorphism (AFLP) was applied to analyze transcript profiles of a Zn-tolerant and a Zn-sensitive isolate of the ectomycorrhizal basidiomycete Suillus luteus, both cultured with and without increased external zinc concentrations. From the obtained transcript profiles that covered approximately 2% of the total expected complement of genes in S. luteus, 144 nonredundant, differentially expressed transcript-derived fragments (TDFs), falling in different classes of expression pattern, were isolated and sequenced. Thirty-six of the represented genes showed homology to function-known genes, whereas 6 matched unknown protein coding sequences, and 102 were possibly novel. Although relatively few TDFs were found to be responsive to the different zinc treatments, their modulated expression levels may suggest a different transcriptional response to zinc treatments in both isolates. Among the identified genes that could be related to heavymetal detoxification or the tolerance trait were genes encoding for homologues of a heat-shock protein, a putative metal transporter, a hydrophobin, and several proteins involved in ubiquitin-dependent proteolysis.

Zn pollution has triggered evolution for adaptive Zn tolerance in populations of Suilloid ectomycorrhizal fungi. The objectives of this study were to determine differential physiological responses that are linked to the Zn tolerance trait and to obtain more insight in the general mechanism responsible for the differential growth in Zn-enriched medium. Therefore, we identified intrinsic growth rates and element profiles in Zn-sensitive and Zn-tolerant genotypes. Isolates from Zn-polluted and unpolluted sites were exposed in vitro to increasing Zn(2+) stress. The Zn concentration which inhibits growth by 50% (EC(50)) was determined, and element (Zn, Fe, Mn, Cu, Mg, Ca and P) profiles in the mycelia were analysed. The intraspecific variation in growth rate and nutrient content of the in vitro grown mycelia is great and was not reduced in Zn-tolerant populations. The Zn resistance was not correlated to the intrinsic mycelial growth rate of the isolates or to the concentrations of the elements analysed, except for Zn. At low external Zn, Zn-resistant genotypes had lower Zn concentrations than sensitive isolates. At high external Zn, the differential Zn accumulation pattern between resistant and sensitive isolates became very prominent. Zn-exclusion mechanisms are most likely involved in the naturally selected adaptive Zn resistance. Other mechanisms of Zn detoxification such as sequestration of Zn on cell wall compounds or intracellular chelation and/or compartmentation are probably active but cannot explain the differential Zn sensitivity of the isolates.

Eight microsatellite loci were isolated from the ectomycorrhizal basidiomycete Suillus luteus using a dual‐suppression‐polymerase chain reaction (PCR) method. In a test sample of 40 isolates, the total number of alleles per locus and the expected heterozygosities ranged from five to 16 and from 0.532 to 0.811, respectively. These highly polymorphic markers allow an accurate description of the genetic diversity and structure of S. luteus populations.