Local Adaptation of Metallicolous and Non‐Metallicolous Anthyllis vulneraria Populations: Their Utilization in Soil Restoration

Restoration of metalliferous mine soils requires using plant species tolerant to high metal concentrations and adapted to nutrient‐poor soil. Legumes can increase plant productivity through N₂‐fixation, but they are often scarce in metalliferous sites. We examined survival, growth, and tolerance of four populations of a legume, Anthyllis vulneraria, from two metalliferous (MET) Zn‐Pb mine sites, Avinières (AV) ([Zn‐EDTA] = 26,000 mg/kg) and Eylie (EY) ([Zn‐EDTA] = 4,632 mg/kg), and two non‐metalliferous (NMET) sites located in the south of France with the aim to select the most appropriate populations for restoration of mined soils. In a common garden experiment, plants from each population were reciprocally grown in soil from the provenance of each population. The two NMET populations exhibited high mortality and low growth rates in soil from the mined sites. The AV MET exhibited a high growth rate in metalliferous soils, but showed high mortality in non‐metalliferous soils. The growth of the EY MET was very low in the AV‐contaminated soil, but was the highest of all populations in moderately and non‐metalliferous soils. Plants from the AV MET population showed a high growth and survival in metalliferous soil and would be appropriate in the restoration of metal‐contaminated sites (>30,000 mg Zn kg^?1). The EY MET population would be adapted to the restoration of moderate metal‐contaminated soils (<30,000 mg Zn kg^?1). Taking into account the broad distribution of A. vulneraria, these two populations could be suitable for the restoration of derelict mine sites in mediterranean and temperate regions of Europe and North America.     

The influence of cadmium,copper, lead,and zinc on the distribution and evolution of metallophytes in the British Isles

Mine spoils and other soils contaminated with cadmium, copper, lead and zinc show natural colonization by species which have strategies of avoidance or tolerance of metal toxicities. The distribution of plants on such substrata in the British Isles is examined in the light of present knowledge of such strategies. Evolutionary processes mediating the selection of tolerant individuals and ecotypic differentiation of adapted populations on metalliferous soils are considered. Other factors determining which species can and which cannot evolve tolerance include constitutional differences in species sensitivity to toxic metals, and phenotypic (environmentally-induced) tolerances. The importance of constitutional properties and phenotypic responses in providing explanations for plant distribution on metalliferous soils is assessed.     

A comparative study of metal levels in plants from two contrasting lead-mine sites

Summary An investigation into the levels of metals in plants growing on metalliferous soils was carried out. The exchangeable metal concentration of soils from Tyndrum and Trelogan was found to be extremely variable from sample to sample and the influence of soil pH on the exchangeable lead and zinc concentration is discussed. Large differences in heavy metal levels were found between species and may be indicative of different mechanisms of tolerance to lead and zinc excess. Large differences in calcium levels between plant tissues were also found. These may result from the use of calcium in ameliorating lead and zinc toxicity in some species.     

Genetic structure of Suillus luteus populations in heavy metal polluted and nonpolluted habitats

The genetic structure of populations of the ectomycorrhizal basidiomycete Suillus luteus in heavy metal polluted and nonpolluted areas was studied. Sporocarps were collected at nine different locations and genotyped at four microsatellite loci. Six of the sampling sites were severely contaminated with heavy metals and were dominated by heavy metal-tolerant individuals. Considerable genetic diversity was found within the geographical subpopulations, but no reduction of the genetic diversity, current or historic, was observed in subpopulations inhabiting polluted soils. The genetic differentiation between the geographical subpopulations was low, and no evidence for clustering of subpopulations from polluted soils vs. subpopulations from nonpolluted soils was found. These results indicate that heavy metal pollution has a limited effect on the genetic structure of S. luteus populations, and this may be due to the high frequency of sexual reproduction and extensive gene flow in S. luteus, which allows rapid evolution of the tolerance trait while maintaining high levels of genetic diversity.     

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.     

Genetic relationships among the torrent suckers, genus Thoburnia

Population samples of the three species of the tetraploid catostomid genus Thoburnia were electrophoretically studied to detect isozyme variability resolving the products of 54 alleles encoded by 34 presumptive loci. Species of Thoburnia express only about one-third of their loci in duplicate suggesting a high level of evolutionary advancement within the Catostomidae. Thoburnia hamiltoni and T. rhothoeca are phenetically close compared to T. atripinne. Cladistic analyses of the genetic data reveal that T. rhothoeca exhibits a relatively high degree of plesiomorphy while T. hamiltoni and T. atripinne represent extremes of separate lineages. A phylogenetic treatment of the genetic data yields a hypothetical phylogeny congruent with that based on morphological criteria.     

Heavy metals in soil affect reproductive processes more than morphological characters in Viola tricolor

Viola tricolor (Violaceae), a species very differentiated morphologically and showing intra- and interpopulation variability, occupies metalliferous (Zn, Pb, Cd, Cu) and nonmetalliferous sites through its geographic range. Here we analyzed morphological and anatomical features and also sexual reproduction in metallicolous and nonmetallicolous populations to determine whether and how they differ.Two-dimensional correspondence analysis based on selected morphological characters of vegetative and generative organs showed that plants from metalliferous soils did not form a compact group separated from those growing on nonmetalliferous soils. SEM of leaf and stipule anatomy showed differences in leaf hair ornamentation. There were significant differences in embryological processes in ovules and anthers: disturbed microsporogenesis (metallicolous 33% vs. nonmetallicolous 18%), lower pollen stainability (75% and 78% vs. 84% and 93%, depending on test), and higher frequency of degeneration in ovules (23% vs. 4%). These ultimately did not impede sexual reproduction of metallicolous populations but they do provide evidence that reproductive processes are sensitive to elevated heavy metals in soil and therefore can be viewed as a cost of metal tolerance.     

Research Priorities for Conservation of Metallophyte Biodiversity and their Potential for Restoration and Site Remediation

Plants that have evolved to survive on metal‐rich soils—metallophytes—have key values that must drive research of their unique properties and ultimately their conservation. The ability of metallophytes to tolerate extreme metal concentrations commends them for revegetation of mines and metal‐contaminated sites. Metallophytes can also be exploited in environmental technologies, for example, phytostabilization, phytoremediation, and phytomining. Actions towards conserving metallophyte species are imperative, as metallophytes are increasingly under threat of extinction from mining activity. Although many hundreds of papers describe both the biology and applications of metallophytes, few have investigated the urgent need to conserve these unique species. This paper identifies the current state of metallophyte research, and advocates future research needs for the conservation of metallophyte biodiversity and the sustainable uses of metallophyte species in restoration, rehabilitation, contaminated site remediation, and other nascent phytotechnologies. Six fundamental questions are addressed: (1) Is enough known about the global status of metallophytes to ensure their conservation? (2) Are metallophytes threatened by the activities of the minerals industry, and can their potential for the restoration or rehabilitation of mined and disturbed land be realized? (3) What problems exist in gaining prior informed consent to access metallophyte genetic resources and how can the benefits arising from their uses be equitably shared? (4) What potential do metallophytes offer as a resource base for phytotechnologies? (5) Can genetic modification be used to “design” metallophytes to use in the remediation of contaminated land? (6) Does the prospect of using metallophytes in site remediation and restoration raise ethical issues?     

The role of the root cell wall in the heavy metal tolerance ofAthyrium yokoscense

Cells of the roots ofA. yokoscense growing on metalliferous habitats were fractionated into their cell wall and cytoplasmic components. About 70–90% of the total copper, zinc and cadmium was located in the cell wall. Copper had a markedly greater affinity for the cell wall than zinc and cadmium, and was prevented from entering the cytoplasm. A large proportion of these heavy metals in the cell wall were exchanged as ions. The capacity of the cell wall for exchanging metal ions inA. yokoscense was higher than in other plants growing on metalliferous habitats. However, compared with different ferns unable to grow on metalliferous habitats, this capacity was not unique toA. yokoscense. Consequetly, the root cell wall ofA. yokoscense is considered to be an important site of metal ion storage and may play the role of an excretory organ for heavy metals. On the other hand, as proportion of the heavy metls was transported to the cytoplasm, where the metal content was much higher than the average for normal ferns. This would suggest thatA. yokoscense has another metabolic mechanism related to metal tolerance.     

Clonal integration and heavy-metal stress: responses of plants with contrasting evolutionary backgrounds

Physiological integration between ramets can ameliorate the growth and survival of clonal plants in spatially-heterogeneous environments, as ramets from favourable patches can provide support to those found in stressful patches. However, the advantage conferred by clonal integration might also depend on the evolutionary history of plants with regards to the presented stress. Here, we compared the benefit of clonal integration in response to the distribution of a heavy metal as a stress factor, and asked if this benefit would differ between ecotypes that have either undergone selection to tolerate heavy metals or not. In a greenhouse experiment, we grew pairs of connected and severed ramets of the metal hyperaccumulator Arabidopsis halleri, which originated from populations of either metalliferous or non-metalliferous soils. The ramets were grown in paired pots, which were contaminated with cadmium (Cd) either heterogeneously (100 or 0 ppm Cd per pot) or homogenously (50 ppm Cd per each pot). A. halleri ecotypes that originated from non-metalliferous soils performed better when ramets were connected and the distribution of Cd was heterogeneous. However, clonal integration had no effect on the performance of genotypes from metalliferous soils, regardless of the distribution of Cd. These results support the hypothesis that clonal integration is beneficial in stressful environments as long as the stress is patchily distributed, and particularly for plants that did not undergo selection to withstand it.     

Evolutionary dynamics of quantitative variation in an adaptive trait at the regional scale: The case of zinc hyperaccumulation in Arabidopsis halleri

Metal hyperaccumulation in plants is an ecological trait whose biological significance remains debated, in particular because the selective pressures that govern its evolutionary dynamics are complex. One of the possible causes of quantitative variation in hyperaccumulation may be local adaptation to metalliferous soils. Here, we explored the population genetic structure of Arabidopsis halleri at fourteen metalliferous and nonmetalliferous sampling sites in southern Poland. The results were integrated with a quantitative assessment of variation in zinc hyperaccumulation to trace local adaptation. We identified a clear hierarchical structure with two distinct genetic groups at the upper level of clustering. Interestingly, these groups corresponded to different geographic subregions, rather than to ecological types (i.e., metallicolous vs. nonmetallicolous). Also, approximate Bayesian computation analyses suggested that the current distribution of A. halleri in southern Poland could be relictual as a result of habitat fragmentation caused by climatic shifts during the Holocene, rather than due to recent colonization of industrially polluted sites. In addition, we find evidence that some nonmetallicolous lowland populations may have actually derived from metallicolous populations. Meanwhile, the distribution of quantitative variation in zinc hyperaccumulation did separate metallicolous and nonmetallicolous accessions, indicating more recent adaptive evolution and diversifying selection between metalliferous and nonmetalliferous habitats. This suggests that zinc hyperaccumulation evolves both ways—towards higher levels at nonmetalliferous sites and lower levels at metalliferous sites. Our results open a new perspective on possible evolutionary relationships between A. halleri edaphic types that may inspire future genetic studies of quantitative variation in metal hyperaccumulation.     

The effects of heavy metal pollution on genetic diversity in zinc/cadmium hyperaccumulator Sedum alfredii populations

The genetic diversity and population structure of seven populations of Sedum alfredii growing in lead/zinc (Pb/Zn) mine spoils or in uncontaminated soils from eastern and southern China were investigated using random amplified polymorphic DNA (RAPD) technology. Four of the sampled sites were heavily contaminated with heavy metals (Zn, Cd, Pb), and extremely high concentrations of Zn, Cd, and Pb were found among these corresponding populations. A significant reduction of genetic diversity was detected in the mining populations. The reduction of genetic diversity could be derived from a bottleneck effect and might also be attributed to the prevalence of vegetative reproduction of the mining populations. Analysis of molecular variance (AMOVA) and the unweighted pair group method with arithmetic mean (UPGMA) tree derived from genetic distances further corroborated that the genetic differentiation between mine populations and uncontaminated populations was significant. Polymorphism with the heavy metal accumulation capability of S. alfredii probably due to the genetic variation among populations and heavy metal contamination could have more impact on the genetic diversity and population structure of S. alfredii populations than geographic distance.     

Investigating heavy-metal hyperaccumulation using Thlaspi caerulescens as a model system

Background

Metal-hyperaccumulating plant species are plants that are endemic to metalliferous soils and are able to tolerate and accumulate metals in their above-ground tissues to very high concentrations. One such hyperaccumulator, Thlaspi caerulescens, has been widely studied for its remarkable properties to tolerate toxic levels of zinc (Zn), cadmium (Cd) and sometimes nickel (Ni) in the soil, and accumulate these metals to very high levels in the shoot. The increased awareness regarding metal-hyperaccumulating plants by the plant biology community has helped spur interest in the possible use of plants to remove heavy metals from contaminated soils, a process known as phytoremediation. Hence, there has been a focus on understanding the mechanisms that metal-hyperaccumulator plant species such as Thlaspi caerulescens employ to absorb, detoxify and store metals in order to use this information to develop plants better suited for the phytoremediation of metal-contaminated soils.

Scope

In this review, an overview of the findings from recent research aimed at better understanding the physiological mechanisms of Thlaspi caerulescens heavy-metal hyperaccumulation as well as the underlying molecular and genetic determinants for this trait will be discussed. Progress has been made in understanding some of the fundamental Zn and Cd transport physiology in T. caerulescens. Furthermore, some interesting metal-related genes have been identified and characterized in this plant species, and regulation of the expression of some of these genes may be important for hyperaccumulation.

Conclusions

Thlaspi caerulescens is a fascinating and useful model system not only for studying metal hyperaccumulation, but also for better understanding micronutrient homeostasis and nutrition. Considerable future research is still needed to elucidate the molecular, genetic and physiological bases for the extreme metal tolerance and hyperaccumulation exhibited by plant species such as T. caerulescens.Key words: Zn, Cd, Ni, Thlaspi caerulescens, hyperacumulator, phytoremediation, heavy metal     

Identification of nickel chelators in three hyperaccumulating plants: an X-ray spectroscopic study

We have investigated the accumulation of nickel in a hyperaccumulating plant from the Brassicacae family Leptoplax emarginata (Boiss.) O.E. Schulz. Two supplementary hyperaccumulating plants, which have been the subject of a high number of publications, Alyssum murale Waldst. & Kit and Thlaspi caerulescens J.&C. Presl, and a nonaccumulating species Aurinia saxatilis were also studied for reference. The plants were grown during 4 months in specific rhizoboxes with Ni-bearing minerals as a source of nickel. Nickel speciation was analyzed through X-ray absorption spectroscopy at Ni K-edge (X-ray absorption near edge spectroscopy and extended X-ray absorption fine structure spectroscopy) in the different parts of the plants (leaves, stems and roots) and compared with aqueous solutions containing different organo-Ni(II) complexes. Carboxylic acids (citrate, malate) appeared as the main ligands responsible of nickel transfer within those plants. Citrate was found as the predominant ligand for Ni in stems of Leptoplax and Alyssum, whereas in leaves of the three plants, malate appeared as the chelating organic acid of accumulated metal. Histidine could not be detected either in leaves, stems nor roots of any studied plant sample.     

IDENTICAL MAJOR GENE LOCI FOR HEAVY METAL TOLERANCES THAT HAVE INDEPENDENTLY EVOLVED IN DIFFERENT LOCAL POPULATIONS AND SUBSPECIES OF SILENE VULGARIS

Heavy metal tolerant Silene vulgaris plants, originating from different metalliferous sites in Germany and one in Ireland, were crossed to each other and to nontolerant plants from a nonmetalliferous site in The Netherlands. Analysis of the crosses suggested that there were two distinct major gene loci for zinc tolerance among a total of five tolerant populations. The tolerance loci for zinc, copper, and cadmium in the Irish plants were shown to be identical with those in the German populations. It is argued that the occurrence of common major genes for tolerance among different geographically isolated populations must have resulted from independent parallel evolution in local nontolerant ancestral populations. Each of the tolerances studied seems to be controlled by only a few specific major genes.     

Characterization of Ni-tolerant methylobacteria associated with the hyperaccumulating plant Thlaspi goesingense and description of Methylobacterium goesingense sp. nov

Various pink-pigmented facultative methylotrophic (PPFM) bacteria (strains iEII3, iEIV1, iEI6, iEII1, iEIII3 iEIII4, iEIII5, iRII1, iRII2, iRIII1, iRIV1 and iRIV2) were obtained from the rhizosphere and endosphere of hyperaccumulating plant Thlaspi goesingense grown in Redschlag, Austria [R. Idris, R. Trifonova, M. Puschenreiter, W.W. Wenzel, A. Sessitsch, Bacterial communities associated with flowering plants of the Ni hyperaccumulator Thlaspi goesingense, Appl. Environ. Microbiol. 70 (2004) 2667–2677]. Due to their unexpected diversity, abundance and nickel tolerance they were further characterized by detailed 16S rRNA gene analysis, DNA–DNA hybridization, fatty acid analysis, heavy metal tolerance, screening for known Ni resistance genes and phenotypic analysis. These strains were found to exhibit different multiple heavy metal resistance characteristics to Ni, Cd, Co, Zn and Cr. On the basis of their physiological and genotypic properties, strains could be grouped with Methylobacterium extorquens and M. mesophilicum. One endophyte, strain iEII3, was found to belong to a novel species for which the name M. goesingense is proposed.     

Zinc and cadmium hyperaccumulation by Thlaspi caerulescens from metalliferous and nonmetalliferous sites in the Mediterranean area: implications for phytoremediation

Growth, tolerance and zinc and cadmium hyperaccumulation of Thlaspi caerulescens populations from three metal contaminated soils and three normal soils were compared under controlled conditions. Individuals of six populations were cultivated on five soils with increasing concentrations of zinc (50–25000 μg g⁻¹) and cadmium (1–170 μg g⁻¹). There was no mortality of normal soil populations in the four metal-contaminated soils, but plant growth was reduced to half that of populations from metal-contaminated soils. However, in noncontaminated soil, the growth of individuals from normal soils was greater than that of individuals from metal-contaminated soils. Individuals from normal soils concentrated three times more zinc in the aboveground biomass than those from metal-contaminated soils, but the latter accumulated twice as much cadmium. We conclude that populations of T. caerulescens from both normal and metal-contaminated soils are interesting material for phytoextraction of zinc and cadmium, but to optimize the process of phytoextraction it is necessary to combine the extraction potentials of both type of populations.     

Genotypic variation among lineages of Trypanosoma cruzi and its geographic aspects

Isozyme analysis with 18 enzyme loci was conducted on 146 isolates of Trypanosoma cruzi from Mexico, Guatemala, Colombia, Ecuador, Peru, Brazil, Bolivia, Paraguay and Chile. Forty-four different MLGs (groups of isolates with identical multilocus genotypes) were identified and a phylogeny was constructed. The phylogenetic tree consisted of two main groups (T. cruzi I, T. cruzi II), and the latter was further divided into two subgroups (T. cruzi IIa, T. cruzi IIb–e). Evidence of hybridization between different MLGs of T. cruzi II was found, which means that genetic exchanges seem to have occurred in South American T. cruzi. On the other hand, the persistence of characteristic T. cruzi I and T. cruzi II isozyme patterns in single small villages in Bolivia and Guatemala suggested that genetic exchange is very rare between major lineages. A significant difference in genetic diversity was shown between T. cruzi I and T. cruzi II from several indices of population genetics. Two possibilities could explain this genetic variation in the population: differences in evolutionary history and/or different tendencies to exchange genetic material. Broad-scale geographic distributions of T. cruzi I and T. cruzi IIb–e were different; T. cruzi I occurred in Central America and south to Bolivia and Brazil, while T. cruzi IIb–e occurred in the central and southern areas of South America, overlapping with T. cruzi I in Brazil and Bolivia.     

金属型植物的研究进展

金属型植物是治理和修复土壤重金属污染的理想材料.综述了金属型植物的耐性机理、生殖生物学及进化生态学方面的研究进展,并对目前研究中存在的问题进行了分析和展望.