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Natural populations thriving in heavy metal contaminated ecosystems are often subjected to selective pressures for an increased resistance to toxic metals. Evolutionary adaptation to heavy metals is a well-documented process in several different groups of organisms including bacteria (Diels & Mergeay, 1990), animals (Levinton et al., 2003), marine algae (Nielsen et al., 2003), mosses (Shaw, 1988), etc. The phenomenon has been particularly studied in higher plants (Bradshaw & McNeilly, 1981; Baker et al., 1986; Schat & Verkleij, 1998). Zn-tolerant plant ecotypes are found on Zn-containing outcrops, on mine spoils, on soils heavily contaminated by Zn smelters or under galvanised metal constructions, such as fences and electricity pylons (Al-Hiyaly et al., 1990, 1993). The potential for the development of Zn tolerance has been demonstrated in monocotyledonous plants, in particular grasses (Al-Hiyaly et al., 1990). Evolution for metal tolerance in grasses can be very rapid because normal, nonadapted populations often contain a low frequency of Zn-tolerant individuals (Walley et al., 1974; Bradshaw & McNeilly, 1981; Al-Hiyaly et al., 1993). Relatively fewer dicots have been found to develop the Zn tolerance trait. These plants are often endemic to metalliferous soils and only a few species, for example Silene vulgaris, Thlaspi caerulescens and Arabidopsis halleri, have metallicolous and nonmetallicolous populations (Ernst, 1990; Assunção et al., 2003a). In trees with long reproductive cycles, the adaptive potential for metal tolerance seems to be low (Meharg & Cairney, 2000). Trees may resist extreme metal toxicity through large phenotypic plasticity and through their association with a small guild of well-adapted ectomycorrhizal (ECM) fungi (Wilkinson & Dickinson, 1995). Plant adaptation to selective pressures is often considered to be regulated by the plant genome, but it is evident that also mutualistic microorganisms can alleviate heavy metal toxicity in plants (Hall, 2002; Adriaensen et al., 2004).
Soil bacteria can adapt relatively quickly to toxic Zn environments (Díaz-Raviña & Bååth, 1996) and this might also be true for those soil-born microorganisms that live in mutualistic symbioses with plant roots (Wu & Lin, 1990; Meharg & Cairney, 2000; Lakzian et al., 2002). In a previous paper, we reported on the presence of a Zn-tolerant population of the ectomycorrhizal basidiomycete, Suillus luteus, in the immediate vicinity of the dismantled Zn smelter of Lommel-Maatheide in Belgium (Colpaert et al., 2000). The occurrence of several genets, some of which were quite large, as well as the yearly production of abundant basidiocarps of S. luteus on the Maatheide site suggest that the Zn-adapted genotypes are quite vigorous under the local toxic conditions. However, at that time, we did not know whether the Zn tolerance trait was present in other populations that were exposed to high Zn stress. Other surveys on Zn-contaminated sites in Europe could not demonstrate an increased Zn tolerance in S. luteus (Blaudez et al., 2000). Overall, there are still few reports that confirm that ECM fungi show evolutionary adaptation to heavy metal pollution (Hartley et al., 1997a; Meharg & Cairney, 2000).
Here, we further elaborate on the frequency and the spatial pattern of the Zn tolerance trait in ECM populations growing along a Zn gradient, which was caused in the previous century by the activities of several Zn smelters, all situated near the Belgian–Dutch border. We investigated fungal populations at 14 collection sites showing different degrees of Zn pollution. Because spores of basidiomycetes may be dispersed over long distances, we were curious to know how far the Zn tolerance trait could spread into populations that are hardly exposed to Zn contamination. Suillus luteus was the prime target species for the investigation, but because there were indications that adaptive Zn tolerance was also present in S. bovinus (Colpaert & Van Assche, 1987), this species was included in the study, as well as two other ECM fungi, Rhizopogon luteolus and Paxillus involutus. The two latter taxa were often present in the same fungal community as the Suillus species and both P. involutus and Rhizopogon sp. were previously reported from other Zn-polluted habitats (Denny & Wilkins, 1987; Turnau et al., 1996; Blaudez et al., 2000).
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The Zn concentrations in pine needles and soil pore waters illustrate that we are indeed studying ECM fungal communities situated along a gradient of Zn pollution. Both parameters were well correlated (Fig. 3). Zn in soil pore waters from the most polluted sites is high enough to cause toxicity in plants and microorganisms. The threshold for acute Zn toxicity in biosensor bacteria lies around 40 µm Zn in pore waters (Chaudri et al., 1999). In hydroponics, 40 µm Zn also causes reductions in root elongation in grasses not adapted to Zn stress (Al-Hiyaly et al., 1988) and 76 µm Zn reduces the growth as well as the N and P uptake capacity of Zn-sensitive S. bovinus associated with pine seedlings (Adriaensen et al., 2004).
The EC50 values for Zn toxicity obtained in this and other in vitro screening studies are very high. These EC50 concentrations as such should not be extrapolated to a field situation. We assume that the rich nutrient composition of most in vitro media used to cultivate and test microorganisms, including ECM fungi, is a major factor that determines the upward shift of the toxicity range. The isolates grow much faster in vitro than in symbiosis. The average relative growth rate of the S. bovinus isolates in the present study was 16% d−1; in an experiment with a host seedling it was only 3.4% d−1, even under optimal conditions (Adriaensen et al., 2004). However, the latter plant experiment also showed that the differential response obtained for a tolerant and a nontolerant isolate in vitro was maintained in symbiosis at much lower, more realistic Zn concentrations. Therefore, these in vitro tests can predict differences in growth when these fungi are exposed to elevated Zn in a contaminated soil.
Impacts of heavy metal pollution on plant communities and populations have received much more attention than effects on fungal communities or populations. Nevertheless, mycotrophic plants colonising extremely metal-polluted sites remain colonised by mycorrhizal fungi (Leyval et al., 1997). One might expect such sites to exert strong selection pressure on the symbiotic partners, a process that might result in genetic differentiation among fungal populations. To study such interpopulation differences, a large number of individuals must be studied, especially because in ECM fungi intraspecific variation as well as within population variation can be very large for many physiological responses (Cairney, 1999; Sawyer et al., 2003). If multiple genotypes are screened, frequency distributions can reveal interpopulation differences. In the present investigation, a strong differential response among the populations was observed in three of the four ECM species studied. The interpopulation differences in the response of S. luteus, S. bovinus and R. luteolus to Zn2+ are clearly related to the Zn status of their natural environment (Figs 4–7). This strong correlation between Zn tolerance and Zn pollution in three fungal species makes a strong case for a causal relationship. The Zn tolerance trait must be genetically determined because frequent subculturing (> 10 X) on basic medium does not cause a shift in the response towards elevated Zn. Physiological acclimation in Zn-exposed mycelia is likely but should disappear after subculturing on control medium.
Highest Zn tolerance was found in the populations of the Suilloid fungi, growing within a 5 km perimeter around the Zn smelters. Only in the recent plantation in Lr, it is not unequivocal whether tolerant isolates dominate (only two S. luteus isolates were tested, one of these being Zn-sensitive). The degree of Zn tolerance had a broad range within tolerant populations (Figs 5a and 6a), similar to what is found in metal-tolerant grass populations (Bradshaw & McNeilly, 1981; Al-Hiyaly et al., 1993). Mixed populations with both Zn-tolerant and nontolerant S. luteus isolates were found in E, Hh and Mm with, respectively, 62%, 32% and 17% of tolerant genotypes. The E and Hh populations are within the sphere of influence of the four northern Zn smelters. The Mm population is more than 30 km away from these Zn smelters but is relatively close to the former Dilsen smelter. We currently do not know whether there are Zn-tolerant Suillus populations in the immediate vicinity of the Dilsen factory. Populations situated more than 15 km from the Zn smelters are dominated by Zn-sensitive genotypes. This pattern in Zn tolerance suggests a gradual change from full Zn tolerance in the vicinity of the smelters to nontolerance in the remote sites. Pennanen et al. (1996) studied the effects of long-term heavy metal deposition on microbial community structure and the level of bacterial community metal tolerance in coniferous forest soils in the surroundings of the Rönnskär metal smelter in Sweden. In this area, an increased bacterial community metal tolerance was found up to 10–15 km from the smelter, a sphere of influence that is similar to the one we found here.
In the transition zone where we found Zn-tolerant and nontolerant Suillus genotypes we assume there is currently little selection pressure for Zn tolerance because plants and soils have almost normal Zn concentrations. Most fungal genets in E, Hh and Mm must have established less than 20 yr ago, in a period when atmospheric Zn deposition was negligible. Therefore, the presence of tolerant individuals might be caused by gene flow, bringing adapted genes for tolerance into normal Zn-sensitive populations. However, we cannot exclude the reverse hypothesis. If Zn deposition in the past was high enough in this zone to exert a temporary selection pressure then it is possible that Zn-sensitive genotypes are now migrating into areas previously occupied by tolerant genotypes. A low frequency of tolerant individuals might be present in normal Suillus populations. A similar phenomenon is known for grass species that can rapidly evolve metal-tolerant populations. Metal-tolerant mutants are present in normal unexposed grass populations at low frequencies, typically between 0.1 and 0.5% of the individuals (Walley et al., 1974; Bradshaw & McNeilly, 1981; Al-Hiyaly et al., 1993; Schat & Verkleij, 1998). A much larger number of fungal isolates should be screened to determine the frequency of Zn-tolerant mutants in normal unexposed populations.
The generation time of the Suilloids studied is relatively short. In primary plantations of pine, S. luteus and S. bovinus sporocarps show up 3–5 yr after planting and there are good indications that Suillus species invest relatively more energy in sexual reproduction than many other ectomycorrhizal fungi (Dahlberg & Finlay, 1999). High sporocarp productivity of R. luteolus has been reported from pine nurseries and its basidiospores have a high survival and inoculation potential (Molina et al., 1999). The short generation time and abundant reproduction may favour rapid selection for genotypes adapted to specific soil conditions.
The widespread distribution of Zn-tolerant S. luteus isolates even into areas with low contamination suggests high migration rates in these pioneer ECM fungi and a relatively low cost for the Zn tolerance. Some Rhizopogon and Suillus species are important colonisers of young conifer trees in pioneer conditions or in secondary successions after a major disturbance, such as stand-replacing fires (Baar et al., 1999; Jones et al., 2003). Spores seem to be the primary means by which these early colonist fungi colonise their hosts (Bruns et al., 2002). The fact that tolerant isolates are not rapidly outcompeted by the sensitive strains or by other pioneer fungi on nontoxic soils suggests that the cost for the tolerance might be relatively low. If Zn-tolerant fungi are less fit than normal fungi in unpolluted soil, then this should keep tolerance genes out of normal populations. If the differences in fitness are very small in clean soil, then tolerance genes can spread much farther away from the pollution source. On uncontaminated soils, metal-tolerant ecotypes are not necessarily inferior to their nontolerant counterparts (Schat & Verkleij, 1998; Rengel, 2000). Nevertheless, the elimination of a selection pressure, for example by cleanup, may lead to the disappearance of metal-tolerant populations as was observed for a Cd-adapted population of an oligochaete worm (Levinton et al., 2003). The speed of the disappearance is probably a function of the effective costs of tolerance, the mating system, gene flow and generation time.
Selection for increased Zn tolerance in Suillus sp. was not found in several other sites, affected by Zn deposition (Blaudez et al., 2000). Two explanations can be proposed for the discrepancy. Possibly selection pressure was not high enough because of low bio-availability of the metal. Determination of the available Zn fraction is a valuable tool to asses soil metal toxicity. Alternatively, local Suillus populations do not have the necessary mutant genes to develop Zn tolerance. Such a genetic constraint has been observed in grasses that normally can adapt to Zn pollution (Al-Hiyaly et al., 1993). However, this would also mean that these Zn-sensitive Suillus genotypes could survive in microsites with low toxicity. Here, we can mention that the Zn concentration in the pore water of the most polluted site (Lm) showed a large variation: from 80 to 190 µm Zn. Our investigation also indicates that P. involutus can survive on polluted sites even without specific genetic adaptation to the pollution, an observation supported by other reports (Denny & Wilkins, 1987; Blaudez et al., 2000). The presence of Zn-sensitive P. involutus genotypes in polluted habitats suggests that this species colonises soil patches with low bio-available Zn. Because organic matter is a good metal immobilising agent, Zn toxicity probably decreases when litter layers start to accumulate in the developing forest. It is likely that microsites with lower Zn concentrations can develop a few years after establishment of the tree seedlings.
The four ECM fungi studied showed a different pattern in Zn tolerance and sensitivity. On polluted sites S. luteus was on average more Zn-tolerant than S. bovinus and R. luteolus (Figs 5a, 6a and 7), and all three were more tolerant than P. involutus (Fig. 8a). However, on sites not under influence of the smelters, P. involutus populations seemed to be less sensitive towards Zn (Fig. 8b) than the Suillus populations (Figs 5c and 6c). EC50 values for P. involutus are in the same range as those observed in the 10 isolates tested by Denny & Wilkins (1987). Although we found adaptive Zn tolerance in three of the four ECM fungi studied, one should not conclude that genetic adaptation to high Zn stress is a very common phenomenon in ECM fungi, as it is also not the case in higher plants. Natural selection can more easily create an adapted population if the necessary variability is present in the original population. In higher plants, metal tolerance seems to be phylogenetically determined. Particular genera of higher plants (e.g. Silene, Agrostis, Festuca, Alyssum, Thlaspi) have a large potential for the development of metal-tolerant species and ecotypes (Ernst, 1990). Is it possible that the Suilloids have a similar genetic potential in the basidiomycetes? Many species in these plant and fungal genera are known as pioneer species. Early colonisers of metal-polluted spoils might have to cope with an extremely toxic environment with very few microsites where they can avoid metal toxicity. Less exposed microsites can develop as soon as vegetation can establish, followed by the immigration of organisms that are less adapted to extreme toxicity (Ernst, 1990). The production of litter and the biotransformations of heavy metals may play an important role in the local reduction of metal availability. Therefore, it is likely that selection pressure for higher tolerance decreases once the forest ecosystem becomes established when ‘late-stage’ fungi take over the dominance of the pioneer fungi. Ernst (1990) also indicates that the very first pioneer plants colonising mine spoils exhibit the highest metal tolerance and that these species create new niches for other organisms with a lower metal tolerance.
Plants with a high potential for metal adaptation often develop ecotypes that show multiple metal tolerances (Assunção et al., 2003b). In most cases multiple tolerance in a particular population is caused by the presence of toxic concentrations of specific metals that are present in the soil that supports this population. In the present investigation, we studied a Zn gradient caused by a number of Zn smelters. However, the Zn production in these factories was accompanied by a significant production, and thus emissions, of Cd, Cu and Pb. Bacteria collected at the Lm site show tolerance against Zn, Cu and Cd (Diels & Mergeay, 1990). At least some of the suilloids collected from the most polluted site also seem to have developed Cd tolerance, but only an exhaustive screening can give a better idea of possible multiple metal tolerances in these fungi.