Metallicolous Populations Research Articles

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.

Variation in copper and cobalt tolerance and accumulation among six populations of the facultative metallophyte Anisopappus chinensis (Asteraceae)

Advances on the ecology and evolution of adaptation to metal toxicity are based on studying metallophytes that are not restricted to soils strongly enriched in trace elements. The evolution of Cu and Co tolerance and accumulation, which principally occurs among the Copperbelt of Central Africa, is poorly known. In this paper, we studied Cu and Co tolerance and accumulation in a facultative metallophyte occupying a very broad ecological niche in southeastern Democratic Republic of Congo: Anisopappus chinensis (Asteraceae). The population variation in Cu and Co tolerance and accumulation was experimentally investigated using four metallicolous and two non-metallicolous populations from contrasted habitats. Surprisingly, Cu tolerance was poorly expressed in metallicolous populations grown in hydroponics, suggesting that specific rhizosphere processes may account for the ability to grow without toxicity symptoms under high Cu concentration on metalliferous soils. Population variation in Co tolerance and accumulation was demonstrated, which positively correlated to the concentration of Co in the native soil. Increased tolerance seems to have evolved in populations originating from Co-enriched soils. Foliar Co accumulation above 300 mg kg−1 dry weight with increased translocation was observed in the most tolerant populations, possibly making such populations promising materials to test for Co-phytomining applications.

Do arsenate reductase activities and oxalate exudation contribute to variations of arsenic accumulation in populations of Pteris vittata?

Although arsenic (As) hyperaccumulation is a constitutive property for Pteris vittata, there is intraspecific variation in As accumulation among metallicolous (from As-contaminated soils) and nonmetallicolous populations (from uncontaminated soils) and the related mechanisms is still not clear. Pot trials, hydroponic culture, and manual simulation were conducted to investigate the roles of arsenate reductase and root exudates in accumulating As in P. vittata, which were collected from two uncontaminated sites including Sun Yat-sen University campus, Guangdong Province (ZD), and a botanical garden in Guangxi Academy of Forestry Sciences, Nanning City, Guangxi Province (NN), and two As and Pb/Zn mining and/or smelting sites located in Shaoguan of Guangdong Province (SG) and Guiyang of Hunan Province (GY). The nonmetallicolous populations (ZD and NN) possessed more efficient uptake of arsenate and arsenite than the metallicolous populations (SG and GY). There were significant (p 67%). The NN population exuded 4.23 times more oxalate than the SG population. Root exudates from the NN population mobilized significantly (p < 0.05) more As from As-contaminated soils than those from the SG population, of which oxalate had the most effective in As mobilization. The present study suggests that higher arsenate reductase activities and oxalate exudation in the nonmetallicolous populations may play an important role in increasing their efficiency in phytoremediation of As-contaminated soils.

Henk Schat.

Henk Schat.

Adaptation to high zinc depends on distinct mechanisms in metallicolous populations of Arabidopsis halleri.

Zinc (Zn) hyperaccumulation and hypertolerance are highly variable traits in Arabidopsis halleri. Metallicolous populations have evolved from nearby nonmetallicolous populations in multiple independent adaptation events. To determine whether these events resulted in similar or divergent adaptive strategies to high soil Zn concentrations, we compared two A.halleri metallicolous populations from distant genetic units in Europe (Poland (PL22) and Italy (I16)). The ionomic (Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES)) and transcriptomic (RNA sequencing (RNA-Seq)) responses to growth at 5 and 150μM Zn were analyzed in root and shoot tissues to examine the contribution of the geographic origin and treatment to variation among populations. These analyses were enabled by the generation of a reference A.halleri transcriptome assembly. The genetic unit accounted for the largest variation in the gene expression profile, whereas the two populations had contrasting Zn accumulation phenotypes and shared little common response to the Zn treatment. The PL22 population displayed an iron deficiency response at high Zn in roots and shoots, which may account for higher Zn accumulation. By contrast, I16, originating from a highly Zn-contaminated soil, strongly responded to control conditions. Our data suggest that distinct mechanisms support adaptation to high Zn in soils among A.halleri metallicolous populations.

Contrasting cadmium resistance strategies in two metallicolous populations of Arabidopsis halleri.

While cadmium (Cd) tolerance is a constitutive trait in the Arabidopsis halleri species, Cd accumulation is highly variable. Recent adaptation to anthropogenic metal stress has occurred independently within the genetic units of A.halleri and the evolution of different mechanisms involved in Cd tolerance and accumulation has been suggested. To gain a better understanding of the mechanisms underlying Cd tolerance and accumulation in A.halleri, ionomic inductively coupled plasma mass spectrometry (ICP-MS), transcriptomic (RNA sequencing) and metabolomic (high-performance liquid chromatography-mass spectrometry) profiles were analysed in two A.halleri metallicolous populations from different genetic units (PL22 from Poland and I16 from Italy). The PL22 and I16 populations were both hypertolerant to Cd, but PL22 hyperaccumulated Cd while I16 behaved as an excluder both insitu and when grown hydroponically. The observed hyperaccumulator vs excluder behaviours were paralleled by large differences in the expression profiles of transporter genes. Flavonoid-related transcripts and metabolites were strikingly more abundant in PL22 than in I16 shoots. The role of novel A.halleri candidate genes possibly involved in Cd hyperaccumulation or exclusion was supported by the study of corresponding A.thaliana knockout mutants. Taken together, our results are suggestive of the evolution of divergent strategies for Cd uptake, transport and detoxification in different genetic units of A.halleri.

Paradoxical effects of density on measurement of copper tolerance in Silene paradoxa L.

This work investigated if the assessment of tolerance to trace metals can depend on plant density in the experimental design. A non-metallicolous and a metallicolous populations of Silene paradoxa were hydroponically cultivated at increasing density and in both the absence (-Cu conditions) and excess of copper (+Cu conditions). In -Cu conditions, the metallicolous population showed a lower susceptibility to plant density in comparison to the non-metallicolous one, explained by a higher capacity of the metallicolous population to exploit resources. In +Cu conditions, an alleviating effect of increasing density was found in roots. Such effect was present to a greater extent in the non-metallicolous population, thus making the populations equally copper-tolerant at the highest density used. In shoots, an additive effect of increasing plant density to copper toxicity was reported. Its higher intensity in the metallicolous population reverted the copper tolerance relationship at the highest plant densities used. In both populations, a density-induced decrease in root copper accumulation was observed, thus concurring to the reported mitigation in +Cu conditions. Our work revealed the importance of density studies on the optimization of eco-toxicological bioassays and of metal tolerance assessment and it can be considered the first example of an alleviating effect of increasing plant number on copper stress in a metallophyte.

Two Silene vulgaris copper transporters residing in different cellular compartments confer copper hypertolerance by distinct mechanisms when expressed in Arabidopsis thaliana.

Silene vulgaris is a metallophyte of calamine, cupriferous and serpentine soils all over Europe. Its metallicolous populations are hypertolerant to zinc (Zn), cadmium (Cd), copper (Cu) or nickel (Ni), compared with conspecific nonmetallicolous populations. These hypertolerances are metal-specific, but the underlying mechanisms are poorly understood. We investigated the role of HMA5 copper transporters in Cu-hypertolerance of a S.vulgaris copper mine population. Cu-hypertolerance in Silene is correlated and genetically linked with enhanced expression of two HMA5 paralogs, SvHMA5I and SvHMA5II, each of which increases Cu tolerance when expressed in Arabidopsis thaliana. Most Spermatophytes, except Brassicaceae, possess homologs of SvHMA5I and SvHMA5II, which originate from an ancient duplication predating the appearance of spermatophytes. SvHMA5II and the A. thaliana homolog AtHMA5 localize in the endoplasmic reticulum and upon Cu exposure move to the plasma membrane, from where they are internalized and degraded in the vacuole. This resembles trafficking of mammalian homologs and is apparently an extremely ancient mechanism. SvHMA5I, instead, neofunctionalized and always resides on the tonoplast, likely sequestering Cu in the vacuole. Adaption of Silene to a Cu-polluted soil is at least in part due to upregulation of two distinct HMA5 transporters, which contribute to Cu hypertolerance by distinct mechanisms.

Photosynthetic Efficiency as Bioindicator of Environmental Pressure in A. halleri.

In earlier ecophysiological studies that were conducted on Arabidopsis halleri plants, scientists focused on the mechanisms of Cd and Zn hyperaccumulation but did not take into consideration the environmental factors that can significantly affect the physiological responses of plants in situ. In this study, we investigated A. halleri that was growing on two nonmetalliferous and three metalliferous sites, which were characterized by different environmental conditions. We compared these populations in order to find differences within the metallicolous and nonmetallicolous groups that have not yet been investigated. The concentrations of several elements in the plant and soil samples also were investigated. To our knowledge, the concentration and fluorescence of chlorophyll were measured for A. halleri in situ for the first time. Our study confirmed the hyperaccumulation of Cd and Zn for each metallicolous population. For the metallicolous populations, the inhibition of parameters that describe the efficiency of the photosynthetic apparatus with increasing accumulations of heavy metals in the shoots also was observed. It was found that the nonmetallicolous plant populations from the summit of Ciemniak Mountain had larger antenna dimensions and chlorophyll content but a lower percentage of active reaction centers. To our knowledge, in this study, the internal high physiological diversity within the populations that inhabit metalliferous and nonmetalliferous sites is presented for the first time.

Effect of short-term Zn/Pb or long-term multi-metal stress on physiological and morphological parameters of metallicolous and nonmetallicolous Echium vulgare L. populations

The aim of the study was to determine the response of metallicolous and nonmetallicolous Echium vulgare L. populations to chronic multi-metal (Zn, Pb, Cd) and acute Zn (200, 400 μM) and Pb (30, 60 μM) stress. Three populations of E. vulgare, one from uncontaminated and two from metal-contaminated areas, were studied. Two types of experiments were performed – a short-term hydroponic experiment with acute Zn or Pb stress and a long-term manipulative soil experiment with the use of soils from the sites of origin of the three populations. Growth parameters, such as shoot and root fresh weight and leaf area, as well as organic acid accumulation were determined. Moreover, the concentration of selected secondary metabolites and antioxidant capacity in the three populations exposed to Pb or Zn excess were measured. Both metallicolous populations generally achieved higher biomass compared with the nonmetallicolous population cultivated under metal stress in hydroponics or on metalliferous substrates. Plants exposed to Pb or Zn excess or contaminated soil substrate exhibited higher malate and citrate concentrations compared with the reference (no metal stress) plants. It was observed that Zn or Pb stress increased accumulation of allantoin, chlorogenic and rosmarinic acids, total phenolics, and flavonoids. Moreover, it was shown that Pb sequestration in the roots or Zn translocation to the shoots may play a role in enhanced metal tolerance of metallicolous populations under acute Pb/Zn stress.

Differential elemental uptake in three pseudo-metallophyte C4 grasses in situ in the eastern USA.

Elemental uptake in serpentine floras in eastern North America is largely unknown. The objective of this study was to determine major and trace element concentrations in soil and leaves of three native pseudo-metallophyte C4 grasses in situ at five sites with three very different soil types, including three serpentine sites, in eastern USA. Pseudo-total and extractible concentrations of 15 elements were measured and correlated from the soils and leaves of three species at the five sites. Element concentrations in soils of pseudo-metallophytes varied up to five orders of magnitude. Soils from metalliferous sites exhibited higher concentrations of their characteristic elements than non-metalliferous. In metallicolous populations, elemental concentrations depended on the element. Concentrations of major elements (Ca, Mg, K) in leaves were lower than typical toxicity thresholds, whereas concentrations of Zn were higher. In grasses, species can maintain relatively low metal concentrations in their leaves even when soil concentrations are richer. However, in highly Zn-contaminated soil, we found evidence of a threshold concentration above which Zn uptake increases drastically. Finally, absence of main characteristics of serpentine soil at one site indicated the importance of soil survey and restoration to maintain serpentinophytes communities and avoid soil encroachment.

Morphometric and phytochemical profile of seeds of metallicolous and nonmetallicolous Echium vulgare populations

The morphometric and phytochemical parameters of seeds of three populations of Echium vulgare L. were compared. Two metallicolous populations (designated as MZ and MC) originated from waste deposits left over ZnPb ore smelting and processing and the reference population originated from an uncontaminated area (NM - nonmetallicolous population). The length, width, weight, and surface area of collected seeds were measured; additionally, the germination ability and vigour index for each population were determined. Moreover, heavy metal, seed oil, and secondary metabolite concentrations were measured. The results obtained indicated differentiation in the seed size and germination ability between the populations. The smaller size of seeds from the metallicolous populations was accompanied by lower germination ability; however, the seeds from the MC population achieved the highest vigour index. Furthermore, the MC population was characterized by the lowest concentration of allantoin and rosmarinic acid; additionally, the seeds of the MC population contained higher content of seed oils and increased contribution of saturated fatty acids and monounsaturated fatty acids by ca. 60 and 45%, respectively, and a decreased (by 16%) fraction of polyunsaturated fatty acids compared with the NM population. The strong differentiation of the MC population coincided with the high seed metal accumulation.

Carlina vulgaris L. as a Source of Phytochemicals with Antioxidant Activity.

The methanol extracts from three populations of Carlina vulgaris L. were examined for the chlorogenic acid content, mineral content, total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activity. Two populations originated from natural nonmetallicolous habitats (NN (populations from Nasiłów) and NP (populations from Pińczów)), and one metallicolous population (MB) was collected from Bolesław waste heap localized at the place of former open-cast mining of Ag-Pb and Zn-Pb ores dating back to the 13th century and 18th century, respectively. The level of Zn, Pb, Cd, Fe, Ni, and Mn was significantly higher in the root and leaves of MB plants as a result of soil contaminations compared to those of the NN and NP ones. The highest antioxidant potency has been showed by the plants growing in a nonmetallicolous habitat. The flower head extracts obtained from the nonmetallicolous populations also contained the largest amount of chlorogenic acid, whereas the lowest was determined in the roots (ca. 2–3.5 mg/g and 0.2–0.4 mg/g of air-dry weight, resp.). These studies provide important information on the influence of a habitat on the quality of herbal materials and the content of the biologically active primary and secondary metabolites.

Demographic history of the trace metal hyperaccumulator Noccaea caerulescens (J. Presl and C. Presl) F. K. Mey. in Western Europe.

Noccaea caerulescens (Brassicaceae) is a major pseudometallophyte model for the investigation of the genetics and evolution of metal hyperaccumulation in plants. We studied the population genetics and demographic history of this species to advance the understanding of among-population differences in metal hyperaccumulation and tolerance abilities. Sampling of seven to 30 plants was carried out in 62 sites in Western Europe. Genotyping was carried out using a combination of new chloroplast and nuclear neutral markers. A strong genetic structure was detected, allowing the definition of three genetic subunits. Subunits showed a good geographic coherence. Accordingly, distant metallicolous populations generally belonged to distinct subunits. Approximate Bayesian computation analysis of demographic scenarios among subunits further supported a primary isolation of populations from the southern Massif Central prior to last glacial maximum, whereas northern populations may have derived during postglacial recolonization events. Estimated divergence times among subunits were rather recent in comparison with the species history, but certainly before the establishment of anthropogenic metalliferous sites. Our results suggest that the large-scale genetic structure of N.caerulescens populations pre-existed to the local adaptation to metalliferous sites. The population structure of quantitative variation for metal-related adaptive traits must have established independently in isolated gene pools. However, features of the most divergent genetic unit (e.g. extreme levels of Cd accumulation observed in previous studies) question the putative relationships between adaptive evolution of metal-related traits and subunits isolation. Finally, admixture signals among distant metallicolous populations suggest a putative role of human activities in facilitating long-distance genetic exchanges.

Functional traits of a facultative metallophyte from tropical Africa: population variation and plasticity in response to cobalt

Divergence in functional traits and their plasticity can occur between metallicolous and non-metallicolous populations, driven by the selective pressure of metal toxicity in metalliferous soils, which is often associated with other edaphic constraints. The intraspecific variation of functional traits and their plasticity in response to cobalt in the soil, among populations of Anisopappus chinensis – a broad-niched metallophyte from southeastern D. R. Congo – have been experimentally investigated. Divergence in shoot height and leaf size was observed according to the ecological status of populations, with higher values occurring in plants from non-metalliferous soils. No clear differentiation in SLA was found between non-metallicolous and metallicolous populations. Metallicolous populations did not diverge in their leaf mineral composition and plastic response, with the exception of a lower leaf Co concentration on Co-contaminated soils. Functional leaf traits seemed to exhibit a homeostatic response to Co, which may account for the broad niche of the species. Further studies should include populations from other metallicolous and non-metallicolous habitats and include root and reproductive traits, to better understand resource acquisition strategies and adaptive response to Co toxicity.

Selected secondary metabolites in Echium vulgare L. populations from nonmetalliferous and metalliferous areas

The aim of this study was to evaluate the effect of severe environmental conditions prevailing on metalliferous waste heaps and heavy metal-contaminated growth substrates on accumulation of selected secondary metabolites, antioxidant capacity, and heavy metal concentration in two metallicolous (MC, MZ) and one nonmetallicolous (NM) populations of Echium vulgare L. The shoots and the roots of the three studied populations were collected from their natural habitats. Additionally, the plants were cultivated on different growth substrates, i.e. a contaminated substrate obtained from the areas of growth of the MZ and MC populations and an uncontaminated one from the NM population site. Several compounds, i.e. allantoin, rutin, rosmarinic acid, chlorogenic acid, and 4-hydroxybenzoic acid were identified in the shoots. Moreover, rosmarinic acid, allantoin, and shikonin were measured in the roots. The adverse environmental conditions contributed to a ca. 10- and 4-fold increase in the concentration of allantoin in the roots and shoots, respectively, as well as a ca. 4-fold and ca. 3-fold increase in the level of 4-hydroxybenzoic acid and shikonin, respectively, in comparison with the plants from the uncontaminated site. Similarly, a great impact of the contaminated substrate on the compounds was demonstrated in the soil experiment. Regardless of the populations, even ca. 20-fold higher levels of allantoin and shikonin were observed in plants grown on the MC and MZ substrates. In contrast, the chlorogenic acid concentration was lower in plants collected from the metalliferous areas and in all populations cultivated on the contaminated substrates in comparison with plants from the uncontaminated soil. Unambiguous results were obtained in the case of rutin, i.e. decreased accumulation in both metallicolous populations from the natural environment and increased accumulation in plants grown on the contaminated substrates. The high concentrations of heavy metals in the substrates contributed to high HM concentrations in plant tissues. However, some differences were observed between the metallicolous and nonmetallicolous populations – the accumulation of metals was lower in the shoots and higher in the roots of the NM population, compared with the MZ and MC populations.

Geography Plays a More Important Role than Soil Composition on Structuring Genetic Variation of Pseudometallophyte Commelina communis.

Pseudometallophytes are excellent models to study microevolution and local adaptation to soil pollution, as they can grow both on metalliferous and contrasting non-metalliferous soils. Although, there has been accumulating evidence for the effects of edaphic conditions and geographical isolation on the genetic structure of pesudometallophytes, it is still a difficult problem in evolutionary biology to assess their relative importance. In this study, we investigated the spatial patterns of genetic variability, population differentiation and genetic groups in pseudometallophyte Commelina communis with 12 microsatellite loci. Eight metallicolous and six non-metallicolous populations of C. communis were sampled from cupriferous sites and surrounding non-contaminated areas in China. Neither significant reduction in genetic diversity nor apparent founder and bottleneck effects were observed in metallicolous populations of C. communis. Based on Bayesian and Neighbor-Joining clustering analyses and a principal coordinates analysis, all sampled populations were found to be mainly separated into three genetic groups, corresponding well to their geographical locations rather than edaphic origins. Moreover, a significant and strong correlation between population genetic divergence and geographical distance were detected by Mantel test (r = 0.33; P < 0.05) and multiple matrix regression with randomization (MMRR; βD = 0.57, P < 0.01). However, the effect of copper concentration on genetic patterns of C. communis was not significant (MMRR; βE = -0.17, P = 0.12). Our study clearly demonstrated that the extreme edaphic conditions in metalliferous areas had limited effects on the genetic variability in C. communis. Geographic distance played a more important role in affecting the genetic structure of C. communis than soil composition did. In C. communis, the geographically disjunctive populations on metalliferous soils had multiple origins and evolved independently from nearby non-metallicolous populations.

Plant adaptation to metal polluted environments—Physiological, morphological, and evolutionary insights from Biscutella laevigata

Polluted soils promote rapid plant adaptation to high concentrations of trace metal elements. Biscutella laevigata is a little investigated pseudometallophyte that appears promising to study these evolutionary processes and advance understanding beyond existing model species, but its metal tolerance is insufficiently understood. We determined the zinc tolerance level and various plant responses to environmentally relevant zinc concentrations in ten metallicolous and non-metallicolous B. laevigata populations from southern Poland. In a two-phase hydroponic experiment, we scored multiple morphological and physiological traits (e.g. biomass, visible stress symptoms, element content in foliage) and assessed phenotypic variability within plant families. The structure of these quantitative traits was compared to that of neutral molecular markers to test, whether natural selection caused population differentiation in zinc tolerance. While zinc tolerance was species-wide (i.e. enhanced in all genotypes compared to a zinc sensitive reference species), we found congruent trends toward higher tolerance in metallicolous compared to non-metallicolous plants. The most indicative parameters for these differences were dry shoot biomass, dry root biomass, and particularly effective photosystem II yield. We found that enhanced zinc tolerance in metallicolous populations is driven by divergent selection in response to metal contamination. In addition, analyses of genotype x environment interaction indicated that this differentiation is heritable. These findings promote diploid accessions of B. laevigata as zinc tolerant but non-hyperaccumulating organisms to study plant adaptation to contaminated environments. Remarkably, tolerance differences between edaphic types emerged already at an environmentally relevant zinc concentration of 150μM. This opens an unusual perspective on plant adaptation that should be tested in other non-hyperaccumulating species.

Pb accumulation, Pb tolerance, antioxidants, thiols, and organic acids in metallicolous and non-metallicolous Peganum harmala L. under Pb exposure

The aim of the present study was to compare the effect of lead (Pb) exposure (0–50mgL−1 Pb) on Pb accumulation, growth, and the concentrations of thiol compounds, organic acids, antioxidant enzyme activities and other biochemical parameters between a non-metallicolous and a metallicolous harmal (Peganum harmala L.) population. Seedlings were grown in hydroponics, and exposed to 0, 5, 10, 25 and 50mgL−1 Pb for 14 days. Lead decreased dry weight to the same extent in both populations, in a concentration-dependent way. Pb accumulation was consistently higher in the metallicolous population than in the non-metallicolous one, both in roots and shoots, showing that the metallicolous plants were more tolerant to plant-internal Pb. Under Pb exposure the concentrations of malondialdehyde (MDA) and H2O2, as well as the LOX and GPX activities, were higher in the non-metallicolous population, albeit inconsiderably. The activities of antioxidant enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), as well as the concentrations of the non-enzymatic antioxidants GSH, ascorbic acid, and proline, were enhanced under Pb exposure, and overall higher in the metallicolous population, though only significantly so under Pb exposure. The concentrations of total thiols, non-protein thiols and protein bound thiols were also increased under Pb exposure. In particular, Pb strongly enhanced the average protein cysteine content in both populations, but much more so in the metallicolous plants than in the non-metallicolous ones. Furthermore, Pb-exposed plants showed significantly increased concentrations of organic acids. Citrate accumulation was strongly induced under Pb exposure, particularly in the metallicolous plants, suggesting that citrate is the most obvious candidate ligand for Pb in the vacuole. Overall, the quantitatively most remarkable differences in Pb response between the populations lie in the degrees of Pb-induced accumulation of proline and protein thiol.

Lower selfing rates in metallicolous populations than in non-metallicolous populations of the pseudometallophyte Noccaea caerulescens (Brassicaceae) in Southern France.

Background and Aims The pseudometallophyte Noccaea caerulescens is an excellent model to study evolutionary processes, as it grows both on normal and on heavy-metal-rich, toxic soils. The evolution and demography of populations are critically impacted by mating system and, yet, information about the N. caerulescens mating system is limited.Methods Mean selfing rates were assessed using microsatellite loci and a robust estimation method (RMES) in five metallicolous and five non-metallicolous populations of N. caerulescens in Southern France, and this measure was replicated for two successive reproductive seasons. As a part of the study, the patterns of gene flow among populations were analysed. The mating system was then characterized at a fine spatial scale in three populations using the MLTR method on progeny arrays.Key Results The results confirm that N. caerulescens has a mixed mating system, with selfing rates ranging from 0·2 to 0·5. Selfing rates did not vary much among populations within ecotypes, but were lower in the metallicolous than in the non-metallicolous ecotype, in both seasons. Effective population size was also lower in non-metallicolous populations. Biparental inbreeding was null to moderate. Differentiation among populations was generally high, but neither ecotype nor isolation by distance explained it.Conclusions The consequences of higher selfing rates on adaptation are expected to be weak to moderate in non-metallicolous populations and they are expected to suffer less from inbreeding depression, compared to metallicolous populations.