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For almost half a century the biogeochemical, physiological, genetic and molecular aspects of metal tolerance, uptake and accumulation by plant species occurring in areas heavily polluted by toxic metals such as zinc, lead, copper, cadmium and nickel have received sustained attention (Baker, 1987; Macnair, 1987; Salt et al., 1998; Hall, 2002). This is linked with the increasing interest in phytoremediation, an environmental technology using heavy-metal-tolerant plants either to clean up polluted soils or to limit dispersal of toxic elements (Salt et al., 1998; Lasat, 2002). By contrast, the population genetics and biology of metal-tolerant plants have been relatively little investigated. If wild species are to be used and improved for phytoremediation, an accurate knowledge of their life history traits and genetic structure (notably breeding system, gene flow and genetic diversity organisation) is needed to estimate the feasibility and efficiency of the process in time and space as well as for developing conservation strategies and maintaining the potential for breeding cultivars.
In this context, previous studies on population genetics and population biology revealed some general features of metal-tolerant populations. In particular, metal-tolerant ecotypes were generally found to be more self-fertile than their nontolerant relatives. Self-fertility was assumed to be an adaptation to limit gene flow from nontolerant populations or to ensure offspring production to the first rare colonizers of the strongly selective metalliferous areas (Antonovics, 1968, 1972; Antonovics et al., 1971; Lefèbvre, 1970, 1973, Macnair, 1987; Ducousso et al., 1990). Genetic differentiation among tolerant populations was higher compared with nonmetal-tolerant ones possibly owing to founder effects coupled with severe selection on toxic soils (Vekemans & Lefèbvre, 1997). In most cases, metal tolerance evolved several times independently in time and space (Bush & Barrett, 1993, Vekemans & Lefèbvre, 1997).
Thlaspi caerulescens is a heavy metal hyperaccumulator considered to be a promising species for phytoextraction (Baker et al., 1994; Lasat et al., 2001; Lombi et al., 2002). It has a wide geographic distribution in Europe, from Scandinavia to the Mediterranean area and from central Europe to the Alps and the Pyrenées mountains (Koch et al., 1998). Remarkably, T. caerulescens occurs on both metalliferous and nonmetalliferous soils. In the Cévennes (southern France), Belgium and Luxembourg, nonmetallicolous populations have lower tolerance and higher zinc accumulation capacity compared with metallicolous ones (Meerts & van Isacker, 1997, Escarréet al., 2000, Reeves et al., 2001, and references therein). Compared with the rapidly growing literature on biogeochemistry and physiology, there has been very little work on the population genetics and biology of T. caerulescens. Koch et al. (1998), studying populations from northern and central Europe and the British Isles, found low levels of allozymic variation. They also provide some evidence based on allozymic polymorphism, that outcrossing rate varies among populations, possibly in relation to population density.
In this paper we use codominant allozymic markers, allozyme variation and pollen : ovule ratios to investigate (1) population genetic structure and (2) breeding system in metallicolous and nonmetallicolous populations from South France, Luxembourg and Belgium. The ratio of the number of pollen grains to the number of ovules per flower (P/O) was shown to decrease with selfing (Cruden, 1977) as predicted by theoretical models (Charlesworth & Charlesworth, 1981). In previous studies, P/O ranges from an average of 28 for strictly autogamous species to an average of 5860 for strictly allogamous species with intermediate values for mixed mating systems (Cruden, 1977).
We examine if the patterns of genetic variation are consistent with the hypothesis that metallicolous populations are more selfing than their nonmetallicolous relatives.
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Two independent indirect methods sets of data (within-population allozymic variation analysis (FIS) and pollen : ovule ratios) provide circumstantial evidence that nonmetallicolous populations of T. caerulescens may have higher selfing rates compared with metallicolous populations, although we cannot exclude the possibility that individuals may be issued of biparental inbreeding. This observation is consistent in the two geographical regions investigated. This observation is consistent in the two geographical regions investigated.
The P/O, as a measure of the relative allocation to male and female functions, is an indicator of the type of reproductive systems of plant species (Cruden, 1977), being lower in autogamous breeding systems compared to allogamous ones. The markedly lower P/O in the NONMET populations thus suggests that they are more selfing than their MET counterparts. Interestingly, the difference in P/O values is in the same direction for the two geographical regions, despite much lower P/O values in southern France. The P/O values of MET populations from Belgium (4000) are typical of strictly allogamous species following Cruden (1977), while the values for the populations from Luxembourg and from South of France (800–2000) are in the range of the ‘alternative allogamous’ category, a mixed mating system where allogamy predominates over autogamy. Koch et al. (1998) provide estimations of outcrossing rates for a few populations, ranging from 0 to 0.881, with widely overlapping ranges for the two ecotypes.
These results are surprising, considering that metallicolous populations are generally found to have higher selfing rates than their nonmetallicolous counterparts; see, for example, Anthoxanthum odoratum and Agrostis capillaris, (Antonovics, 1968, 1972; McNeilly & Antonovics, 1968), Armeria maritima (Lefèbvre, 1970, 1973) and Arrhenatherum elatius (Cuguen et al., 1989). Different mutually nonexclusive hypotheses have been put forward to explain these results. Antonovics (1968) suggested that selfing might evolve in metallicolous populations as a selected mechanism preventing breakdown of coadapted characters through gene flow from adjacent nontolerant populations. From a study on metallicolous isolated populations of A. maritima, Lefèbvre (1970) inferred that self-fertility in this species was more probably selected as a mechanism ensuring a reliable seed production in colonizing situations when individuals are at low density (Herlihy & Eckert, 2002). In a recent study, Vekemans & Lefèbvre (1997) concluded that the evolution of metal tolerance was not associated with reproductive barriers, as the pattern of partial reproductive barriers was related to gene flow and was similar for MET and NONMET populations. In Mimulus guttatus, Macnair & Christie (1983) found that reproductive barriers evolved as a pleiotropic effect of metal tolerance.
Our results for T. caerulescens are not well explained by Antonovics’ (1968) hypothesis for three main reasons. First, it is worth noting that MET and NONMET populations are not closely adjacent, being a few kilometres apart from each other in France and up to 80 km in Belgium and Luxembourg. Therefore, barriers to gene flow are not clearly advantageous at the present time for MET populations although this could have been the case at the time of their foundation. Second, Koch et al. (1998) found some evidence for a positive correlation between outcrossing rates and population density in T. caerulescens. The NONMET populations of T. caerulescens in South France mostly do consist of very few, sparse individuals (from 20 to less than 100 individuals in the five populations studied). In the same region, mine populations are generally much larger and denser (> 50 individuals m−2). Relatively small population size is also typical of NONMET populations from Luxembourg. By contrast, the MET population of Prayon is very large (> 100 000 individuals) and dense (> 10 individuals m−2). Plombières is also a large population, although it recently decreased in size because of reclamation of the old mine. Although it is not demonstrated in this study, these observations suggest that inbreeding selfing rate may conceivably have evolved in NONMET populations as a response to low density and/or small population size. Finally, NONMET populations typically occur in unstable habitats such as road verges while MET populations probably occur in habitat which that have remained unchanged since mining works ceased.
Very high FST values (compare our data with those compiled by Hamrick & Godt, 1989), indicative of restricted gene flow, were found for pairwise comparisons among the 10 French populations. Remarkably, this differentiation cannot be accounted for by interecotypic divergence, since FST values for interecotypic comparisons were generally low (FST = 0.097). This indicates that the two ecotypes, although differentiated for metal tolerance and accumulation (Escarréet al., 2000) are not distinct for allozymes which are assumed to represent neutral alleles. This may indicate that the ecotypic differentiation is relatively recent and/or that gene flows are still effective and reproductive isolation between MET and NONMET populations is not achieved.
In French populations, the positive correlation between the FST and the geographical distance is significant for the NONMET origin, in agreement with the isolation by distance model. This is not the case with for the MET populations. The relatively strong differentiation of the metallicolous population MG can be related to the long period of time elapsed since the mine was abandoned (fifteenth century; Bailly Maître, 1989) so that MG has evolved in isolation for several centuries. Populations from mining sites abandoned in the nineteenth and twentieth century are less differentiated from each other.
Seed dispersal by sheep may be the main factor of gene flow between the NONMET populations from the flat Causses plateaus, where T. caerulescens is most often found along the verges of paths and roads. Despite their geographical vicinity (less than 5 km in a straight line), MET populations are separated by landscape discontinuities such as steep hills and deep valleys, even if the landscape at the beginning of the twentieth century was open, mainly comprising cultivated terraces (Debussche et al., 1999). In addition, mine sites probably were not frequently visited by cattle and men because of the well-known toxicity of the plants and the soil. For this reason, gene flow among MET populations has probably been very limited and increased the isolation among the different mine sites.
The pattern of genetic differentiation is quite distinct in Belgium and Luxembourg. There, most of the variation can be ascribed to the ecotypic differentiation, most likely owing to the large geographic distance between MET and NONMET populations (80 km). The weak differentiation between Plombières and Prayon may be surprising, considering the relatively large distance between them (28 km). It is possible that population of Prayon, which started to be heavily polluted 150 yr ago, was founded by seeds transported from Plombières, a mining site known since the Roman period.
In conclusion, based on indirect evidence we have shown that metallicolous populations of T. caerulescens have a more allogamous reproductive system than nonmetallicolous populations. A more autogamous breeding system may have evolved in nonmetallicolous populations as a reproductive assurance mechanism, because these populations most often have a lower number of individuals and a lower density compared with metallicolous populations in the same region.