Nickel hyperaccumulation, elemental profiles and agromining potential of three species of Odontarrhena from the ultramafics of Western Iran.

The profiles of trace and major elements in three Odontarrhena species from the ultramafics of Western Iran (O. callichroa, O. penjwinensis and O. inflata) were evaluated to provide detailed information on their soil-plant relationships and potentials for agromining. The mean concentrations of Ni in leaf dry matter of these three species were 877, 3,270 and 2,720 mg kg-1, respectively. The mean concentrations of total soil Ni at sites Mazi Ban, Kamyaran and Ghala Ga were 1,470, 2,480, 1,030 mg kg-1, respectively. The Bioconcentration Factor (BCF) for Ni was >1 in O. penjwinensis and O. inflata, but not in O. callichroa. A positive relationship between shoot Ni and soil pH was found for all three species. They display Ni hyperaccumulation in the leaves, but with pronounced variation in the Ni concentrations attained. Odontarrhena penjwinensis emerged as the most promising potential candidate for future Ni agromining. The progress made in this study will enable further consideration of the three Odontarrhena species, especially O. penjwinensis, for any future commercial Ni agromining of the serpentinic ultramafic soils in Western Iran.

This pioneering study in a remote part of Western Iran is the first to evaluate nickel hyperaccumulating species of Odontarrhena (Alyssum) in relation to their soil-plant relationships and potential for agromining.

Intensive cycling of nickel in a New Caledonian forest dominated by hyperaccumulator trees.

The hyperaccumulator Pycnandra acuminata is a New Caledonian rainforest tree known to have the highest concentration of nickel in any living organism, with 25 wt% nickel in its latex. All trees (with a diameter of >10 cm) and soil profiles in a 0.25-hectare permanent plot were sampled to assess the biogeochemical compartmentalisation of nickel in a dense stand of P. acuminata trees. Nickel stable isotope analysis permitted insights into the cycling of nickel in this ecosystem. The total tree biomass of the plot was calculated to be 281 tonnes ha-1 , which contained 0.44 kg of cobalt, 49.1 kg of manganese, 257 kg of nickel and 6.76 kg of zinc. Nickel stable isotope analysis identified the biotic origin of the nickel in the soil upper layers, with P. acuminata shoots enriched in lighter nickel isotopes. The δ60 Ni latex signature suggests that long-distance transport, radial xylem and phloem loading are at play in P. acuminata.

Isotopic signatures reveal zinc cycling in the natural habitat of hyperaccumulator Dichapetalum gelonioides subspecies from Malaysian Borneo.

BACKGROUND: Some subspecies of Dichapetalum gelonioides are the only tropical woody zinc (Zn)-hyperaccumulator plants described so far and the first Zn hyperaccumulators identified to occur exclusively on non-Zn enriched 'normal' soils. The aim of this study was to investigate Zn cycling in the parent rock-soil-plant interface in the native habitats of hyperaccumulating Dichapetalum gelonioides subspecies (subsp. pilosum and subsp. sumatranum). We measured the Zn isotope ratios (δ66Zn) of Dichapetalum plant material, and associated soil and parent rock materials collected from Sabah (Malaysian Borneo). RESULTS: We found enrichment in heavy Zn isotopes in the topsoil (δ66Zn 0.13 ‰) relative to deep soil (δ66Zn -0.15 ‰) and bedrock (δ66Zn -0.90 ‰). This finding suggests that both weathering and organic matter influenced the Zn isotope pattern in the soil-plant system, with leaf litter cycling contributing significantly to enriched heavier Zn in topsoil. Within the plant, the roots were enriched in heavy Zn isotopes (δ66Zn ~ 0.60 ‰) compared to mature leaves (δ66Zn ~ 0.30 ‰), which suggests highly expressed membrane transporters in these Dichapetalum subspecies preferentially transporting lighter Zn isotopes during root-to-shoot translocation. The shoots, mature leaves and phloem tissues were enriched in heavy Zn isotopes (δ66Zn 0.34-0.70 ‰) relative to young leaves (δ66Zn 0.25 ‰). Thisindicates that phloem sources are enriched in heavy Zn isotopes relative to phloem sinks, likely because of apoplastic retention and compartmentalization in the Dichapetalum subspecies. CONCLUSIONS: The findings of this study reveal Zn cycling in the rock-soil-plant continuum within the natural habitat of Zn hyperaccumulating subspecies of Dichapetalum gelonioides from Malaysian Borneo. This study broadens our understanding of the role of a tropical woody Zn hyperaccumulator plant in local Zn cycling, and highlights the important role of leaf litter recycling in the topsoil Zn budget. Within the plant, phloem plays key role in Zn accumulation and redistribution during growth and development. This study provides an improved understanding of the fate and behaviour of Zn in hyperaccumulator soil-plant systems, and these insights may be applied in the biofortification of crops with Zn.

The potential of Blepharidium guatemalense for nickel agromining in Mexico and Central America.

The aim of this study was to assess the potential of the woody nickel hyperaccumulator species Blepharidium guatemalense (Standl.) Standl. for agromining in southeastern Mexico. Pot trials consisting of nickel dosing (0, 20, 50, 100, and 250 mg Ni kg-1), and synthetic and organic fertilization were conducted. Field trials were also undertaken with different harvesting regimes of B. guatemalense. Foliar nickel concentrations increased significantly with rising nickel additions, with a 300-fold increase at 250 mg Ni kg-1 treatment relative to the control. Synthetic fertilization strongly increased nickel uptake without any change in plant growth or biomass, whereas organic fertilization enhanced plant shoot biomass with a negligible effect on foliar nickel concentrations. A 5-year-old stand which was subsequently harvested twice per year produced the maximum nickel yield tree-1 yr-1, with an estimated total nickel yield of 142 kg ha-1 yr-1. Blepharidium guatemalense is a prime candidate for nickel agromining on account of its high foliar Ni concentrations, high bioconcentration (180) and translocation factors (3.3), fast growth rate and high shoot biomass production. Future studies are needed to test the outcomes of the pot trials in the field. Extensive geochemical studies are needed to identify potential viable agromining locations. Novelty Statement Our research team is a pioneer in the discovery of metal hyperaccumulator plants in Mesoamerica with at least 13 species discovered in the last 2 years. This study is the first to assess the potential of nickel agromining (phytomining) in Mexico (and in all the American continent), using one of the strongest nickel hyperaccumulators reported so far. The promising results of this study are the basis for optimal agricultural management of Blepharidium guatemalense.

Hyperaccumulator Plants from China: A Synthesis of the Current State of Knowledge.

Hyperaccumulator plants are the material basis for phytoextraction research and for practical applications in decontaminating polluted soils and industrial wastes. China's high biodiversity and substantial mineral resources make it a global hotspot for hyperaccumulator plant species. Intensive screening efforts over the past 20 years by researchers working in China have led to the discovery of many different hyperaccumulators for a range of elements. In this review, we present the state of knowledge on all currently reported hyperaccumulator species from China, including Cardamine hupingshanensis (selenium, Se), Dicranopteris dichotoma (rare earth elements, REEs), Elsholtzia splendens (copper, Cu), Phytolacca americana (manganese, Mn), Pteris vittata (arsenic, As), Sedum alfredii, and Sedum plumbizincicola (cadmium/zinc, Cd/Zn). This review covers aspects of the ecophysiology and molecular biology of tolerance and hyperaccumulation for each element. The major scientific advances resulting from the study of hyperaccumulator plants in China are summarized and synthesized.

Two Blast-independent tools, CyPerl and CyExcel, for harvesting hundreds of novel cyclotides and analogues from plant genomes and protein databases.

MAIN CONCLUSION: Two high-throughput tools harvest hundreds of novel cyclotides and analogues in plants. Cyclotides are gene-encoded backbone-cyclized polypeptides displaying a diverse range of bioactivities associated with plant defense. However, genome-scale or database-scale evaluations of cyclotides have been rare so far. Here, a novel time-efficient Perl program, CyPerl, was developed for searching cyclotides from predicted ORFs of 34 available plant genomes and existing plant protein sequences from Genbank databases. CyPerl-isolated sequences were further analyzed by removing repeats, evaluating their cysteine-distributed regions (CDRs) and comparing with CyBase-collected cyclotides in a user-friendly Excel (Microsoft Office) template, CyExcel. After genome-screening, 186 ORFs containing 145 unique cyclotide analogues were identified by CyPerl and CyExcel from 30 plant genomes tested from 10 plant families. Phaseolus vulgaris and Zea mays were the richest two species containing cyclotide analogues in the plants tested. After screening protein databases, 266 unique cyclotides and analogues were identified from seven plant families. By merging with 288 unique CyBase-listed cyclotides, 510 unique cyclotides and analogues were obtained from 13 plant families. In total, seven novel plant families containing cyclotide analogues and 202 novel cyclotide analogues were identified in this study. This study has established two Blast-independent tools for screening cyclotides from plant genomes and protein databases, and has also significantly widened the plant distribution and sequence diversity of cyclotides and their analogues.

Agromining: farming for metals in the future?

Phytomining technology employs hyperaccumulator plants to take up metal in harvestable plant biomass. Harvesting, drying and incineration of the biomass generates a high-grade bio-ore. We propose that "agromining" (a variant of phytomining) could provide local communities with an alternative type of agriculture on degraded lands; farming not for food crops, but for metals such as nickel (Ni). However, two decades after its inception and numerous successful experiments, commercial phytomining has not yet become a reality. To build the case for the minerals industry, a large-scale demonstration is needed to identify operational risks and provide "real-life" evidence for profitability.

Irrigation water quality influences heavy metal uptake by willows in biosolids.

Phytoextraction is an effective method to remediate heavy metal contaminated landscapes but is often applied for single metal contaminants. Plants used for phytoextraction may not always be able to grow in drier environments without irrigation. This study investigated if willows (Salix x reichardtii A. Kerner) can be used for phytoextraction of multiple metals in biosolids, an end-product of the wastewater treatment process, and if irrigation with reclaimed and freshwater influences the extraction process. A plantation of willows was established directly onto a tilled stockpile of metal-contaminated biosolids and irrigated with slightly saline reclaimed water (EC ∼2 dS/cm) at a wastewater processing plant in Victoria, Australia. Biomass was harvested annually and analysed for heavy metal content. Phytoextraction of cadmium, copper, nickel and zinc was benchmarked against freshwater irrigated willows. The minimum irrigation rate of 700 mm per growing season was sufficient for willows to grow and extract metals. Increasing irrigation rates produced no differences in total biomass and also no differences in the extraction of heavy metals. The reclaimed water reduced both the salinity and the acidity of the biosolids significantly within the first 12 months after irrigation commenced and after three seasons the salinity of the biosolids had dropped to <15% of initial values. A flushing treatment to remove excess salts was therefore not necessary. Irrigation had an impact on biosolids attributes such as salinity and pH, and that this had an influence on metal extraction. Reclaimed water irrigation reduced the biosolid pH and this was associated with reductions of the extraction of Ni and Zn, it did not influence the extraction of Cu and enhanced the phytoextraction of Cd, which was probably related to the high chloride content of the reclaimed water. Our results demonstrate that flood-irrigation with reclaimed water was a successful treatment to grow willows in a dry climate. However, the reclaimed water can also change biosolids properties, which will influence the effectiveness of willows to extract different metals.

Organic-mineral colloids regulate the migration and fractionation of rare earth elements in groundwater systems impacted by ion-adsorption deposits mining in South China.

Ion-adsorption rare earth element (REE) deposits distributed in the subtropics provide a rich global source of REEs, but in situ injection of REEs extractant into the mine can result in leachate being leaked into the surrounding groundwater systems. Due to the lack of understanding of REE speciation distribution, particularly colloidal characteristics in a mining area, the risks of REEs migration caused by in situ leaching of ion-adsorption REE deposits has not been concerned. Here, ultrafiltration and asymmetric flow field-flow fractionation coupled with inductively coupled plasma mass spectrometry (AF4-ICP-MS) were integrated to characterize the size and composition of REEs in leachate and groundwater from mining catchments in South China. Results show that REEs were associated with four fractions: 1) the <1 kDa fraction including dissolved REEs; 2) the 1 - 100 kDa nano-colloidal fraction containing organic compounds; 3) the 100 kDa - 220 nm fine colloids including organic-mineral (Fe, Mn and Al (oxy)hydroxides and clay minerals); 4) the >220 nm coarse colloids and acid soluble particles (ASPs) comprising minerals. Influenced by the ion exchange effect of in situ leaching, REEs in leachate were mostly dissolved (79 %). The pH of the groundwater far from the mine site was increased (5.8 - 7.3), the fine organic-mineral colloids (46 % - 80 %) were the main vectors of transport for REEs. Further analysis by AF4 revealed that the fine colloids can be divided into mineral-rich (F1, 100 kDa - 120 nm) and organic matter-rich (F2, 120 - 220 nm) populations. The main colloids associated with REEs shifted from F1 (64 % ∼ 76 %) to F2 (50 % ∼ 52 %) away from the mining area. For F1 and F2, the metal/C molar ratio decreased away from the mining area and middle to heavy REE enrichment was presented. According to the REE fractionation, organic matter was the predominant component capable of binding REEs in fine colloids. Overall, our results indicate that REEs in the groundwater system shifted from the dissolved to the colloidal phase in a catchment affected by in situ leaching, and organic-mineral colloids play an important role in facilitating the migration of REEs.

Thallium hyperaccumulation status of the violets of the Allchar arsenic-thallium deposit (North Macedonia) confirmed through synchrotron µXRF imaging.

The abandoned Allchar Mine in the Republic of North Macedonia is a globally unique deposit with the highest known grades of thallium (Tl) and arsenic (As) mineralization. We aimed to determine the distribution of As and Tl in whole dehydrated shoots of the three Viola taxa using synchrotron micro-X-ray fluorescence analysis. Additionally, soil and plant organ samples were collected from all three Viola taxa at the Allchar site and analysed using inductively coupled plasma-atomic emission spectrometry. Concentrations of Tl were extremely high in all three Viola taxa (up to 58 900 mg kg-1), but concentrations of As were highly variable with V. tricolor subsp. macedonica and V. allchariensis having low As (up to 20.2 and 26.3 mg kg-1, respectively) and V. arsenica having the highest concentrations (up to 381 mg kg-1). The extremely high Tl in all three species is endogenous and not a result of contamination. Arsenic in V. tricolor subsp. macedonica and V. allcharensis is strongly affected by contamination, but not in V. arsenica where it appears to be endogenous. The pattern of As enrichment in V. arsenica is very unusual and coincides with Ca-oxalate deposits and Br hotspots. The results of this study could form the basis for more detailed investigations under controlled conditions, including plant dosing experiments.

Heavy metal contamination affects the core microbiome and assembly processes in metal mine soils across Eastern China.

Mining activities in metal mine areas cause serious environmental pollution, thereby imposing stresses to soil ecosystems. Investigating the ecological pattern underlying contaminated soil microbial diversity is essential to understand ecosystem responses to environment changes. Here we collected 624 soil samples from 49 representative metal mines across eastern China and analyzed their soil microbial diversity and biogeographic patterns by using 16 S rRNA gene amplicons. The results showed that deterministic factors dominated in regulating the microbial community in non-contaminated and contaminated soils. Soil pH played a key role in climatic influences on the heavy metal-contaminated soil microbial community. A core microbiome consisting of 25 taxa, which could be employed for the restoration of contaminated soils, was identified. Unlike the non-contaminated soil, stochastic processes were important in shaping the heavy metal-contaminated soil microbial community. The largest source of variations in the soil microbial community was land use type. This result suggests that varied specific ecological remediation strategy ought to be developed for differed land use types. These findings will enhance our understanding of the microbial responses to anthropogenically induced environmental changes and will further help to improve the practices of soil heavy metal contamination remediation.

Plant homeostasis of foliar manganese sinks: specific variation in hyperaccumulators.

Plant manganese (Mn) hyperaccumulation provides unusual insight into homeostasis of this essential micronutrient, in particular its excessive storage in shoot tissues. The compartmentation of hyperaccumulated foliar Mn appears exceptional among metal hyperaccumulators, since it occurs via specific microdistribution patterns. Here, three associated Mn hyperaccumulators, Virotia neurophylla, Maytenus fournieri, and Garcinia amplexicaulis exhibiting distinctly different Mn detoxification strategies were examined. Non-invasive sample preparation in conjunction with cryo scanning electron microscopy (SEM) was used to obtain in vivo quantitative microprobe X-ray and anatomical data from fully hydrated cells. Highly vacuolated large palisade mesophyll cells in V. neurophylla leaves were found to contain around 650 mM Mn. The large non-photosynthetic hypodermal cells of M. fournieri leaves, also with high vacuolar content, and the main site for Mn disposal, had an estimated mean vacuolar Mn concentration of around 600 mM. Previous qualitative X-ray mapping had shown Mn to be almost evenly sequestered across the entire leaf cross section of G. amplexicaulis. However, quantitative data obtained here showed a marked variation in localised concentrations that ranged between ~15 and >800 mM. Notable among these were mean values of >600 mM in spongy mesophyll cells, and ~800 mM within cells of a narrow sub epidermal layer preceding the palisade mesophyll. This study demonstrated the extraordinary Mn carrying capacities of different types of leaf cell vacuoles.

Phytoextraction of Cd-Contaminated Soils: Current Status and Future Challenges.

Cadmium (Cd) is one of the most toxic and widely distributed pollutants in the environment. Cadmium contamination of soils has posed a serious threat to safe food production in many parts of the world. The authors present a comprehensive review of present status of phytoextraction technology for cleaning up Cd-contaminated soils, based primarily on the data resulting from both laboratory and field-scale studies that have been conducted to assess or improve the Cd phytoextraction potential of various plant species in the past decade. The encouraging results of field-scale studies have provided a fundamental basis to usher phytoextraction technology into practical use to remediate slightly to moderately Cd-contaminated soils in Europe and Asia, although this technology is not yet ready for widespread application. Chelators and microorganisms tested so far seem not to contribute to the applicability of Cd phytoextraction. The major challenges for the large-scale application of Cd phytoextraction are (a) how to further improve the efficiency of Cd phytoextraction, (b) how to cut the overall costs of Cd phytoextraction, and (c) how to get greater stakeholders' acceptance of Cd phytoextraction as a reliable option.

Strategies of accumulation of potentially toxic elements in Minuartia recurva and M. bulgarica.

The aim of this study was to determine the concentrations of potentially toxic elements in soil samples and plant tissues of Minuartia recurva and M. bulgarica, predominantly or exclusively calcifuge species. Biological concentration (BCs) and translocation factors (TFs) were used to evaluate their accumulation potential. Considerable differences were observed between M. recurva and M. bulgarica assessions in terms of accumulation strategies of potentially toxic elements (PTEs). In M. recurva, most of the elements analyzed (Mn, Cu, Zn, Cd, and Co) were transported to the shoot, whereas in M. bulgarica, these elements remained predominantly in the roots. The Cu concentrations in the shoot samples of M. recurva from an abandoned iron-copper mine at Mt. Kopaonik were clearly above the notional hyperaccumulation threshold, characterizing this species as a possible Cu hyperaccumulator. Additionally, strong accumulation potential for Cr, Ni, Zn, Pb, and Cd was observed in M. recurva assessions, but without significant accumulation due to the low concentrations of these elements in the soils. The strong accumulation capacity and the different strategies in tolerance to PTEs indicate a potential of the two species for an application in phytoremediation: M. recurva for phytoextraction and M. bulgarica for phytostabilization.

Root responses to localised soil arsenic enrichment in the fern Pityrogramma calomelanos var. austroamericana grown in rhizoboxes.

The terrestrial fern Pityrogramma calomelanos, a cosmopolitan tropical species, is one of the strongest known arsenic (As) hyperaccumulator plants. This study aimed to determine whether P. calomelanos preferentially forages for arsenite (As3+) or arsenate (As5+) in As-contaminated soils, and whether a positive root response to As enhances accumulation in P. calomelanos. Therefore, an experiment using rhizoboxes divided in two halves were constructed with a control soil (C) and As3+ or As5+ dosed soil at either 50 and 100 µg g-1 As. Micro-X-ray Fluorescence elemental mapping (µXRF) was employed to analyze the distribution of As in roots and fronds, and Scanning Electron Microscopy with Energy Dispersive Spectroscopy (SEM-EDS) was used to determine As distribution in the reproductive tissues of P. calomelanos. The results showed that Pityrogramma roots do not specifically forage for As-contaminated soil; the area based on pixel counts was similar across all the treatments with no statistical differences. However, frond biomass was slightly higher in the treatments C ǀ As3+ and C ǀ As5+, and the highest accumulation of As in fronds was in the As5+ ǀ As3+ (100 µg g-1) treatment, with 3418 and 2370 µg g-1 in old and young fronds respectively. Arsenic cycling across the roots was observed by the µXRF mapping; in C ǀ As5+ (100) the As was higher and evenly distributed in both sections, whilst in C ǀ As3+ (50), the As was higher in the As3+ side. The µXRF mapping showed a broader As distribution in older fronds, where As was highest in the rachis and extended into the pinnule through the midrib. Pityrogramma calomelanos does not specifically root forage for As-enriched zones in the soil and grows healthily without signs of toxicity at lower (50 µg g-1) and higher (100 µg g-1) concentrations of As3+ and As5+ in the soil.

Are Grasses Really Useful for the Phytoremediation of Potentially Toxic Trace Elements? A Review.

The pollution of soil, water, and air by potentially toxic trace elements poses risks to environmental and human health. For this reason, many chemical, physical, and biological processes of remediation have been developed to reduce the (available) trace element concentrations in the environment. Among those technologies, phytoremediation is an environmentally friendly in situ and cost-effective approach to remediate sites with low-to-moderate pollution with trace elements. However, not all species have the potential to be used for phytoremediation of trace element-polluted sites due to their morpho-physiological characteristics and low tolerance to toxicity induced by the trace elements. Grasses are prospective candidates due to their high biomass yields, fast growth, adaptations to infertile soils, and successive shoot regrowth after harvest. A large number of studies evaluating the processes related to the uptake, transport, accumulation, and toxicity of trace elements in grasses assessed for phytoremediation have been conducted. The aim of this review is (i) to synthesize the available information on the mechanisms involved in uptake, transport, accumulation, toxicity, and tolerance to trace elements in grasses; (ii) to identify suitable grasses for trace element phytoextraction, phytostabilization, and phytofiltration; (iii) to describe the main strategies used to improve trace element phytoremediation efficiency by grasses; and (iv) to point out the advantages, disadvantages, and perspectives for the use of grasses for phytoremediation of trace element-polluted soils.

Nitrogen supply and cyanide concentration influence the enrichment of nitrogen from cyanide in wheat (Triticum aestivum L.) and sorghum (Sorghum bicolor L.).

Cyanide assimilation by the beta-cyanoalanine pathway produces asparagine, aspartate and ammonium, allowing cyanide to serve as alternate or supplemental source of nitrogen. Experiments with wheat and sorghum examined the enrichment of (15)N from cyanide as a function of external cyanide concentration in the presence or absence of nitrate and/or ammonium. Cyanogenic nitrogen became enriched in plant tissues following exposure to (15)N-cyanide concentrations from 5 to 200 microm, but when exposure occurred in the absence of nitrate and ammonium, (15)N enrichment increased significantly in sorghum shoots at solution cyanide concentrations of > or =50 microm and in wheat roots at 200 microm cyanide. In an experiment with sorghum using (13)C(15)N, there was also a significant difference in the tissue (13)C:(15)N ratio, suggestive of differential metabolism and transport of carbon and nitrogen under nitrogen-free conditions. A reciprocal (15)N labelling study using KC(15)N and (15)NH(4)(+) and wheat demonstrated an interaction between cyanide and ammonium in roots in which increasing solution ammonium concentrations decreased the enrichment from 100 microm cyanide. In contrast, with increasing solution cyanide concentrations there was an increase in the enrichment from ammonium. The results suggest increased transport and assimilation of cyanide in response to decreased nitrogen supply and perhaps to ammonium supply.

Population Genetics of Odontarrhena (Brassicaceae) from Albania: The Effects of Anthropic Habitat Disturbance, Soil, and Altitude on a Ni-Hyperaccumulator Plant Group from a Major Serpentine Hotspot.

Albanian taxa and populations of the genus Odontarrhena are most promising candidates for research on metal tolerance and Ni-agromining, but their genetic structure remains unknown. We investigated phylogenetic relationships and genetic differentiation in relation to distribution and ploidy of the taxa, anthropic site disturbance, elevation, soil type, and trace metals at each population site. After performing DNA sequencing of selected accessions, we applied DNA-fingerprinting to analyze the genetic structure of 32 populations from ultramafic and non-ultramafic outcrops across Albania. Low sequence divergence resulted in poorly resolved phylograms, but supported affinity between the two diploid serpentine endemics O. moravensis and O. rigida. Analysis of molecular variance (AMOVA) revealed significant population differentiation, but no isolation by distance. Among-population variation was higher in polyploids than in diploids, in which genetic distances were lower. Genetic admixing at population and individual level occurred especially in the polyploids O. chalcidica, O. decipiens, and O. smolikana. Admixing increased with site disturbance. Outlier loci were higher in serpentine populations but decreased along altitude with lower drought and heat stress. Genetic variability gained by gene flow and hybridization at contact zones with "resident" species of primary ultramafic habitats promoted expansion of the tetraploid O. chalcidica across anthropogenic sites.