Assessing the taxonomic status of closely related taxa is crucial in plant systematics and can have important implications for conservation and human plant use. Ericaandevalensis Cabezudo & Rivera is a metallophyte endemic species from highly metal-polluted soils of SW Iberian Peninsula, an area with a mining history going back more than 5,000 years. Ericaandevalensis is closely related to Ericamackayana Bab., a northern Iberian species also present in western Ireland. The status of E.andevalensis as a species or subspecies subordinated to E.mackayana is subject to debate. Here, we assessed the genetic and phenotypic relationship between both species, including the population structure of E.andevalensis. We used high throughput sequencing to determine genome-wide Single Nucleotide Polymorphisms (SNPs), and morphometric analyses from 35 reproductive and vegetative traits. The morphological analysis showed at least eight characters that can discriminate the two species, from which ovary hairiness and the size of leaf glandular hairs were the most informative. Genetic analyses showed that each species formed a monophyletic cluster with full support, separated by an interspecific genetic distance >4-fold higher than intra-specific distance. Population genetic analyses of E.andevalensis shows that populations are highly structured, with the Portuguese one as the most isolated and less variable. These results support the recognition of E.andevalensis as a distinct species with a highly constrained ecological requirements and a narrow geographic distribution, but with a limited gene flow between populations. We discuss the implications of these outcomes in conservation policies and potential uses of E.andevalensis such as decontamination of polluted soils.

Self-sustaining vegetation in metal-contaminated areas is essential for rebuilding ecological resilience and community stability in degraded lands. Metal-tolerant plants originating from contaminated post-mining areas may hold the key to successful plant establishment and growth. Yet, little is known about the impact of metal toxicity on reproductive strategies, metal accumulation, and allocation patterns at the seed stage. Our research focused on the metal tolerant Atriplex lentiformis. Specifically, we examined the effects of toxic metal(loid) concentration in soils on variability in its reproductive strategies, including germination patterns, elemental uptake, and allocation within the seeds. We employed advanced imaging techniques like synchrotron X-ray fluorescence microscopy (2D scans and 3D tomograms) combined with inductively coupled plasma mass spectrometry to reveal significant differences in metal(loid) concentration and distribution within the seed structures of A. lentiformis from contrasting habitats. Exclusive Zn hotspots of high concentrations were found in the seeds of the metallicolous accession, primarily in the sensitive tissues of shoot apical meristems and root zones of the seed embryos. Our findings offer novel insights into phenotypic variability and metal tolerance and accumulation in plants from extreme environments. This knowledge can be applied to enhance plant survival and performance in land restoration efforts.

This study investigated the impact of several priming agents on metal-tolerant and sensitive Silene vulgaris ecotypes exposed to environmentally relevant cadmium dose. We analyzed how priming-induced changes in the level of lipid, protein, and DNA oxidation contribute to calamine (Cal) and non-calamine (N-Cal) ecotype response to Cd toxicity, and whether the oxidative modifications interrelate with Cd tolerance. In non-primed ecotypes, the levels of DNA and protein oxidation were similar whereas Cal Cd tolerance was manifested in reduced lipid peroxidation. In both ecotypes protective action of salicylic acid (SA) and nitric oxide (NO) priming was observed. SA stimulated growth and reduced lipid and DNA oxidation at most, while NO protected DNA from fragmentation. Priming with hydrogen peroxide reduced biomass and induced DNA oxidation. In N-Cal, priming diminished Cd accumulation and oxidative activity, whereas in Cal, it merely affected Cd uptake and induced protein carbonylation. The study showed that priming did not stimulate extra stress resistance in the tolerant ecotype but induced metabolic remodeling. In turn, the lack of adaptive tolerance made the sensitive ecotype more responsive to the benefits of the primed state. These findings could facilitate priming exploitation with a view of enhancing metallophyte and non-metallophyte suitability for phytoremediation and land revegetation.

Pistacia lentiscus L. is an excluder metallophyte proposed for the revegetation and phytostabilization of metal-contaminated sites in the Mediterranean area. The present study aims at evaluating the linking between bacterial communities and plants spontaneously growing in ecosystems chronically impacted by mining activities. Environmental properties and metal accumulation into hypogeal and epigeal tissues were analyzed in wild plants of two contrasting habitats with extreme metal contamination (> 2300 mg/kg for Zn, > 1100 mg/kg for Pb, > 10 mg/kg for Cd). The community structures of rhizospheric and root endophytic bacteria were fingerprinted by terminal restriction fragment length polymorphism of the 16S rRNA gene. The wild shrubs efficiently restrict the accumulation of the three major contaminants to the epigeal tissues in the two habitats under study (249 ± 68 mg/kg dw for Zn, 43 ± 21 mg/kg dw for Pb, and 1.4 ± 0.5 mg/kg dw for Cd). Evidence was provided that the combined but not individual effect of environmental conditions (moisture, inorganic carbon, pH) and proportion between Zn and Cd in the mine substrate play a role in structuring rhizosphere bacterial communities. The observed changes in community structures of root endophytes were found to be strongly associated with Pb level in roots and substrate properties (inorganic carbon and Zn/Cd ratio). Overall, our study highlights the importance of the analysis of multifactorial interactions among mine substrate, plant, and microbes for understanding how the environmental context affects phytoremediation under real conditions.

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.

Former mine sites can provide habitat for many rare specialised bryophyte species that have adapted to metal-rich soil conditions that are toxic to most other plant species. Some of the bryophyte species found in this habitat are facultative metallophytes, and others are regarded as strict metallophytes, the so-called \'copper mosses\'. It is a general assumption in the literature that Cephaloziella nicholsonii and C. massalongoi, both categorised as Endangered in the IUCN Red List for Europe, are also strict metallophytes and obligate copper bryophytes. This in vitro experiment investigated the growth and gemma production of these two species from different sites in Ireland and Britain on treatment plates of 0 ppm, 3 ppm, 6 ppm, 12 ppm, 24 ppm, 48 ppm and 96 ppm copper. Results show that elevated copper is not an obligate requirement for optimum growth. Differences in response to the copper treatment levels among populations evident within both species could possibly be due to ecotypic variation. A case is also made for the taxonomic revision of the Cephaloziella genus. Implications for the species\' conservation are discussed.

Tolerance to heavy metals in plants is a model process used to study adaptations to extremely unfavorable environments. One species capable of colonizing areas with high contents of heavy metals is Armeria maritima (Mill.) Wild. A. maritima plants growing in metalliferous areas differ in their morphological features and tolerance levels to heavy metals compared to individuals of the same species growing in non-metalliferous areas. The A. maritima adaptations to heavy metals occur at the organismal, tissue, and cellular levels (e.g., the retention of metals in roots, enrichment of the oldest leaves with metals, accumulation of metals in trichomes, and excretion of metals by salt glands of leaf epidermis). This species also undergoes physiological and biochemical adaptations (e.g., the accumulation of metals in vacuoles of the root\'s tannic cells and secretion of such compounds as glutathione, organic acids, or HSP17). This work reviews the current knowledge on A. maritima adaptations to heavy metals occurring in zinc-lead waste heaps and the species\' genetic variation from exposure to such habitats. A. maritima is an excellent example of microevolution processes in plants inhabiting anthropogenically changed areas.

Serpentine soils are rich in heavy metals and poor in nutrients, limiting plant species\' performance and survival. Nevertheless, specificities of such limitations as well as adaptability features required for thriving in serpentine environments are barely known. The Barberton Greenstone Belt in South Africa is an example of an area containing serpentine soil with adapted vegetation. In this study, a pot experiment was performed to compare development features (i.e., germination rates, leaf count, leaf length, biomass and photosynthetic capacity) during the early development of the non-serpentine species Berkheya radula, a genus consisting of known metal hyperaccumulators from serpentine areas in South Africa. B. radula was grown in serpentine soils taken from the Barberton region. B. radula leaves had heavy metals in concentrations that confirmed the species as a phytoextractor. There were trends for enhanced productivity and photosynthesis in the serpentine treatments compared to the control. Leaf count, leaf length, electron transport efficiency (ψEo/(1 - ψEo), density of reaction centers and PIABS,total were significantly and positively correlated with at least one of the heavy metals in the leaves. Germination rates were positively influenced by K, whereas biomass and the density of reaction centers were negatively affected by Ca and P, and only Ca, respectively. The heavy metals Zn, Ni and Co were positively correlated with each other, whereas they were negatively correlated with the macronutrients K, Ca and P. The latter correlated positively with each other, confirming higher fertility of the control soil. Our study suggests that B. radula exhibits metallophyte characteristics (i.e., preadapted), despite not naturally occurring on metal-enriched soil, and this provides evidence that the potential for bioaccumulation and phytoremediation is shared between serpentine and non-serpentine species in this genus.

Heavy metal-rich toxic soils and ordinary soils are both natural habitats of Arabidopsis halleri, a diploid perennial and obligate outcrosser in the sister clade of the genetic model plant Arabidopsis thaliana. The molecular divergence underlying survival in sharply contrasting environments is unknown. Here we comparatively address metal physiology and transcriptomes of A. halleri originating from the most highly heavy metal-contaminated soil in Europe, Ponte Nossa, Italy (Noss), and from non-metalliferous (NM) soils. Plants from Noss exhibit enhanced hypertolerance and attenuated accumulation of cadmium (Cd), and their transcriptomic Cd responsiveness is decreased, compared to plants of NM soil origin. Among the condition-independent transcriptome characteristics of Noss, the most highly overrepresented functional class of \'meiotic cell cycle\' comprises 21 transcripts with elevated abundance in vegetative tissues, in particular Argonaute 9 (AGO9) and the synaptonemal complex transverse filament protein-encoding ZYP1a/b. Increased AGO9 transcript levels in Noss are accompanied by decreased long terminal repeat retrotransposon expression. Similar to Noss, plants from other highly metalliferous sites in Poland and Germany share elevated somatic AGO9 transcript levels in comparison to plants originating from NM soils in their respective geographic regions. Transcript levels of Iron-Regulated Transporter 1 (IRT1) are very low and transcript levels of Heavy Metal ATPase 2 (HMA2) are strongly elevated in Noss, which can account for its altered Cd handling. We conclude that in plants adapted to the most extreme abiotic stress, broadly enhanced functions comprise genes with likely roles in somatic genome integrity maintenance, accompanied by few alterations in stress-specific functional networks.

The tolerance of Mentha crispa L. (garden mint) cultivated in cadmium-contaminated oxisol for 120 days was analyzed using plant growth variables such as height, the number of leaves and shoots in different Cd exposure periods, as well as assessing the metal concentration absorbed and accumulated in the plant parts (root, stem, and leaves). The maximum adsorption capacity was estimated at 9220 mg kg-1 and used as a reference to establish the different Cd concentrations to be applied in the soil. M. crispa showed tolerance and revealed a reduction of height, the number of leaves and shoots, root development, and secondary toxicity signs such as chlorosis and leaf wilting. Comparing to the stems and leaves, Cd was retained mainly in the roots. PERMANOVA showed that plant growth variables and Cd concentrations in the plant\'s part were affected by the Cd exposure time. The canonical discriminant analysis demonstrated height as the most affected variable until 45 growing days, and different responses were observed after 75 days. However, the number of shoots was the variable most affected by higher Cd concentrations. The bioaccumulation and translocation factors for all treatments were lower than one, indicating that M. crispa can be considered as an excluder plant and applied for a phytostabilization strategy.