Genetic diversity of heavy metal-tolerant populations in Silene paradoxa L. (Caryophyllaceae): a chloroplast microsatellite analysis

Eight populations of Silene paradoxa L. (Caryophyllaceae) growing in copper mine deposits, in serpentine outcrops or in uncontaminated soil in central Italy were studied. Genetic diversity was estimated using five polymorphic chloroplast microsatellite loci (cpSSR), identifying 27 different chloroplast haplotypes. The effective number of alleles, the haplotypic diversity and a stepwise mutational model-based parameter (DSH2) were computed. The effective number of alleles observed within populations from copper mine deposits was 20% that of the serpentine neighbouring populations, suggesting the occurrence of a founder effect. Moreover, 13 of the 27 different haplotypes scored were exclusive to only one population, indicating genetic isolation for all tolerant populations. Even the copper-tolerant populations appeared to have evolved independently. Finally, analysis of molecular variance (AMOVA) of the cpSSR markers gave statistical significance to the grouping of populations according to their geographical location. This study demonstrates that cpSSR markers could be a useful complementary tool to isoenzymes or random amplified polymorphic DNA markers for elucidating the pattern of genetic differentiation in heavy metal-tolerant populations.     

Serpentine and non-serpentine Silene dioica plants do not differ in nickel tolerance

Serpentine soils are hostile to plant life. They are dry, contain high concentrations of nickel and have an unfavorable calcium/magnesium ratio. The dioecious plant Silene dioica (L.) Clairv. (Caryophyllaceae) is the most common herb on serpentine soils in the Swedish mountains. It also commonly grows on non-serpentine soils in the subalpine and coastal area. I have compared the germination frequency, plant establishment and growth of serpentine and subalpine non-serpentine populations in serpentine soil under greenhouse conditions. Further more I have studied the specific effect of nickel on root and shoot growth of serpentine and non-serpentine plants from the subalpine and coastal area in solutions with different concentrations of nickel. Plants from serpentine and non-serpentine populations grew well and in a similar fashion in serpentine soil. Moreover, S. dioica plants, irrespective of original habitat, tolerated enhanced concentrations of nickel when grown in solutions. An analysis of metal content in serpentine plants from natural populations shows that S. dioica has a higher nickel concentration in the roots than in the shoots. The growth studies show that S. dioica is constitutively adapted to serpentine, and that all populations have the genetic and ecological tolerance to grow on serpentine.     

ECOLOGICAL GENETICS OF SERPENTINE TOLERANCE IN THE MOSS,FUNARIA FLAVICANS: VARIATION WITHIN AND AMONG HAPLOID SIB FAMILIES

Many mosses occur both on and off serpentine soils, but experiments designed to test whether serpentine populations of widespread species display genetic adaptations for growth on serpentines have not been reported. Toward that end, two populations of the moss, Funaria flavicans, were grown on nutrient media varying in nickel and chromium concentration and in the ratio of magnesium to calcium. Three haploid siblings from each of five sporophytic families from the two populations were grown on five experimental media. There was no evidence that serpentine plants were more tolerant of nickel, chromium, high magnesium/calcium, or high nickel combined with high magnesium/calcium. In fact, plants from the nonserpentine population produced more protonemal growth than the serpentine plants on every medium except the control, on which plants from the two populations were indistinguishable. Large differences in nickel tolerance among haploid sib families (families of meiotic progeny derived from the same sporophyte) from the nonserpentine site provided evidence of genetic polymorphism in that population.     

Responses of two populations of an Iranian nickel-hyperaccumulating serpentine plant,Alyssum inflatum Nyar., to substrate Ca/Mg quotient and nickel

We selected two geographically close serpentine and non-serpentine populations of a Ni-hyperaccumulating plant (Alyssum inflatum) to investigate the influence of two common factors of serpentine soils: high Ni concentrations and low Ca/Mg quotients. Soils and plants were sampled from serpentine and non-serpentine substrates, and concentrations of Ca, Mg and Ni were measured. A hydroponic culture was used to compare growth and elemental composition responses of serpentine and non-serpentine plants to different Ca/Mg quotients and Ni concentrations in the nutrient solution. The Ca/Mg quotient for non-serpentine soils was 15 times higher than for serpentine soils, but there was no difference in the Ca/Mg quotient of plants from the two populations. In hydroponic culture, plants from both populations were able to survive at high Ca/Mg quotients. This result suggests that serpentine plants of A. inflatum do not necessarily need a substrate with a low Ca/Mg quotient for survival. Decreases in the Ca/Mg quotient in hydroponics decreased growth. The magnitude of this decrease was significantly greater in non-serpentine plants, suggesting a greater resistance of serpentine plants to low Ca/Mg quotients. Total Ni concentration in serpentine soils was 13 times higher than in non-serpentine soils, but ammonium nitrate-extractable concentrations of Ni in both soil types were similar. Ni concentrations in non-serpentine plants from their natural habitat were significantly lower than in serpentine plants, but there was no significant difference in Ni accumulation by plants of the two populations in hydroponic culture. However, increased concentrations of Ni in the hydroponic medium caused similar decreases in growth of both populations, indicating that Ni tolerance of the two populations was similar.     

Species adaptation in serpentine soils in Lesbos Island (Greece): metal hyperaccumulation and tolerance

Serpentine (ultramafic) soils, containing relatively high nickel and other metal concentrations, present a stressful environment for plant growth but also a preferred substrate for some plants which accumulate nickel in their tissues. In the present study we focused on: (1) the relationships between serpentine soils of Lesbos Island (Greece) and serpentinophilic species in order to test their adaptation to the ‘serpentine syndrome’, and (2) the Ni-hyperaccumulation capacity of Alyssum lesbiacum, a serpentine endemic, Ni-hyperaccumulating species, recorded over all its distribution for the first time. We sampled soil and the most abundant plant species from the four serpentine localities of Lesbos Island. Soil and leaf elemental concentrations were measured across all the sites. Our results confirmed our hypothesis that serpentinophilic species are adapted to elevated heavy metal soil concentrations but restricting heavy metal concentration in their leaves. We demonstrated that different A. lesbiacum populations from Lesbos Island present differences in Ni hyperaccumulation according to soil Ni availability. Our results highlighted the understanding of serpentine ecosystems through an extensive field study in an unexplored area. Alyssum lesbiacum and Thlaspi ochroleucum emerge as two strong Ni hyperaccumulators with the former having a high potential for phytoextraction purposes.     

Genetic diversity inferred from AFLP fingerprinting in populations of Onosma echioides (Boraginaceae) from serpentine and calcareous soils

Abstract

Onosma echioides is a non-obligate serpentinophytic borage occurring discontinuously on calcareous and serpentine outcrops at the northwest limit of its range. Mean concentrations of Ca, Mg and heavy metals in root and shoot samples of eight populations from the two soil types were first determined. Subsequently, the genetic polymorphism of the same accessions was estimated by means of Amplified Fragment Length Polymorphism (AFLP) fingerprinting technique. Root and shoot samples from serpentine outcrops showed higher levels of Ni, Cr and Mg, and lower Ca/Mg ratios compared with those from calcareous soils. Based on 353 polymorphic AFLP bands, the two edaphic groups of populations showed comparable levels of genetic diversity. A remarkable genetic differentiation between populations and a high level of within-population genetic variance were found. Results of Mantel's test supported a significant correlation between genetic and geographical distances, while no difference in relation to the edaphic factor was detected. Molecular data suggested isolation as the key factor shaping the infraspecific genetic structure of O. echioides, which may be in relation with the short-distance, zoochorous systems of seed dispersal and pollination of this species.     

Phytoextraction potential of the nickel hyperaccumulators Leptoplax emarginata and Bornmuellera tymphaea

Leptoplax emarginata and Bornmuellera tymphaea are nickel hyperaccumulators of the Brassicaceae family endemic to serpentine soils in Greece. The aims of this work were to compare the growth and uptake behavior of these plants with the Ni hyperaccumulator species Thlaspi caerulescens and Alyssum murale, and to evaluate their effect on soil Ni availability. Plants were grown for 3 mo on three soils that differ in Ni availability. Ni availability in soils was measuredby isotopic exchange kinetics and DTPA-TEA extractions. Results showed that L. emarginata produced significantly more biomass than other plants. On the serpentine soil, B. tymphaea showed the highest Ni concentration in shoots. However, Niphytoextraction on the three soils was maximal with L. emarginata. The high initial Ni availability of soil Serp (470.5 mg kg(-1)) was the main explanation for the high Ni concentrations measured in plant shoots grown on this soil, compared to those grown on soils Calc and Silt A. murale was the least efficient in reducing Ni availability on the serpentine soil L. emarginata appeared as the most efficient species for Ni phytoextraction and decrease of the Ni available pool.     

Soil calcium and plant disease in serpentine ecosystems: a test of the pathogen refuge hypothesis

Ecologists have long sought mechanistic explanations for the patterns of plant distribution and endemism associated with serpentine soils. We conducted the first empirical test of the serpentine pathogen refuge hypothesis, which posits that the low levels of calcium found in serpentine soils provide associated plants with a refuge from attack by pathogens. We measured the range of soil calcium concentrations experienced by 16 wild population of California dwarf flax (Hesperolinon californicum) and experimentally recreated part of this range in the greenhouse by soaking serpentine soils in calcium chloride solutions of varying molarity. When flax plants grown in these soils were inoculated with spores of the rust fungus Melampsora lini we found a significant negative relationship between infection rates and soil calcium concentrations. This result refutes the pathogen refuge hypothesis and suggests that serpentine plants, by virtue of their association with low calcium soils, may be highly vulnerable to attack by pathogens. This interaction between plant nutrition and disease may in part explain demographic patterns associated with serpentine plant populations and suggests scenarios for the evolution of life history traits and the distribution of genetic resistance to infection in serpentine plant communities.     

Distribution and Metal-Accumulating Behavior of Thlaspi caerulescens and Associated Metallophytes in France

The heavy metal hyperaccumulator Thlaspi caerulescens is widespread in France on many kinds of sites and substrates, including Zn/Pb/Cd mine and smelter wastes, Ni-rich serpentine outcrops and a variety of nonmetalliferous soils. Thlaspi caerulescens is remarkable among the metallophytes of France because it accumulates Zn to high concentrations (almost always >0.1%, and often >1% in the dry matter) regardless of the total Zn concentration of the substrate. The extraordinary uptake of Zn from soils of normal Zn concentration draws attention to the need for studies of the mechanisms by which such mobilization and uptake can occur. Different populations of Thlaspi caerulescens in France show considerable variation in their ability to accumulate Cd; individuals in some populations contain as much as 0.1 to 0.4% Cd, the highest levels recorded in vascular plants. The hyperaccumulation of Ni (sometimes exceeding 1%) from serpentine soils in France is also noteworthy. Despite the generally low biomass, some very large individuals occur, giving good potential for selective breeding to improve the value of Thlaspi caerulescens for phytoremediation, especially of Cd. The high Zn uptake from all kinds of soils is a property shared by the related T. brachypetalum, and T. alpinum shows dual Zn- and Ni uptake, depending on the substrate. The extent to which other species of Thlaspi occurring in France exhibit metal accumulation is also discussed.     

Patterns of introduction and adaptation during the invasion of Aegilops triuncialis (Poaceae) into Californian serpentine soils

Multiple introductions can play a prominent role in explaining the success of biological invasions. One often cited mechanism is that multiple introductions of invasive species prevent genetic bottlenecks by parallel introductions of several distinct genotypes that, in turn, provide heritable variation necessary for local adaptation. Here, we show that the invasion of Aegilops triuncialis into California, USA, involved multiple introductions that may have facilitated invasion into serpentine habitats. Using microsatellite markers, we compared the polymorphism and genetic structure of populations of Ae. triuncialis invading serpentine soils in California to that of accessions from its native range. In a glasshouse study, we also compared phenotypic variation in phenological and fitness traits between invasive and native populations grown on loam soil and under serpentine edaphic conditions. Molecular analysis of invasive populations revealed that Californian populations cluster into three independent introductions (i.e. invasive lineages). Our glasshouse common garden experiment found that all Californian populations exhibited higher fitness under serpentine conditions. However, the three invasive lineages appear to represent independent pathways of adaptation to serpentine soil. Our results suggest that the rapid invasion of serpentine habitats in California may have been facilitated by the existence of colonizing Eurasian genotypes pre‐adapted to serpentine soils.     

Genomic analysis of differentiation between soil types reveals candidate genes for local adaptation in Arabidopsis lyrata

Serpentine soil, which is naturally high in heavy metal content and has low calcium to magnesium ratios, comprises a difficult environment for most plants. An impressive number of species are endemic to serpentine, and a wide range of non-endemic plant taxa have been shown to be locally adapted to these soils. Locating genomic polymorphisms which are differentiated between serpentine and non-serpentine populations would provide candidate loci for serpentine adaptation. We have used the Arabidopsis thaliana tiling array, which has 2.85 million probes throughout the genome, to measure genetic differentiation between populations of Arabidopsis lyrata growing on granitic soils and those growing on serpentinic soils. The significant overrepresentation of genes involved in ion transport and other functions provides a starting point for investigating the molecular basis of adaptation to soil ion content, water retention, and other ecologically and economically important variables. One gene in particular, calcium-exchanger 7, appears to be an excellent candidate gene for adaptation to low CaratioMg ratio in A. lyrata.     

Nickel and copper tolerance and toxicity in three Tuscan populations of Silene paradoxa

Three Tuscan ecotypes of Silene paradoxa L. were studied to evaluate the occurrence of multiple tolerance or co-tolerance mechanisms and to underline some tolerance strategies in plants naturally adapted to toxic concentrations of heavy metals. Seeds were collected from non-toxic calcareous soil, a serpentine outcrop with high nickel content and a copper mine dump. The evaluation of the toxic effects of the metals on root growth showed the copper-tolerant population as nickel co-tolerant, whereas the opposite was not the case. This suggests the occurrence of a non-reciprocal co-tolerance mechanism.
The nickel-tolerant population seemed able to tolerate nickel by limiting its inhibiting effect on the peroxisomal H₂O₂ scavenging enzymes since, in the sensitive population, this inhibition revealed itself as one of the causes of nickel-induced oxidative stress. A very low copper root and shoot concentration seemed to be characteristic of the copper-tolerant population, combined with a low susceptibility to metal-induced oxidative stress.     

Gene flow inAgrostis tenuis (Poaceae)

The copper tolerance of both adult plants and their seedlings, ofAgrostis tenuis from sites within and outside, but neighbouring the Parys Mountain mine (North Wales) were studied. Intensive sampling was carried out along a line starting from the mine boundaries to a distance of five miles away in the direction of the prevailing wind, and of two miles in an upwind direction. The test have shown that mine populations are very tolerant. In outside populations on soils with reduced of very low copper content the mean tolerance level of the seedlings is considerably higher than that of corresponding adult plants, while both show a gradual decrease in tolerance with distance from the mine. The gene flow moves in the direction of the prevailing wind transfering tolerant characters from the mine to the normal populations. The selective pressures on the toxic soil of the mine are quite strong and permit ± tolerant plants only, while on normal soils these pressures are not so severe, so that populations may be rather heterogenous regarding their tolerance towards copper.     

Plant life on gypsum: a review of its multiple facets

The adaptation of plants to particular soil types has long intrigued biologists. Gypsum soils occupy large areas in many regions of the world and host a striking biological diversity, but their vegetation has been much less studied than that developing over serpentine or saline soils. Herein, we review all aspects of plant life on gypsum ecosystems, discuss the main processes driving their structure and functioning, and highlight the main conservation threats that they face. Plant communities in gypsum habitats typically show distinctive bands at very small spatial scales, which are mainly determined by topography. Plants living on gypsum soils can be classified into three categories: (i) wide gypsophiles are specialists that can penetrate the physical soil crust during early life stages and have physiological adjustments to cope with the chemical limitations imposed by gypsum soils; (ii) narrow gypsophiles are refugee plants which successfully deal with the physical soil crust and can tolerate these chemical limitations but do not show specific adaptations for this type of soils; and (iii) gypsovags are non‐specialist gypsum plants that can only thrive in gypsum soils when the physical crust is absent or reduced. Their ability to survive in gypsum soils may also be mediated by below‐ground interactions with soil microorganisms. Gypsophiles and gypsovags show efficient germination at low temperatures, seed and fruit heteromorphism within and among populations, and variation in seed dormancy among plants and populations. In gypsum ecosystems, spatio‐temporal changes in the composition and structure of above‐ground vegetation are closely related to those of the soil seed bank. Biological soil crusts (BSCs) dominated by cyanobacteria, lichens and mosses are conspicuous in gypsum environments worldwide, and are important drivers of ecosystem processes such as carbon and nitrogen cycling, water infiltration and run‐off and soil stability. These organisms are also important determinants of the structure of annual plant communities living on gypsum soils. The short‐distance seed dispersal of gypsophiles is responsible for the high number of very narrow endemisms typically found in gypsum outcrops, and suggests that these species are evolutionarily old taxa due to the time they need to colonize isolated gypsum outcrops by chance. Climate change and habitat fragmentation negatively affect both plants and BSCs in gypsum habitats, and are among the major threats to these ecosystems. Gypsum habitats and specialists offer the chance to advance our knowledge on restrictive soils, and are ideal models not only to test important evolutionary questions such as tolerance to low Ca/Mg proportions in soils, but also to improve the theoretical framework of community ecology and ecosystem functioning.     

Tolerance assessment of Cistus ladanifer to serpentine soils by developmental stability analysis

Developmental instability is the result of random environmental perturbations during development. Its absence (developmental stability) depends on an organism's ability to buffer environmental disturbances. Both genotype and environment influence the phenotypic expression of developmental instability and it is susceptible to selection pressure. We studied developmental instability (as indicated by increased within-individual asymmetry of repeated traits) in vegetative and reproductive structures of three populations of Cistus ladanifer L. living in different soil substrates (serpentine, siliceous and contact zone) to detect tolerance to serpentine soils. Serpentine soils, characterized by high concentrations of heavy metals (Ni, Cr, and Co), low levels of Ca/Mg ratio and high water deficit, can adversely affect plant performance. In this study we demonstrated that asymmetry and within-plant variance were higher in the contact zone population than either the silica or serpentine populations, proving the adaptation of C. ladanifer to serpentine soils. Within-population estimates of developmental instability were concordant for both vegetative and reproductive traits. There was little or no within-individual correlation among estimates of developmental instability based on different structures, i.e., plants that had highly asymmetric leaves always had high developmental instability in translational symmetry. Radial asymmetry of petals was negatively correlated with petal size, especially in silica soil plants, providing evidence of selection for symmetric and large petals. While leaf size was positively correlated with absolute fluctuating asymmetry, suggesting selection for small or intermediate size leaves. Serpentine soils presented the largest foliar and floral traits, as well as shoot elongation, while silica soil plants had the smallest scores. On the contrary, aboveground plant biomass was larger in silica soil plants, while the contact zone plants had the lowest biomass.     

Plant-by-Plant Variations of Bacterial Communities Associated with Leaves of the Nickel Hyperaccumulator Alyssum bertolonii Desv.

Bacteria associated with tissues of metal-hyperaccumulating plants are of great interest due to the multiple roles they may play with respect to plant growth and resistance to heavy metals. The variability of bacterial communities associated with plant tissues of three populations of Alyssum bertolonii, a Ni hyperaccumulator endemic of serpentine outcrops of Central Italy, was investigated. Terminal-restriction fragment length polymorphism (T-RFLP) analysis of bacterial 16S rRNA genes was applied to DNA extracted from leaf tissues of 30 individual plants from three geographically separated serpentine outcrops. Moreover, T-RFLP fingerprinting was also performed on DNA extracted from the same soils from which the plants were collected. Fifty-nine unique terminal-restriction fragments (TRFs) were identified, with more than half of the taxonomically interpreted TRFs assigned to Alpha- and Gamma-Proteobacteria and Clostridia. Data were then used to define the extent of variation of bacterial communities due to single plants or to plant populations. Results indicated a very high plant-by-plant variation of leaf-associated community (more than 93% of total variance observed). However, a core (numerically small) of plant-specific TRFs was found. This work demonstrates that plant-associated bacterial communities represent a large reservoir of biodiversity and that the high variability existing between plants, even from the same population, should be taken into account in future studies on association between bacteria and metal-hyperaccumulating plants.     

Genetic diversity and population structure of the serpentine endemic Calystegia collina (Convolvulaceae) in northern California

We used enzyme electrophoresis to evaluate genetic diversity in 32 populations of Calystegia collina, a clonal plant species endemic to serpentine outcrops in northern California (USA). Of 34 loci examined 56% were polymorphic, but on average only 17% were polymorphic within local populations. Neither the total number of alleles nor the number of multilocus genotypes differed significantly between populations in small vs. large serpentine outcrops. Genetic and geographic distances between populations were positively correlated, but this relationship was not significantly affected by the isolation of serpentine outcrops. Populations were highly differentiated (F(st) = 0.417) and little genetic variation was explained by geographic region or serpentine outcrop.Observed heterozygosity within populations almost always exceeded Hardy-Weinberg expectations. In many populations, all 30 sample ramets were uniformly heterozygous at one or more loci yet were genetically variable at other loci. These results imply that many C. collina populations originate from one or a few genetic founders, with little recruitment from seeds. Genetic variation within uniformly heterozygous populations must be the product of multiple, closely related founders or somatic mutations within the population. We conclude that vegetative reproduction, perhaps coupled with somatic mutation, helps maintain genetic diversity in these isolated but long-lived populations.     

Metal concentration and metal mass of metallicolous, non metallicolous and serpentine Noccaea caerulescens populations, cultivated in different growth media

Aims

Evaluate the genetic and environmental variability of metal concentration and metal mass of Noccaea caerulescens, from metalliferous (MET), non metalliferous (NMET) and serpentine (SERP) soils.

Methods

18 populations were cultivated in 18 different growth conditions, such as a soil mine tailing, soils amended with zinc (Zn), cadmium (Cd) and nickel (Ni) salts (in mixtures or in monometallic salts) and a hydroponic solution with two Zn concentrations.

Results

MET populations had Zn concentrations lower than NMET and SERP in the different soils but higher Cd mass (the product of aerial biomass and foliar metal concentration). SERP had the highest Ni concentration and Ni mass values. The addition of Cd or Ni to a Zn-contaminated soil significantly decreases Zn concentration. In hydroponics, MET and NMET had equivalent Zn concentrations but these were three times higher than those obtained in soil experiments. Zn mass of NMET was significantly lower than MET with the latter having Zn mass values largely above those obtained in mine soil.

Conclusions

Results showed a large heterogeneity of responses among populations depending on the substrate used, and it was not possible to correctly assign a single population to its accurate origin with only one experiment. Finally, data on metal concentration obtained in culture soils are closer to those in field soils than those from hydroponics so that they could give a more accurate information on the accumulating capacity of Noccaea caerulescens and its use in phytoextraction of metals in field conditions.     

Mutations in CAX1 produce phenotypes characteristic of plants tolerant to serpentine soils

Plant tolerance of serpentine soils is potentially an excellent model for studying the genetics of adaptive variation in natural populations. A large-scale viability screen of Arabidopsis thaliana mutants on a defined nutrient solution with a low Ca(2+) : Mg(2+) ratio (1 : 24 mol : mol), typical of serpentine soils, yielded survivors with null alleles of the tonoplast calcium-proton antiporter CAX1. cax1 mutants have most of the phenotypes associated with tolerance to serpentine soils, including survival in solutions with a low Ca(2+) : Mg(2+) ratio; requirement for a high concentration of Mg(2+) for maximum growth; reduced leaf tissue concentration of Mg(2+); and poor growth performance on 'normal' levels of Ca(2+) and Mg(2+). A physiological model is proposed to explain how loss-of-function cax1 mutations could produce all these phenotypes characteristic of plants adapted to serpentine soils, why 'normal' plants are unable to survive on serpentine soil, and why serpentine-adapted plants are unable to compete on 'normal' soils.     

Chloroplast microsatellites reveal that metallicolous populations of the Mediterranean shrub Cistus ladanifer L have multiple origins

Cistus ladanifer L. (Cistaceae) is a Mediterranean shrub covering different kinds of soils in the Western Mediterranean area. This species has colonised several metalliferous areas (serpentine outcrops as well as human-polluted sites) throughout its distribution range, and is therefore an interesting species to study the possible effects on genetic diversity and differentiation produced by the colonisation of areas polluted with heavy metals. The genetic structure of 33 natural populations distributed across its entire natural distribution range (Morocco, Portugal and Spain) and growing on either metalliferous or non-metalliferous soils was investigated using chloroplast microsatellites. Population genetic parameters were estimated and genetic groups were identified using Bayesian inference. In addition, we compared the genetic diversity and differentiation among metallicolous and non-metallicolous populations within each Bayesian-defined group. The cpSSR data suggested that metallicolous populations of Cistus ladanifer have arisen through multiple independent evolutionary origins within two different chloroplast lineages. Evidence that the soil type provoked genetic bottlenecks in metallicolous populations or genetic differentiation among metallicolous and non-metallicolous populations was not observed. Historical factors are the main cause of the present genetic structure of C. ladanifer. The nature of tolerance to heavy metals as a species-wide trait in this shrub is discussed.