Functional Trait-Based Screening of Zn-Pb Tolerant Wild Plant Species at an...

[Full title: Functional Trait-Based Screening of Zn-Pb Tolerant Wild Plant Species at an Abandoned Mine Site in Gard (France) for Rehabilitation of Mediterranean Metal-Contaminated Soils]

International Journal of Environmental Research and Public Health Article Functional Trait-Based Screening of Zn-Pb Tolerant Wild Plant Species at an Abandoned Mine Site in Gard (France) for Rehabilitation of Mediterranean Metal-Contaminated Soils Isabelle La ff ont-Schwob 1, * , Jacques Rabier 2 , V é ronique Masotti 2 , H é l è ne Folzer 2 , Lor è ne Tosini 1,2 , Laurent Vassalo 3 , Marie-Dominique Salducci 2 and Pascale Prudent 3 1 Aix Marseille University, IRD, LPED, IRD UMR 151, 13331 Marseille, France; lorene.tosini@univ-amu.fr 2 Aix Marseille University, Avignon Universit é , CNRS, IRD, IMBE UMR 7263, 13331 Marseille, France; rabier.jacques 3@gmail.com (J.R.); veronique.masotti@imbe.fr (V.M.); helene.folzer@imbe.fr (H.F.); marie-dominique.salducci@imbe.fr (M.-D.S.) 3 Aix Marseille University, CNRS, LCE, UMR 7376, 13331 Marseille, France; Laurent.Vassalo@univ-amu.fr (L.V.); pascale.prudent@univ-amu.fr (P.P.) * Correspondence: isabelle.schwob@univ-amu.fr Received: 22 June 2020; Accepted: 27 July 2020; Published: 30 July 2020 Abstract: The selection of plant species at mine sites is mostly based on metal content in plant parts. Recent works have proposed referring to certain ecological aspects. However, plant traits for plant metal-tolerance still need to be accurately assessed in the field. An abandoned Zn-Pb mine site in Gard (France) o ff ered the opportunity to test a set of ecological criteria. The diversity of micro-habitats was first recorded through floristic relev é s and selected categorical and measured plant traits were compared for plant species selection. The floristic composition of the study site consisted in 61 plant species from 31 plant families. This approach enabled us to focus on seven wild plant species naturally growing at the mining site. Their ability to form root symbioses was then observed with a view to phytostabilization management. Four species were considered for phytoextraction: Noccaea caerulescens (J. et C. Presl) FK Meyer, Biscutella laevigata L., Armeria arenaria (Pers.) Schult and Plantago lanceolata L. The metal content of their aerial and root parts was then determined and compared with that of soil samples collected at the same site. This general approach may lead to the development of a knowledge base for assessment of the ecological restoration trajectory of the site and can help in plant selection for remediation of other metal-rich soils in the Mediterranean area based not only on metal removal but on ecological restoration principles Keywords: passive ecological restoration; native plant selection; phytoremediation strategy; metal tolerance 1. Introduction Heavy metal contaminated soils raise major environmental and human health issues, which may be partially solved by phytoremediation technologies. This cost-e ff ective plant-based approach to remediation takes advantage of the remarkable ability of plants to concentrate trace elements from the environment and to metabolize a large number of molecules in their tissues to reduce their toxicity [ 1 , 2 ]. Consequently, on-site management of heavy metal contaminated soils can be achieved either by using metal hyperaccumulators that are plant species accumulating exceptionally large amounts of heavy metals in their tissues [ 3 ] or by using a biocontainment method such as phytostabilization [ 4 ]. In most of the cases, plant selection is assessed for commercial phytoremediation (preferentially phytoextraction but not excluding phytostabilization) and is designed on agronomy principles using Int. J. Environ. Res. Public Health 2020 , 17 , 5506; doi:10.3390 / ijerph 17155506 www.mdpi.com / journal / ijerph

Int. J. Environ. Res. Public Health 2020 , 17 , 5506 2 of 22 crops and amendments [ 5 – 7 ] and far less on the basis of ecological concepts such as trait-based selection [ 8 – 10 ]. Initially attracting attention for their potential use in phytomining, hyperaccumulators have been increasingly of interest from an evolutionary or ecological perspective. Studies on hyperaccumulators and more generally on metallophytes have led to a taxonomic approach with a focus on botanical families hosting numerous metal-tolerant species such as Brassicaceae, Poaceae, Fabaceae and Asteraceae in Mediterranean areas. This first selection grid is of interest but remains at a preliminary stage and is not always accurate (i.e., Plantaginaceae). Moreover, facultative hyperaccumulators are dominant in metalliferous soils, and for the purpose of the accurate selection of plant species for metalliferous soil revegetation, it is worthwhile to distinguish species-wide versus population-specific metal tolerance following Pollard et al. [ 3 ]. Plant functional traits have been considered as a promising tool for the selection of plants for the purpose of ecological restoration [ 10 ]. Life cycle is one of the main traits to be considered for phytostabilization, perennial species being preferred. Most of those recent studies dealt with above-ground-traits and very few with below-ground-traits, which are less easy to access. However, the very first interaction between plant species and trace metals and metalloids (TMM)-rich soil is focused at the root and rhizosphere level. Deep root system and root symbioses are of great importance in the mechanisms of plant tolerance to heavy metals [ 11 , 12 ]. The analysis of the spontaneous processes of plant colonization of abandoned mine sites may contribute to developing better key selection criteria for suitable plant species for on-site TMM management. Moreover, plant ecotypes in the mining sites are tolerant to mixed toxic metals in the soil solution and not only of a single metal such as Zn. For example, it has been recently demonstrated that Silene vulgaris ecotypes from calamine soils were fully adapted to heavy metal mixture (Zn, Pb and Cd) compared to ecotypes from both serpentine and non-metallicolous soils [ 13 ]. These adaptations depend on their capacity for reactive oxygen species scavenging and TMM compartmentalization in plant parts to avoid their toxicity [ 2 , 6 ]. Selecting ecotypes from a Zn-Pb mining site may be of interest for rehabilitation of multi-metal-contaminated soils Our hypothesis is that the flora spontaneously colonizing an abandoned mine site shares common above-ground and below-ground plant traits, i.e., Raunkiær life form, Grime strategy, root system type and, mycorrhizal status. We also considered botanical family as a functional trait, as phylogenetic relatedness is a strong driver for associations with fungal rhizosphere communities [ 14 ]. These common features may help in plant species selection for mine rehabilitation. For that purpose, a Zn-Pb-rich abandoned mine site without any previous rehabilitation implemented was selected. The diversity of micro-habitats was recorded through floristic relev é s and the selected traits and plant characteristics were compared for plant species selection. A second hypothesis is that phytostabilization and phytoextraction processes may occur concomitantly in abandoned mine sites due to the diversity of plant adaptations in the plant community. This will be discussed through the literature data on plant species known for their hyperaccumulation, phytoaccumulation or phytostabilization abilities Using a plant selection grid, seven plant species were selected to study their ability to form root symbioses for the purpose of phytostabilization management. Four plant species were also considered for phytoextraction. The metal content of their aerial and root parts was then determined and compared with that of soil samples collected at the same site. This general approach has led to the development of a knowledge base for the assessment of the ecological restoration trajectory of the site and can help in plant selection for on-site remediation of other metal-rich soil in the Mediterranean area 2. Materials and Methods 2.1. Site Location The study site is located in the town of Rousson in Gard (Figure 1 ), and more specifically at a place called La Gardie. It is an abandoned mine site which was used for the extraction of zinc. Established in 1876, the Rousson concession (310 ha) included the sites of Landas, Font de Rouve, and Croix de Fauvie

Int. J. Environ. Res. Public Health 2020 , 17 , 5506 3 of 22 (study site). These deposits, located in interlocking limestones, were superficial deposits made up of pieces of shale in ferruginous soil which contained 20 to 40% of zinc. The ore was processed in calamine furnaces. At the time when this small surface mining operation was closed down in 1910, it employed 8 miners and 5 laborers. Today, there are only a few excavations left that spontaneous vegetation colonization is gradually filling up. These red soils, containing wastes from the mine rich in metals, are potentially colonized by a specific flora adapted to metal-rich soils Int. J. Environ. Res. Public Health 2020 , 17 , x 3 of 21 2. Materials and Methods 2.1. Site Location The study site is located in the town of Rousson in Gard (Figure 1), and more specifically at a place called La Gardie. It is an abandoned mine site which was used for the extraction of zinc. Established in 1876, the Rousson concession (310 ha) included the sites of Landas, Font de Rouve, and Croix de Fauvie (study site). These deposits, located in interlocking limestones, were superficial deposits made up of pieces of shale in ferruginous soil which contained 20 to 40% of zinc. The ore was processed in calamine furnaces. At the time when this small surface mining operation was closed down in 1910, it employed 8 miners and 5 laborers. Today, there are only a few excavations left that spontaneous vegetation colonization is gradually filling up. These red soils, containing wastes from the mine rich in metals, are potentially colonized by a specific flora adapted to metal-rich soils. Figure 1. Site location in Gard (France) nearby Rousson and a view of the spontaneous vegetation at the abandoned mine site (March 2020). 2.2. Soil Sampling and Metal Analyses Samples of soil at the mine site were collected randomly from the top 20 cm after litter removal, then sieved to 2 mm and pooled in three composite samples for metal analysis (Cr, Cu, Fe, Mn, Ni, Pb, Zn). In the laboratory, soil composite aliquots were air-dried at room temperature and then ground to pass through a 0.2 mm titanium sieve (RETSCH zm 1000 with tungsten blades) before analyses. Trace and major metal element concentrations were determined on 3 analytical replicates for each of the three soil composite samples. Soils were mineralized in a microwave mineralizer (Milestone Start D) using aqua regia (1/3 HNO 3 + 2/3 HCl). The mineralization products were filtered with a 0.45 μm mesh and the metal concentrations were determined by ICP-AES (inductively coupled plasma atomic emission spectroscopy, Spectra 2000, Jobin Yvon Horiba group, Longjumeau, France). Quality controls and accuracy were checked using standard soil reference materials (CRM 049–050, from RTC-USA) with accuracies within 100 ± 10%. 2.3. Experimental Approach for Plant Selection The main aim is to assess the efficiency of a trait-based approach for phytoremediation plant selection in the field. To achieve this aim, a hierarchical method was designed based on analysis of literature data with experimental measurements to fill the gaps in knowledge, as described in Figure 2. 2.4. Floristic Analysis and Habitat Inventory The study was done during spring in a defined area of 30 m 2 representative of an excavation in the abandoned mining area with spontaneous vegetation colonization Vertical (herbaceous, shrub and tree strata) and horizontal (global plant cover) structures of plant communities were used to distinguish the different micro-habitats (Figures 1–3). Within each type of micro-habitat, N France Gard Figure 1. Site location in Gard (France) nearby Rousson and a view of the spontaneous vegetation at the abandoned mine site (March 2020) 2.2. Soil Sampling and Metal Analyses Samples of soil at the mine site were collected randomly from the top 20 cm after litter removal, then sieved to 2 mm and pooled in three composite samples for metal analysis (Cr, Cu, Fe, Mn, Ni, Pb, Zn). In the laboratory, soil composite aliquots were air-dried at room temperature and then ground to pass through a 0.2 mm titanium sieve (RETSCH zm 1000 with tungsten blades) before analyses Trace and major metal element concentrations were determined on 3 analytical replicates for each of the three soil composite samples. Soils were mineralized in a microwave mineralizer (Milestone Start D) using aqua regia (1 / 3 HNO 3 + 2 / 3 HCl). The mineralization products were filtered with a 0.45 µ m mesh and the metal concentrations were determined by ICP-AES (inductively coupled plasma atomic emission spectroscopy, Spectra 2000, Jobin Yvon Horiba group, Longjumeau, France). Quality controls and accuracy were checked using standard soil reference materials (CRM 049–050, from RTC-USA) with accuracies within 100 ± 10% 2.3. Experimental Approach for Plant Selection The main aim is to assess the e ffi ciency of a trait-based approach for phytoremediation plant selection in the field. To achieve this aim, a hierarchical method was designed based on analysis of literature data with experimental measurements to fill the gaps in knowledge, as described in Figure 2 . 2.4. Floristic Analysis and Habitat Inventory The study was done during spring in a defined area of 30 m 2 representative of an excavation in the abandoned mining area with spontaneous vegetation colonization. Vertical (herbaceous, shrub and tree strata) and horizontal (global plant cover) structures of plant communities were used to distinguish the di ff erent micro-habitats (Figures 1 – 3 ). Within each type of micro-habitat, representative areas characterized by the homogeneity of vegetation were selected for floristic analysis. The micro-habitats were classified based on plant species composition (presence / absence). Finally, a global analysis on 6 relev é s was done on the four selected micro-habitats in terms of plant species occurrence.

Int. J. Environ. Res. Public Health 2020 , 17 , 5506 4 of 22 Int. J. Environ. Res. Public Health 2020 , 17 , x 4 of 21 representative areas characterized by the homogeneity of vegetation were selected for floristic analysis. The micro-habitats were classified based on plant species composition (presence/absence). Finally, a global analysis on 6 relevés was done on the four selected micro-habitats in terms of plant species occurrence. Figure 2. Trait-based grid for local plant selection for phytoremediation of Zn-Pb-rich soils from an ecological restoration perspective. 2.5. Bibliographical Functional Traits Functional traits such as type of root, Grime strategy, Raunkiær biological type, and mycorrhizal status were collected from a review of scientific papers for each of the identified plant species in the field. The functional response traits were selected with the aim of better understanding the influence that these plant species may have on the passive phytoremediation processes. 2.6. Plant Analyses 2.6.1. Root Symbiosis Assessment Seven plant species were selected, i.e., Armeria arenaria (Pers.) Schult., Biscutella laevigata L., Brachypodium phoenicoides (L.) Roem. & Schult, Trifolium pratense L., Mibora minima (L.) Desv., Noccaea caerulescens (J. et C. Presl) FK Meyer and Thymus vulgaris L. for assessment of their root symbioses in the field. The selection was done after literature review, and only plant species for which there was a lack of knowledge, scarce data or controversial status were chosen. Thin roots were randomly selected in the root system of 3 individuals of each of the selected plant species. The root samples were first rinsed under tap water then with deionized water and stored in alcohol (60%, v/v) at room temperature until proceeding. The occurrence of symbiont colonization was estimated by visual observation of fungal colonization after clearing roots in 10% KOH and staining with lactophenol blue solution, according to Phillips and Hayman [15]. Mosaic of habitats in Zn-Pb-rich soils Pool of local plant species for each selected micro-habitat Categorical traits available in literature and/or databases Trait measurements to fill missing data Trait-based plant species selection Type of root system Root symbioses Soil preferendum Root symbioses Life form Grime strategy Zn-tolerance Aboveground Belowground Zn and Pb content in aerial parts Zn and Pb content in root parts Potential efficiency in Zn and Pb phytoextraction vs Potential efficiency in Zn and Pb phytostabilization Identification and selection of micro-habitats Soil surface Figure 2. Trait-based grid for local plant selection for phytoremediation of Zn-Pb-rich soils from an ecological restoration perspective Int. J. Environ. Res. Public Health 2020 , 17 , x 6 of 21 (8 species), then the Brassicaceae and Rosaceae with 5 species each. Asteraceae occurred with 3 species, followed by Fabaceae, Caryophyllaceae, Rubiaceae and Ranunculaceae (Table 2). In terms of estimated plant cover, the dominant plant species were Brassicaceae with mainly two species ( B. laevigata and N. caerulescens ), Poaceae ( B. phoenicoides, M. minima and Festuca ovina L.), Plumbaginaceae ( A. arenaria ), Fabaceae ( T. pratense ). and Lamiaceae ( T. vulgaris ). Figure 3. Cartography of the mosaic of micro-habitats at an abandoned mine site at La Gardie. Icons with number indicate the number of the relevé and its location in the field (bar = 2 m). Table 2. Floristic list established during springtime and occurrence of each plant species in the 6 relevés out of the 4 selected micro-habitats encountered at the former mine site. * (1): Metalliferous grassland; (2) and (3): Herbaceous formation on rocky raised areas; (4) and (5): Matorral with majorly shrubs; (6): Tree stands and frequency out of the 6 relevés. Latin Name Botanical Family Occurrence in Each Relevé * Frequency (1) (2) (3) (4) (5) (6) - Amelanchier ovalis Medik. Rosaceae - - - - - X 1/6 Aphyllantes monspeliensis L . Liliaceae - X - - X - 2/6 Arenaria serpyllifolia L. Caryophyllaceae - - X X X X 4/6 Argyrolobium zanonii (Turra) P.W.Ball Fabaceae - X - X - X 3/6 Armeria arenaria subsp bupleuroides (Godr. & Gren.) Greuter & Burdet Plumbaginaceae X X X X - - 4/6 Asparagus acutifolius L. Asparagaceae - - - X - - 1/6 Asplenium ruta-muraria L. Aspleniaceae - X - - - - 1/6 Biscutella laevigata L. Brassicaceae X X X X X X 6/6 Brachypodium phoenicoides (L.) Roem. & Schult Poaceae - - - - - X 1/6 Brachypodium retusum (Pers.) P.Beauv. Poaceae - - - - - X 1/6 Bromus madritensis L. Poaceae - - - X - - 1/6 Buxus sempervirens L. Buxaceae - X X X - X 4/6 Carex halleriana Asso Cyperaceae - - - - X - 1/6 Centaurea pectinata L. Asteraceae X X X X - - 4/6 Cerastium pumilum Curtis Caryophyllaceae - X X - - X 3/6 Clematis vitalba L. Ranunculaceae - - - - - X 1/6 Clinopodium nepeta (L.) Kuntze Lamiaceae - - - - X X 1/6 Dactylis glomerata L. Poaceae - - - - X X 2/6 Dioscorea communis (L.) Caddick & Wilkin Dioscoreaceae - - - - - X 1/6 Metalliferous grassland North Location of the 6 floristic relevés Herbaceous formation on rocky raised areas Lichen cover with scarce herbaceous vegetation Matorral with majorly shrubs Tree stands Matorral with many ruderals Path Departmental road Stones Contour Uneven surface 2 meters 1 3 2 5 4 6 Figure 3. Cartography of the mosaic of micro-habitats at an abandoned mine site at La Gardie. Icons with number indicate the number of the relev é and its location in the field (bar = 2 m).

Int. J. Environ. Res. Public Health 2020 , 17 , 5506 5 of 22 2.5. Bibliographical Functional Traits Functional traits such as type of root, Grime strategy, Raunkiær biological type, and mycorrhizal status were collected from a review of scientific papers for each of the identified plant species in the field. The functional response traits were selected with the aim of better understanding the influence that these plant species may have on the passive phytoremediation processes 2.6. Plant Analyses 2.6.1. Root Symbiosis Assessment Seven plant species were selected, i.e., Armeria arenaria (Pers.) Schult., Biscutella laevigata L., Brachypodium phoenicoides (L.) Roem. & Schult, Trifolium pratense L., Mibora minima (L.) Desv., Noccaea caerulescens (J. et C. Presl) FK Meyer and Thymus vulgaris L. for assessment of their root symbioses in the field. The selection was done after literature review, and only plant species for which there was a lack of knowledge, scarce data or controversial status were chosen. Thin roots were randomly selected in the root system of 3 individuals of each of the selected plant species. The root samples were first rinsed under tap water then with deionized water and stored in alcohol (60%, v / v ) at room temperature until proceeding. The occurrence of symbiont colonization was estimated by visual observation of fungal colonization after clearing roots in 10% KOH and staining with lactophenol blue solution, according to Phillips and Hayman [ 15 ]. 2.6.2. Metal Content in Below-Ground and Above-Ground Plant Parts Four plant species were selected for metal analysis, i.e., N. caerulescens , B. laevigata , A. arenaria and Plantago lanceolata L. Dried plant samples (root and aerial parts, separately) were ground to pass a 0.2 mm mesh titanium sieve, and three aliquots were analyzed per sample. About 0.5 g of dry matter was digested with the microwave digestion system Milestone start D with a HNO 3 -HCl mixture (volume proportion ratio 2 / 1). After filtration (0.45 µ m), acid digests were analyzed for metal content by ICP-AES (JY 2000 Jobin Yvon Horiba group, Longjumeau, France). Standard plant reference material (DC 73,349) from the China National Analysis Centre for Iron and Steel (NCS), was analyzed as a part of the quality control protocol (accuracies within 100 ± 10%) 2.7. Statistical Analysis Statistical analyses were performed for all data using JMP 12 statistical software (SAS Institute, Cary, NC, USA) using the non-parametric Kruskal-Wallis test because data did not follow a normal distribution pattern. The nonparametric pairwise multiplecomparison Dunn’s test was used when the null hypothesis was rejected with the Kruskal–Wallis test 3. Results and Discussion 3.1. Soil Contamination Soils contained high concentrations of zinc (ca.11.7%) and lead (ca.1.7%) as expected in this type of abandoned mining area (Table 1 ). Moreover, elevated concentrations in Fe, Mn, and Cr were also detected. Zn and Pb soil concentrations were higher than those previously reported in mine tailings in Spain, i.e., highest concentrations ranging between ca. 2360, 7000 and 11,600 mg / kg of Zn in the Alcudia Valley, San Jos é heaps and El Lirio tailing, respectively and ca. 7000 and 10,000 mg / kg of Pb in Belleza tailing and the Alcudia Valley, respectively [ 16 – 18 ]. However higher Zn soil content was detected at M á nforas with 135,080 mg / kg [ 17 ]. In Portugal, up to 9330 mg / kg of Pb were observed in abandoned Pb mines [ 19 ]. Compared to mine sites in France, at the Les Malines and Les Avini è res near the present study site, the authors detected lower concentrations in Zn and greater in Pb, i.e., 59,040 mg / kg of Zn and 62,051 mg / kg of Pb [ 20 ]. In Poland, in reclaimed soils of Zn-Pb mine wastes, up to 7.57% of Zn and 0.46% of Pb were found [ 21 ]. Such heavy metal-rich soils may not be used for agriculture

Int. J. Environ. Res. Public Health 2020 , 17 , 5506 6 of 22 purposes, and the spontaneous plant colonization that occurred after operation ceased was a great opportunity to reduce the transfer of elements into the food web and to reduce the environmental and human exposure Table 1. Average soil metal content (mg / kg dry weight) at La Gardie mine site Element (mg / kg) Cr Cu Fe Mn Ni Pb Zn 103 ± 38 39 ± 17 173,377 ± 58,177 3000 ± 936 89 ± 30 17,230 ± 6804 117,321 ± 33,770 Values are means of triplicates 3.2. Diversity of Habitats and Plant Species A total of 4 micro-habitats were identified after preliminary cartography of the 30 m 2 study site, consisting of (i) grassland colonizing metal-rich soil, (ii) herbaceous colonization of rocky soils, (iii) matorral with shrub dominating and (iv) woody vegetation (Figure 3 ). Two supplementary micro-habitats were removed from the inventory. The first concerned an area in which the major part was covered by lichens belonging to 2 species, i.e., Cladonia rengiformis and Cladonia foliacea s.l., beyond the scope of the study. A second area near the road was mostly constituted of ruderals due to the modified substrate of the road foundation The floristic composition of the study site consisted in 61 plant species growing wild on these 4 micro-habitat types and identified during the spring period (Table 2 ). It included 31 botanical families at the period of the relev é s. In terms of species diversity, the Poaceae family was the most frequent (8 species), then the Brassicaceae and Rosaceae with 5 species each. Asteraceae occurred with 3 species, followed by Fabaceae, Caryophyllaceae, Rubiaceae and Ranunculaceae (Table 2 ). Table 2. Floristic list established during springtime and occurrence of each plant species in the 6 relev é s out of the 4 selected micro-habitats encountered at the former mine site. * (1): Metalliferous grassland; (2) and (3): Herbaceous formation on rocky raised areas; (4) and (5): Matorral with majorly shrubs; (6): Tree stands and frequency out of the 6 relev é s Latin Name Botanical Family Occurrence in Each Relev é * Frequency (1) (2) (3) (4) (5) (6) - Amelanchier ovalis Medik Rosaceae - - - - - X 1 / 6 Aphyllantes monspeliensis L Liliaceae - X - - X - 2 / 6 Arenaria serpyllifolia L Caryophyllaceae - - X X X X 4 / 6 Argyrolobium zanonii (Turra) P.W.Ball Fabaceae - X - X - X 3 / 6 Armeria arenaria subsp bupleuroides (Godr. & Gren.) Greuter & Burdet Plumbaginaceae X X X X - - 4 / 6 Asparagus acutifolius L Asparagaceae - - - X - - 1 / 6 Asplenium ruta-muraria L Aspleniaceae - X - - - - 1 / 6 Biscutella laevigata L Brassicaceae X X X X X X 6 / 6 Brachypodium phoenicoides (L.) Roem. & Schult Poaceae - - - - - X 1 / 6 Brachypodium retusum (Pers.) P.Beauv Poaceae - - - - - X 1 / 6 Bromus madritensis L Poaceae - - - X - - 1 / 6 Buxus sempervirens L Buxaceae - X X X - X 4 / 6 Carex halleriana Asso Cyperaceae - - - - X - 1 / 6 Centaurea pectinata L Asteraceae X X X X - - 4 / 6 Cerastium pumilum Curtis Caryophyllaceae - X X - - X 3 / 6 Clematis vitalba L Ranunculaceae - - - - - X 1 / 6 Clinopodium nepeta (L.) Kuntze Lamiaceae - - - - X X 1 / 6 Dactylis glomerata L Poaceae - - - - X X 2 / 6 Dioscorea communis (L.) Caddick & Wilkin Dioscoreaceae - - - - - X 1 / 6

Int. J. Environ. Res. Public Health 2020 , 17 , 5506 7 of 22 Table 2. Cont Latin Name Botanical Family Occurrence in Each Relev é * Frequency (1) (2) (3) (4) (5) (6) - Draba verna L Brassicaceae - X X - - - 2 / 6 Eryngium campestre L Apiaceae - - - X - - 1 / 6 Euphorbia cyparissias L Euphorbiacee - X X X - - 3 / 6 Festuca ovina sl L Poaceae X X X - X X 5 / 6 Galium aparine L Rubiaceae - X X X X - 4 / 6 Galium corrudifolium Vill Rubiaceae - - - - X X 2 / 6 Helleborus foetidus L Ranunculaceae - - - - - X 1 / 6 Hordeum murinum L Poaceae - - - X - - 1 / 6 Hornungia petraea (L.) ex Rchb Brassicaceae - X X - - - 2 / 6 Hypericum perforatum L Hypericaceae - X - - - - 1 / 6 Juniperus oxycedrus L Cupressaceae - - - X - X 2 / 6 Lactuca perennis L Asteraceae - - - - - X 1 / 6 Lepidium draba L Brassicaceae - - - X - - 1 / 6 Lysimachia arvensis (L.) U. Manns & Anderb Primulaceae - - - X X - 2 / 6 Mibora minima (L.) Desv Poaceae X X X X - - 4 / 6 Noccaea caerulescens (J.Presl & C.Presl) F.K.Mey Brassicaceae X X X X X - 5 / 6 Pilosella o ffi cinarum Vaill Asteraceae - X X X - - 3 / 6 Pinus sylvestris L Pinaceae - X X - - X 3 / 6 Pistacia terebinthus L Anacardiaceae - - - - - X 1 / 6 Plantago lanceolata L Plantaginaceae - - - - - X 1 / 6 Poa annua L Poaceae - - - X - - 1 / 6 Poterium sanguisorba L Rosaceae - - - - - X 1 / 6 Pyrus spinosa Forssk Rosaceae - - - - - X 1 / 6 Quercus ilex L Fagaceae - X X X X X 5 / 6 Quercus pubescens Willd Fagaceae - - - - X X 2 / 6 Ranunculus bulbosus L Ranunculaceae - - - - X X 2 / 6 Reseda lutea L Resedaceae X X X X - - 4 / 6 Rosa canina L Rosaceae - - - - - X 1 / 6 Rubia peregrina L Rubiaceae - - - X - X 2 / 6 Rubus ulmifolius Schott Rosaceae - - - X - X 2 / 6 Rumex intermedius D.C Polygonaceae - X X - - - 2 / 6 Ruscus aculeatus L Asparagaceae - - - - X X 2 / 6 Scabiosa atropurpurea L Caprifoliaceae X X X - - - 3 / 6 Scrophularia lucida L Scrophulariaceae - X X - - - 2 / 6 Sedum acre L Crassulaceae - - - X - - 1 / 6 Sedum annuum L Crassulaceae - X X - - - 2 / 6 Senecio vulgaris L Asteraceae - - - X - - 1 / 6 Silene vulgaris (Moench) Garcke Caryophyllaceae - - - X - - 1 / 6 Smilax aspera L Smilacaceae - X - X - X 3 / 6 Thymus vulgaris L Lamiaceae X X X X X X 6 / 6 Trifolium pratense L Fabaceae - - - - X X 2 / 6 Ulex parviflorus Pourr Fabaceae - X - X - - 2 / 6 X: found on the plot In terms of estimated plant cover, the dominant plant species were Brassicaceae with mainly two species ( B. laevigata and N. caerulescens ), Poaceae ( B. phoenicoides, M. minima and Festuca ovina L.), Plumbaginaceae ( A. arenaria ), Fabaceae ( T. pratense ). and Lamiaceae ( T. vulgaris ) 3.3. Analysis of Plant Traits Linked with TMM Tolerance From the literature, di ff erent plant traits commonly used in di ff erent floristic analyses of spontaneous colonization of mining sites were assessed for all the 61 identified plant species (Table A 1 ). Those categorical traits may help us to understand the distribution pattern of plant species along

Int. J. Environ. Res. Public Health 2020 , 17 , 5506 8 of 22 environmental gradients [ 22 ]. However, their local variability needs to be discussed, and continuous traits need to be locally assessed to test the robustness of this approach 3.3.1. Categorical Trait Analysis of the 61 Identified Plant Species Considering below-ground parts, 61% of the plant species were characterized by fibrous root systems, 26% by rhizome or tuberous root system and only 13% by slender roots. A hypothesis has been formulated by Sardans and Peñuelas [ 23 ] and confirmed by Pierret et al. [ 24 ] considering deep roots as able to access water and nutrients in deep layers of soil and / or fractured bedrock, which are unavailable to surface roots. This mechanism will potentially help to maintain higher moisture levels in the upper soil layers and could be a factor explaining the high plant diversity in these dry habitats, despite the water stress due to both Mediterranean conditions and high salt concentration in the water linked to the metalliferous soils The mycorrhizal status of most of these 61 plant species has already been described [ 25 – 27 ]. Out of all the plant species, 67% were associated with arbuscular endomycorrhizal fungi, ca. 5% with ectomycorrhizal fungi and 8% were reputed to share no mycorrhizal interactions. For ca. 20% of the identified plant species, no information regarding their mycorrhizal status has been reported to the best of our knowledge Concerning the preferential type of soil, 44% of the plant species were adapted to dry and rocky soils, ca. 20% specific to calcareous soils, ca. 20% preferred sandy or well-drained soils and 16% were mostly found in disturbed or cultivated soils The dominant life form (Figure 4 ) were hemicryptophytes with an average percentage of 51%, then therophytes (19%) and phanerophytes (15%). Chamaephytes only represented an average of 9%. Hemicryptophytes and therophytes were found in all of the 6 relev é s although the occurrence of phanerophytes varied with none of this life form in the center of the area (open dry grasslands) and 31% in the tree stand (Tables 2 and A 1 ). Geophytes and lianas were only identified in 2 and 3 out of 6 relev é s, respectively Int. J. Environ. Res. Public Health 2020 , 17 , x 8 of 21 been formulated by Sardans and Peñuelas [23] and confirmed by Pierret et al. [24] considering deep roots as able to access water and nutrients in deep layers of soil and/or fractured bedrock, which are unavailable to surface roots. This mechanism will potentially help to maintain higher moisture levels in the upper soil layers and could be a factor explaining the high plant diversity in these dry habitats, despite the water stress due to both Mediterranean conditions and high salt concentration in the water linked to the metalliferous soils. The mycorrhizal status of most of these 61 plant species has already been described [25–27]. Out of all the plant species, 67% were associated with arbuscular endomycorrhizal fungi, ca. 5% with ectomycorrhizal fungi and 8% were reputed to share no mycorrhizal interactions. For ca. 20% of the identified plant species, no information regarding their mycorrhizal status has been reported to the best of our knowledge. Concerning the preferential type of soil, 44% of the plant species were adapted to dry and rocky soils, ca. 20% specific to calcareous soils, ca. 20% preferred sandy or well-drained soils and 16% were mostly found in disturbed or cultivated soils. The dominant life form (Figure 4) were hemicryptophytes with an average percentage of 51%, then therophytes (19%) and phanerophytes (15%). Chamaephytes only represented an average of 9 %. Hemicryptophytes and therophytes were found in all of the 6 relevés although the occurrence of phanerophytes varied with none of this life form in the center of the area (open dry grasslands) and 31% in the tree stand (Table 2 and A 1). Geophytes and lianas were only identified in 2 and 3 out of 6 relevés, respectively. Concerning plant strategy, 33% of species were considered as CS, i.e., competitive/stress tolerant, and 22% CSR, i.e., competitive, stress tolerant, ruderal (Figure 5). Figure 4. Average percentage of each life form in the studied mosaic of habitats at the former mine site. Ph: Phanerophytes; He: Hemicryptophytes; Ch: Chamaephytes; L: Liana; Ge: Geophytes; Th: Therophytes. 9% 3% 51% 3% 15% 19% Ch Ge He L Ph Th Figure 4. Average percentage of each life form in the studied mosaic of habitats at the former mine site. Ph: Phanerophytes; He: Hemicryptophytes; Ch: Chamaephytes; L: Liana; Ge: Geophytes; Th: Therophytes Concerning plant strategy, 33% of species were considered as CS, i.e., competitive / stress tolerant, and 22% CSR, i.e., competitive, stress tolerant, ruderal (Figure 5 ).

Int. J. Environ. Res. Public Health 2020 , 17 , 5506 9 of 22 Int. J. Environ. Res. Public Health 2020 , 17 , x 9 of 21 Figure 5. Average percentage of each Grime strategy in the studied mosaic of habitats at the former mine site. CS: competitive, stress tolerant; CSR: competitive, stress tolerant, ruderal; SR: stress tolerant, ruderal; S: stress tolerant; R: ruderal; CR: competitive ruderal; C: competitive. 3.3.2. Analysis of Plant Traits by TMM Tolerance Strategy The identified plant species were grouped in hyperaccumulators, phytoaccumulators, phytostabilizators or not known in the literature. Using this grid of comparison, the above-cited traits were analyzed (Figure 6). Even if the number of species considered as hyperaccumulators (2) or as phytoaccumulators (3) is limited in the study field, some traits are typical of the plant strategy. The dominant root system was fibrous for the two hyperaccumulators (Figure 6 a) and deep root system for phytoaccumulators (Figure 6 b). No specific root type could be defined for phytostabilizators (Figure 6 c). For phytostabilization strategy (43), identified plant species in the study site had mainly rhizomes or tuberous roots (35%, Figure 6 c), ca. all of them were known to be endomycorrhized (78%, Figure 6 g), the dominant Grime strategy was CS (49%, Figure 6 k), and the main life form was hemicryptophyte (46%, Figure 6 o). These results are in accordance with many previous studies demonstrating that only a few spontaneous plant species colonizing mine sites may favor heavy metal translocation in the aerial parts and may be useful for phytoextraction. Most of the metal tolerant plant species may accumulate heavy metals in their roots and limit their transfer to the aerial parts, being potentially useful in phytostabilization. Without any human intervention by plant harvesting, the dominant natural process is therefore phytostabilization. Hemicryptophyte life form was strongly present in the three groups (Figure 6 m–o). Hemicryptophytes such as biannuals or with thin root systems may limit the phytostabilization potential. On the other hand, the non-perennial aerial parts of hemicryptophytes may also limit the phytoextraction ability. The traits that most discriminated hyperaccumulators from both phytoaccumulators and phytostabilizators were the ability to form arbuscular mycorrhizal (AM) associations (50% non-mycorrhizal for the first and 33% and 78% AM for the other two; Figure 6 e, f and g). It is noteworthy that ectomycorrhizal (EC) type was dominant (67%) in phytoaccumulators. It is congruent with the 67% of phanerophyte type. Quercus ilex and Pinus sylvestris are known to be predominantly associated with ectomycorrhizal fungi [26]. Figure 5. Average percentage of each Grime strategy in the studied mosaic of habitats at the former mine site. CS: competitive, stress tolerant; CSR: competitive, stress tolerant, ruderal; SR: stress tolerant, ruderal; S: stress tolerant; R: ruderal; CR: competitive ruderal; C: competitive 3.3.2. Analysis of Plant Traits by TMM Tolerance Strategy The identified plant species were grouped in hyperaccumulators, phytoaccumulators, phytostabilizators or not known in the literature. Using this grid of comparison, the above-cited traits were analyzed (Figure 6 ). Even if the number of species considered as hyperaccumulators (2) or as phytoaccumulators (3) is limited in the study field, some traits are typical of the plant strategy The dominant root system was fibrous for the two hyperaccumulators (Figure 6 a) and deep root system for phytoaccumulators (Figure 6 b). No specific root type could be defined for phytostabilizators (Figure 6 c). For phytostabilization strategy (43), identified plant species in the study site had mainly rhizomes or tuberous roots (35%, Figure 6 c), ca. all of them were known to be endomycorrhized (78%, Figure 6 g), the dominant Grime strategy was CS (49%, Figure 6 k), and the main life form was hemicryptophyte (46%, Figure 6 o). These results are in accordance with many previous studies demonstrating that only a few spontaneous plant species colonizing mine sites may favor heavy metal translocation in the aerial parts and may be useful for phytoextraction. Most of the metal tolerant plant species may accumulate heavy metals in their roots and limit their transfer to the aerial parts, being potentially useful in phytostabilization. Without any human intervention by plant harvesting, the dominant natural process is therefore phytostabilization. Hemicryptophyte life form was strongly present in the three groups (Figure 6 m–o). Hemicryptophytes such as biannuals or with thin root systems may limit the phytostabilization potential. On the other hand, the non-perennial aerial parts of hemicryptophytes may also limit the phytoextraction ability. The traits that most discriminated hyperaccumulators from both phytoaccumulators and phytostabilizators were the ability to form arbuscular mycorrhizal (AM) associations (50% non-mycorrhizal for the first and 33% and 78% AM for the other two; Figure 6 e, f and g). It is noteworthy that ectomycorrhizal (EC) type was dominant (67%) in phytoaccumulators. It is congruent with the 67% of phanerophyte type Quercus ilex and Pinus sylvestris are known to be predominantly associated with ectomycorrhizal fungi [ 26 ].

Int. J. Environ. Res. Public Health 2020 , 17 , 5506 10 of 22 Int. J. Environ. Res. Public Health 2020 , 17 , x 10 of 21 Figure 6. Distribution pattern of root type traits ( a–d ), root symbiosis potential occurrence ( e–h ), Grime strategy ( i–l ) and Raunkiær life forms ( m–p ) by TMM tolerance strategy, i.e., hyperaccumulators ( N. caerulescens ( Zn/Cd/Pbhyperaccumulator) and B. leavigata ( Tl/Pb/Cdhyperaccumulator)), Zn-phytoaccumulators ( Plantago lanceolata , Quercus ilex and, Pinus sylvestris ), phytostabilizators or not known in the literature (see Table A 1). 3.3.3. Measured Trait Analysis on Selected Plant Species Mycorrhizal status of 7 selected plant species: an efficient tool for plant species selection for phytostabilization strategy? The mycorrhizal status of A. arenaria, B. laevigata, B. phoenicoides, T. pratense, M. minima, N. caerulescens and T. vulgaris was assessed N. caerulescens (Figure 7 a) and B. laevigata (not shown) were not mycorrhized at this site. However, there is no consensus regarding the mycorrhizal status of B. laevigata and N. caerulescens , long considered not to be mycorrhized; some authors detected AM associations with both species [26 – 28] A total of 20% of root length colonization by AM fungi was detected for A. arenaria (Figure 7 b) although no AM colonization was revealed for M. minima (not shown). No previous data regarding the mycorrhizal status of A. arenaria and M. minima have been published. To the best of our knowledge, this is the first report of AM association with A. arenaria. With regard to M. minima , the smallest Poaceae occurring in France, it is also the first report concerning its mycorrhizal status. Its very short life cycle (few months) could be linked to a lack of AM association Wang and Qiu [26] and Pawlowska et al. [27] gave discordant results for T. pratense. At the present site, AM mycelia were abundant, and many spores were detected in roots of T. pratense with an overall colonization ca. 80% of root length (Figure 7 c) B. phoenicoides mycorrhizal status has previously been analyzed [29]. However, data are scarce. In our study, this species appeared to be endomycorrhized (Figure 7 d). T. vulgaris was the only Lamiaceae identified at our study site and since Lamiaceae are usually good candidates for phytostabilization, we endeavored to confirm its mycorrhizal status. This perennial developed AM association and a dense web of mycelia was observed with many vesicles (Figure 7 e) 7% 23% 31% 31% 8% Ch Th Ph He L Ge 7% 21% 12% 46% 5% 9% 50% 50% 67% 33% 67% 33% 78% 15% 7% 49% 15% 9% 15% 12% 23% 23% 8% 23% 15% 8% CS S CR R SR 54% 8% 38% AM EC Non mycorhizal Not determined 33% 67% 100% Hyperaccumulators (n=2) 23% 16% 35% 26% Phytostabilizators (n=43) 54% 7% 8% 31% Not known (n=13) Deep root system Slender roots Rhizome or tuberous roots Fibrous root system 67% 33% Zn-phytoaccumulators (n=3) 50% 50% Distribution of root type traits 100% Root symbiose potential occurrence Grime strategy Raunkier life forms a b c d e i m f g h j k l n o p Figure 6. Distribution pattern of root type traits ( a – d ), root symbiosis potential occurrence ( e – h ), Grime strategy ( i – l ) and Raunkiær life forms ( m – p ) by TMM tolerance strategy, i.e., hyperaccumulators ( N. caerulescens (Zn / Cd / Pbhyperaccumulator) and B. leavigata (Tl / Pb / Cdhyperaccumulator)), Zn-phytoaccumulators ( Plantago lanceolata , Quercus ilex and, Pinus sylvestris ), phytostabilizators or not known in the literature (see Table A 1 ). 3.3.3. Measured Trait Analysis on Selected Plant Species Mycorrhizal status of 7 selected plant species: an e ffi cient tool for plant species selection for phytostabilization strategy? The mycorrhizal status of A. arenaria , B. laevigata , B. phoenicoides , T. pratense , M. minima , N. caerulescens and T. vulgaris was assessed N. caerulescens (Figure 7 a) and B. laevigata (not shown) were not mycorrhized at this site However, there is no consensus regarding the mycorrhizal status of B. laevigata and N. caerulescens , long considered not to be mycorrhized; some authors detected AM associations with both species [ 26 – 28 ]. N. caerulescens (Figure 7 a) and B. laevigata (not shown) were not mycorrhized at this site However, there is no consensus regarding the mycorrhizal status of B. laevigata and N. caerulescens , long considered not to be mycorrhized; some authors detected AM associations with both species [ 26 – 28 ]. A total of 20% of root length colonization by AM fungi was detected for A. arenaria (Figure 7 b) although no AM colonization was revealed for M. minima (not shown). No previous data regarding the mycorrhizal status of A. arenaria and M. minima have been published. To the best of our knowledge, this is the first report of AM association with A. arenaria With regard to M. minima , the smallest Poaceae occurring in France, it is also the first report concerning its mycorrhizal status. Its very short life cycle (few months) could be linked to a lack of AM association.

Int. J. Environ. Res. Public Health 2020 , 17 , 5506 11 of 22 Int. J. Environ. Res. Public Health 2020 , 17 , x 11 of 21 The occurrence of AM symbioses is a strong advantage for phytostabilization but not specific to this type of process. Mycorrhizal associations may favor TMM extraction by enhancing metal mobilization [30]. Therefore, this trait alone would not be a good criterium for plant species selection for the purpose of phytostabilization strategy. Figure 7. Microphotographs of roots stained for mycorrhizal association observation: root parts from N. caerulescens ( a ), A. arenaria ( b ), T. pratense ( c ), B. phoenicoides ( d ) and, T. vulgaris ( e ). AM mycelium and vesicles appear in dark blue. Scale bars are directly on the microphotographs. Plant colonization at mining sites may be favored by AM fungi, the latter, which may lower metal toxicity and improve nutrient availability for plants. However, in a Zn-, Cd-, Pb-, and Cupolluted field study, no evidence for an effect of AM symbioses has been found on plant metal uptake [31]. Therefore, with regard to a phytoextraction strategy, the authors suggest not channeling efforts exclusively towards reinforcing AM symbiosis. In a recent review paper dealing with AM associations at mining sites, it was observed that more than 80% of the plant species from metallic mines were endomycorrhized suggesting adaptive strategies in coevolved fungal strains and plant b 50 µ m d 50 µ m c 50 µ m e 50 µ m a 50 µ m Figure 7. Microphotographs of roots stained for mycorrhizal association observation: root parts from N. caerulescens ( a ), A. arenaria ( b ), T. pratense ( c ), B. phoenicoides ( d ) and, T. vulgaris ( e ). AM mycelium and vesicles appear in dark blue. Scale bars are directly on the microphotographs Wang and Qiu [ 26 ] and Pawlowska et al. [ 27 ] gave discordant results for T. pratense At the present site, AM mycelia were abundant, and many spores were detected in roots of T. pratense with an overall colonization ca. 80% of root length (Figure 7 c). B. phoenicoides mycorrhizal status has previously been analyzed [ 29 ]. However, data are scarce In our study, this species appeared to be endomycorrhized (Figure 7 d). T. vulgaris was the only Lamiaceae identified at our study site and since Lamiaceae are usually good candidates for phytostabilization, we endeavored to confirm its mycorrhizal status. This perennial developed AM association and a dense web of mycelia was observed with many vesicles (Figure 7 e). The occurrence of AM symbioses is a strong advantage for phytostabilization but not specific to this type of process. Mycorrhizal associations may favor TMM extraction by enhancing metal

Int. J. Environ. Res. Public Health 2020 , 17 , 5506 12 of 22 mobilization [ 30 ]. Therefore, this trait alone would not be a good criterium for plant species selection for the purpose of phytostabilization strategy Plant colonization at mining sites may be favored by AM fungi, the latter, which may lower metal toxicity and improve nutrient availability for plants. However, in a Zn-, Cd-, Pb-, and Cu-polluted field study, no evidence for an e ff ect of AM symbioses has been found on plant metal uptake [ 31 ]. Therefore, with regard to a phytoextraction strategy, the authors suggest not channeling e ff orts exclusively towards reinforcing AM symbiosis. In a recent review paper dealing with AM associations at mining sites, it was observed that more than 80% of the plant species from metallic mines were endomycorrhized suggesting adaptive strategies in coevolved fungal strains and plant species [ 32 ]. Rehabilitation of metal-rich soils without metal removal may be achieved by selecting plant species with their co-evolved mycorrhizal symbionts 3.3.4. Metal Content in 4 Plant Species for Phytoextraction Strategy In agreement with the literature, N. caerulescens was the best accumulating species out of the 4 selected for zinc and lead (Table 3 ), and it is defined as a facultative Zn-hyperaccumulator [ 3 ]. This species also accumulated high levels of Fe in its aerial parts. Furthermore, B. laevigata and A. arenaria seemed to be suitable as Zn-phytoaccumulators with more than 1000 mg / kg (dry matter, DM) of zinc accumulated in their aerial parts. Those species have already been identified as valuable temperate zone phytoaccumulators of Zn and Cd P. lanceolata , in a lesser way, appeared as a good Zn phytoaccumulators. Zn content up to 430 mg / kg in shoots of P. lanceolata was previously reported in a mining area of Southern Poland [ 33 ] although 946 mg / kg were detected in P. lanceolata aerial parts in the present study. All these four plant species are also potentially good candidates in Mediterranean areas. Although average soil Pb concentration was high (Table 1 ), this element was moderately accumulated in the aerial parts of N. caerulescens (ca. 496 mg / kg) and ranged from 48 to 75 mg / kg in the aerial parts of the three other plant species Table 3. Aerial and root part metal content (mg / kg dry weight) at La Gardie mine site Element (mg / kg) Plant Species A. arenaria B. laevigata N. caerulescens P. lanceolata In aerial parts Cr 9.6 ± 4.3 10.7 ± 5.3 11.4 ± 4.2 11.4 ± 4.2 Cu 4.6 ± 0.3 3.9 ± 0.5 5.0 ± 1.3 7.3 ± 0.4 Fe 966.3 ± 375.4 325.6 ± 186.2 5780 ± 1878 728.5 ± 167.1 Mn 160.3 ± 99.1 204.9 ± 80.2 105.3 ± 25.0 36.6 ± 4.0 Ni 1.8 ± 0.2 ab 1.8 ± 0.3 ab 3.6 ± 0.8 a 0.5 ± 0.2 b Pb 74.9 ± 6.8 ab 48.2 ± 6.3 b 496.2 ± 104.6 a 69.2 ± 15.1 ab Zn 1,916 ± 201 ab 1,775 ± 125 ab 10,664 ± 563 a 946 ± 88 b In root parts Cr 4.3 ± 1.9 2.5 ± 0.8 29.9 ± 9.1 10.1 ± 0.5 Cu 8.6 ± 1.7 6.9 ± 0.1 27.2 ± 6.7 21.3 ± 1.2 Fe 7,719 ± 3,565 5,472 ± 1,009 56,779 ± 24,995 15,210 ± 1,035 Mn 303.0 ± 89.7 223.2 ± 46.3 1,143.2 ± 427.7 405.7 ± 37.9 Ni 5.2 ± 2.5 5.9 ± 2.5 30.6 ± 13.7 7.8 ± 0.6 Pb 1,769 ± 49 1,393 ± 129 5,123 ± 1,879 1,630 ± 124 Zn 10,659 ± 2,763 6,902 ± 547 66,381 ± 24,010 12,217 ± 996 Values are means of triplicates. Means followed by di ff erent letters ( a,b, ab ) are significantly di ff erent (Dunn test, p ≤ 0.05) Apart from Fe, Zn and Pb were the studied elements most translocated to the aerial parts of the four plant species. However, Pb, as a non-essential element, may be transferred to aerial parts via transporters of other elements essential to plants. This type of elemental competition between nutrients and toxic metals is most of the time antagonistic. However, the present results show concomitant

Int. J. Environ. Res. Public Health 2020 , 17 , 5506 13 of 22 translocation of Zn and Pb. Our results demonstrated similar results for N. caerulescens , A. arenaria , B. laevigata and P. lanceolata N. caerulescens is sometimes considered as a Zn-hyperaccumulator and Pb co-accumulator [ 34 ]. A previous work on N. caerulescens also shows a negative correlation between Zn content in shoot parts and Ca and Mg concentrations in shoot parts due to competition between these di ff erent cations [ 13 ]. Synergistic and antagonistic interactions for element absorption in plants is a challenging field of research and there is still a need for more knowledge. However, these preliminary results showed the interest of these plant species in Zn and Pb-rich soils 3.3.5. Spontaneous Plant Colonization at Abandoned Zn-Pb Mine Sites: A Matter of Geographical Situation or Plant Traits? Out of the 61 plant species identified at the abandoned mine site, 47 were already known to grow in Zn-rich soils. Among the 14 other plant species, 4 are phanerophytes and may have slowly colonized the site. Five have a reduced biomass and a low cover potential. Four plant species are typical of Mediterranean environments, and the others have a wider range of distribution. Some previous studies, notably at an abandoned Zn-mine site and in metalliferous soils in Greece, Poland, Spain, Italy, Portugal and France, have detailed the floristic composition and some of their plant traits [ 17 , 19 , 20 , 27 , 35 – 37 ] enabling a comparison of occurrence of plant species even though these studies were conducted under varying biogeographical conditions. Out of the seven relev é s from the cited literature, Rubus ulmifolius Schott and P. lanceolata were the most frequently identified species on these sites B. leavigata, D. glomerata and F. ovina occurred in 4 out of the 7 relev é s in France, Poland and / or Portugal N. caerulescens only occurred in the 2 relev é s from France. Hemicryptophytes were dominant in most of the studies [ 20 , 38 ] including in the present study. No systematic selection could be made solely on the basis of plant traits, but common features are shared by the spontaneous vegetation of the European and Mediterranean abandoned Zn-mine sites that it might be useful to identify with a view to the ecological restoration of Zn-rich soils It is worth noticing that, in many studies, the occurrence of certain exotic phanerophytes was recurrent, such as Eucalyptus globulus [ 19 ], but these species are not to be encouraged in ecological restoration processes; it would be better to focus on native plant species On the basis of all the information collected through the diverse relev é s, it appeared that some ubiquitous Zn-tolerant plant species in Mediterranean and European areas are potentially good candidates for the first stages of ecological restoration of Zn-Pb-rich soils. Previous restoration programs in Poland have used D. glomerata and T. pratense, also identified at the present study site However, this was done with selected cultivars and not seedlings of wild origin [ 39 ]. This previous integrative study has suggested various potential lines of research including endomycorrhizal inoculation before transplantation with selected fungal strains and also selection of xerothermic plant species to cope with water stress. Their feedback shows how important the development of rhizosphere microorganisms consortia is for successful site restoration [ 39 ]. AM colonization was also a dominant trait as previously reported [ 27 ]. However, local plant associations should be favored and the geographical situation needs to be taken into account. Moreover, more than the below-ground parts of the plants, rhizosphere micro-organisms are potentially the key factor for plant colonization in these metal-rich soils following the mycorrhizal fungal diversity–ecosystem function concept of Hazard and Johnson [ 40 ]. Plant functioning (nutrition, stress-tolerance, etc.) is intrinsically dependent on associated micro-organisms [ 41 ]. 4. Conclusions This work enabled the implementation of a plant database consisting of above-ground and below-ground plant traits of plant species able to grow on Zn-Pb rich soils. This preliminary work may be a useful tool for plant selection in rehabilitation programs for Zn-Pb-rich soils. It also highlighted the need for more information regarding below-ground traits and rhizosphere microbial consortia.

Int. J. Environ. Res. Public Health 2020 , 17 , 5506 14 of 22 The trait-based analysis provided a basis for drawing a general picture of plant communities in a Mediterranean abandoned Zn-Pb mine site. Below-ground traits appeared as important features for phytostabilization. Ectomycorrhizae were dominant in the Zn-phytoaccumulators species and AM, in the phytostabilizators. Together, these plant strategies may favor fungal interaction diversity and enhance the sustainability of the plant-fungal communities The four plant species selected for phytoextraction, i.e., N. caerulescens , B. laevigata , A. arenaria and P. lanceolata showed interesting Pb and Zn accumulation capacities in their aerial parts However, these plant species are of reduced biomass and a phytoextraction process with those species would not be e ffi cient. Those plant species are however interesting in the early plant succession stage after mines are abandoned. Among the identified potential phytoaccumulators, Quercus ilex and Pinus sylvestris , both phanerophytes, are long-term colonizers and persistent plant species However, management strategies based on tree plantation would create more litter with reduced undergrowth. With long-term Zn potential accumulation in the aerial parts, there is a need for further knowledge regarding the risk of Zn transfer into the food web Plant communities at the mine site mostly favored a passive phytostabilization that is maintained over time by seasonal turnover of therophytes and persistence of belowground parts of hemicryptophytes and geophytes and both belowground and aboveground parts of chamaephytes and phanerophytes. Only few plant species were potentially able to accumulate Zn and Pb in the aerial parts. Moreover, a phytoextraction process requires human intervention by removal of metal-rich aerial parts. Consequently, even if both strategies occurred in these plant communities, the overall trend is the immobilization of heavy metals in the soils and root systems Author Contributions: Conceptualization, I.L.-S., J.R. and P.P. methodology, I.L.-S., J.R., P.P., V.M., L.V. and M.-D.S.; validation, I.L.-S., P.P., J.R., H.F. and L.T.; investigation, I.L.-S., P.P. and J.R.; resources, I.L.-S., J.R. and P.P.; writing—original draft preparation, I.L.-S.; writing—review and editing, J.R., P.P., V.M., M.-D.S., H.F. and L.T.; supervision, I.L.-S. and P.P.; project administration, I.L.-S.; funding acquisition, I.L.-S., J.R. and P.P. All authors have read and agreed to the published version of the manuscript Funding: This research received no external funding Acknowledgments: We thank Claude Roux for the lichen identification; Daniel E Silva, Mathieu Baroncini and Gr é gory Dron for their help in mycorrhizal observations and Baptiste Benita, Gr é gory Dron and Jean-Baptiste Portier for their technical assistance in field sampling and sample preparation. Many thanks to Daniel Pavon for his help in plant determination and to Manuel Cartereau for his helpful discussions about plant traits and notably Grime strategies. We also thank Michael Paul for revising the English of this text Conflicts of Interest: The authors declare no conflict of interest.

Int. J. Environ. Res. Public Health 2020 , 17 , 5506 15 of 22 Appendix A Table A 1. Floristic list established during springtime from the mosaic of habitats encountered in the former mine site Traits selected from literature: Root type; Root symbioses: AM: Arbuscular mycorrhizae, EC: Ectomycorrhizae; NM: Non mycorrhizal; Soil preferendum; Life forms [ 42 ]: Ph: Phanerophytes; He: Hemicryptophytes; Ch: Chamaephytes; L: Liana; Ge: Geophytes, Th: Therophytes; Grime strategy: CS: Competitive stress tolerant; CSR: Competitive, stress tolerant, ruderal; SR: Stress tolerant, ruderal; S: Stress tolerant; R: Ruderal; CR: Competitve ruderal; C: Competitive; Zn tolerance Latin Name Botanical Family Root System Root Symbioses Soil Preferendum Life Form Grime Strategy Zn Tolerance Amelanchier ovalis Medik Rosaceae Deep root system AM [ 43 ] Rocky dry soils Ph CS Not known Aphyllantes monspeliensis L Liliaceae Fibrous root system Not known Dry soils He S Not known Arenaria serpyllifolia L Caryophyllaceae Thin root system NM [ 44 , 45 ] Sandy and rocky soils, calcareous soils Th R Population-specific Zn tolerance [ 46 ] Argyrolobium zanonii (Turra) P.W.Ball Fabaceae Fibrous root system AM [ 47 ] Calcareous soils Ch S Not known Armeria arenaria subsp bupleuroides (Godr. & Gren.) Greuter & Burdet Plumbaginaceae Fibrous root system Not known Sandy and rocky soils He S Population-specific Zn tolerance [ 48 ] Asparagus acutifolius L Asparagaceae Rhizome AM [ 26 ] Dry sand arid oils He CS Population-specific Zn tolerance [ 20 ] Asplenium ruta-muraria L Aspleniaceae Rhizome AM + NM [ 26 ] Cli ff s, rocks He CS Population-specific Zn tolerance [ 49 ] Biscutella laevigata L Brassicaceae Fibrous root system NM [ 26 ] / AM [ 27 , 28 ] Rocky soils He SR Species-wide Zn tolerance [ 8 ], Tl-, Cd-hyperaccumulator [ 50 ] Brachypodium phoenicoides (L.) Roem. & Schult Poaceae Rhizome AM [ 29 ] Dry soils He CS Population-specific Zn tolerance [ 19 ] Brachypodium retusum (Pers.) P.Beauv Poaceae Rhizome AM [ 26 ] Dry soils He CS Population-specific Zn tolerance [ 8 ] Bromus madritensis L Poaceae Fibrous root system AM [ 26 ] Sandy soils, cultivated soils Th SR Population-specific Zn tolerance [ 51 ] Buxus sempervirens L Buxaceae Deep root system AM [ 26 ] Dry calcareous soils Ph CS Population-specific Zn tolerance [ 35 ] Carex halleriana Asso Cyperaceae Rhizome NM [ 52 ] Dry calcareous soils He CSR Population-specific Zn tolerance [ 37 ] Centaurea pectinata L Asteraceae Deep root system Not known Rocky soils He S Population-specific Zn tolerance [ 20 ] Cerastium pumilum Curtis Caryophyllaceae Slender root system Not known Sandy soils Th SR Population-specific Zn tolerance [ 53 ] Clematis vitalba L Ranunculaceae fibrous root system AM [ 26 ] calcareous to acidic soils L CS Population-specific Zn tolerance [ 54 ]

Int. J. Environ. Res. Public Health 2020 , 17 , 5506 16 of 22 Table A 1. Cont Latin Name Botanical Family Root System Root Symbioses Soil Preferendum Life Form Grime Strategy Zn Tolerance Clinopodium nepeta (L.) Kuntze Lamiaceae fibrous root system AM [ 55 ] Dry rocky soils He S Population-specific Zn tolerance [ 56 , 57 ] Dactylis glomerata L Poaceae Fibrous root system AM [ 26 ] Cultivated soils He CSR Population-specific Zn tolerance [ 20 ] Dioscorea communis (L.) Caddick & Wilkin Dioscoreaceae Rhizome AM [ 25 ] Calcareous, well-drained soils L CS Not known Draba verna L Brassicaceae Fibrous root system Not known Sandy and calcareous soils Th CR Not known Eryngium campestre L Apiaceae Rhizome AM [ 26 ] Sandy soils Ge SR Population-specific Zn tolerance [ 16 ] Euphorbia cyparissias L Euphorbiacee Fibrous root system AM [ 26 , 58 ] Calcareous soils He S Population-specific Zn tolerance [ 58 ] Festuca ovina sl L Poaceae Fibrous root system AM [ 26 , 27 ] Dry soils He CSR Population-specific Zn tolerance [ 59 ] Galium aparine L Rubiaceae Slender root system AM + NM [ 26 ] Cultivated soils Th CR Population-specific Zn tolerance [ 60 ] Galium corrudifolium Vill Rubiaceae Deep and fibrous root system Not known Dry soils He CR Not known Helleborus foetidus L Ranunculaceae Rhizome AM [ 26 ] Welldrained, calcareous soil Ge CSR Population-specific Zn tolerance [ 17 ] Hordeum murinum L Poaceae Fibrous root system AM [ 26 ] Dry and sandy soils, disturbed soils Th R Population-specific Zn tolerance [ 61 ] Hornungia petraea (L.) ex Rchb Brassicaceae Fibrous root system Not known Sandy and rocky soils Th R Not known Hypericum perforatum L Hypericaceae Rhizome AM [ 26 ] Roadside soils, dry soils He SR Population-specific Zn tolerance [ 62 ] Juniperus oxycedrus L Cupressaceae Deep root system AM [ 26 ] Arid soils Ph CS Population-specific Zn tolerance [ 63 ] Lactuca perennis L Asteraceae Deep root system Not known Rocky and calcareous soils He S Not known Lepidium draba L Brassicaceae Rhizome NM [ 64 ] Roadside soils Ge CR Population-specific Zn tolerance [ 65 ] Lysimachia arvensis (L.) U Manns & Anderb Primulaceae Fibrous root system AM [ 25 ] Cultivated and sandy soils Th R Not known Mibora minima (L.) Desv Poaceae Slender root Not known Sandy soils Th R Population-specific Zn tolerance [ 66 ] Noccaea caerulescens (J.Presl & C.Presl) F.K.Mey Brassicaceae Fibrous root system AM [ 26 ] Well-drained soils, cultivated soils He CS Species-wide Zn tolerance [ 20 ], Zn / Cd / Ni hyperaccumulator [ 3 ]

Int. J. Environ. Res. Public Health 2020 , 17 , 5506 17 of 22 Table A 1. Cont Latin Name Botanical Family Root System Root Symbioses Soil Preferendum Life Form Grime Strategy Zn Tolerance Pilosella o ffi cinarum Vaill Asteraceae Rhizome AM [ 67 ] Dry soils He CS Population-specific Zn tolerance [ 68 ] Pinus sylvestris L Pinaceae Deep root system EC [ 26 ] Dry soils, sandy and rocky soils Ph CS Population-specific Zn tolerance [ 69 ], Zn accumulator [ 69 ] Pistacia terebinthus L Anacardiaceae Deep root system AM [ 26 ] Calcareous soils Ph CS Population-specific Zn tolerance [ 70 ] Plantago lanceolata L Plantaginaceae Fibrous root system AM [ 25 ] Neutral to calcareous soils, rangelands He CSR Population-specific Zn tolerance [ 20 ], Znaccumulator [ 62 ] Poa annua L Poaceae Fibrous root system AM + NM [ 26 ] Well-drained soil Th R Population-specific Zn tolerance [ 71 ] Poterium sanguisorba L Rosaceae deep-root system AM [ 72 ] Roadside soils, Rocky soils He CS Population-specific Zn tolerance [ 20 ] Pyrus spinosa Forssk Rosaceae deep-root system AM + NM [ 26 ] Dry soils, Rocky soils Ph C Not known Quercus ilex L Fagaceae deep-root system EC [ 26 ] Calcareous soils, well-drained soils Ph CS Population-specific Zn tolerance [ 18 ], Znaccumulator [ 18 ] Quercus pubescens Willd Fagaceae deep-root system EC [ 26 ] Well-drained soils, Calcareous soils Ph C Not known Ranunculus bulbosus L Ranunculaceae Tuberous root system AM [ 26 , 27 ] Calcareous soils, Stony soil Ge CSR Population-specific Zn tolerance [ 21 ] Reseda lutea L Resedaceae deep-root system AM + NM [ 26 , 27 ] Sandy soils, roadside soils, rangelands He CSR Population-specific Zn tolerance [ 20 ] Rosa canina L Rosaceae deep-root system AM [ 26 ] Poor soils, sandy soils Ph CS Population-specific Zn tolerance [ 73 ] Rubia peregrina L Rubiaceae Rhizome AM [ 26 ] Dry soils, well-drained soils He CS Population-specific Zn tolerance [ 19 ] Rubus ulmifolius Schott Rosaceae Rhizome AM [ 26 ] Calcareous soils Ph CS Population-specific Zn tolerance [ 19 ] Rumex intermedius D.C Polygonaceae deep-root system AM [ 26 ] Rocky soils, dry soils He CSR Not known Ruscus aculeatus L Asparagaceae Rhizome AM [ 25 ] Dry soils, rocky soils Ch CS Population-specific Zn tolerance [ 19 ] Scabiosa atropurpurea L Caprifoliaceae Deep root system Not known Sandy soils, rangelands He CSR Population-specific Zn tolerance [ 74 ] Scrophularia lucida L Scrophulariaceae Slender roots Not known Dry calcareous soils He CSR Population-specific Zn tolerance [ 75 ]

Int. J. Environ. Res. Public Health 2020 , 17 , 5506 18 of 22 Table A 1. Cont Latin Name Botanical Family Root System Root Symbioses Soil Preferendum Life Form Grime Strategy Zn Tolerance Sedum acre L Crassulaceae Slender roots AM [ 26 ] Rocky soils Ch S Population-specific Zn tolerance [ 36 ] Sedum annuum L Crassulaceae Slender roots Not known Dry soils Th R Not known Senecio vulgaris L Asteraceae Deep root system AM [ 26 , 76 ] Cultivated soils Th R Population-specific Zn tolerance [ 36 ] Silene vulgaris (Moench) Garcke Caryophyllaceae Slender roots NM [ 27 ] Well-drained soils, rangelands He CSR Population-specific Zn tolerance [ 8 ] Smilax aspera L Smilacaceae Rhizome AM [ 25 ] Dry soils L CS Population-specific Zn tolerance [ 77 ] Thymus vulgaris L Lamiaceae Fibrous root system AM [ 26 ] Dry soils Ch CS Population-specific Zn tolerance [ 20 ] Trifolium pratense L Fabaceae Deep root system AM + NM [ 26 , 27 ] Meadows, woods He CSR Population-specific Zn tolerance [ 27 ] Ulex parviflorus Pourr Fabaceae Deep root system AM [ 26 ] Dry, rocky soils, poor soils Ph CS Not known

Int. J. Environ. Res. Public Health 2020 , 17 , 5506 19 of 22 References 1 Bolan, N.; Kunhikrishnan, A.; Thangarajan, R.; Kumpiene, J.; Park, J.; Makino, T.; Kirkham, M.B.; Scheckel, K. Remediation of Heavy Metal(Loid)s Contaminated Soils—To Mobilize or to Immobilize? J. Hazard. Mater 2014 , 266 , 141–166. [ CrossRef ] [ PubMed ] 2 A ff holder, M.-C.; La ff ont-Schwob, I.; Coulomb, B.; Rabier, J.; Borla, A.; Boudenne, J.-L.; Demelas, C.; Prudent, P Implication of Phytometabolites on Metal Tolerance of the Pseudo-Metallophyte - Rosmarinus o ffi cinalis - in a Mediterranean Brownfield Chemosphere 2020 , 249 , 126159. [ CrossRef ] [ PubMed ] 3 Pollard, A.J.; Reeves, R.D.; Baker, A.J.M. Facultative Hyperaccumulation of Heavy Metals and Metalloids Plant Sci 2014 , 217 , 8–17. [ CrossRef ] [ PubMed ] 4 Mendez, M.O.; Maier, R.M. Phytostabilization of Mine Tailings in Arid and Semiarid Environments—An Emerging Remediation Technology Environ. Health Perspect 2008 , 116 , 278–283. [ CrossRef ] 5 Desjardins, D.; Brereton, N.J.B.; Marchand, L.; Brisson, J.; Pitre, F.E.; Labrecque, M. Complementarity of Three Distinctive Phytoremediation Crops for Multiple-Trace Element Contaminated Soil Sci. Total Environ 2018 , 610–611 , 1428–1438. [ CrossRef ] 6 Antoniadis, V.; Levizou, E.; Shaheen, S.M.; Ok, Y.S.; Sebastian, A.; Baum, C.; Prasad, M.N.V.; Wenzel, W.W.; Rinklebe, J. Trace Elements in the Soil-Plant Interface: Phytoavailability, Translocation, and Phytoremediation—A Review Earth-Sci. Rev 2017 , 171 , 621–645. [ CrossRef ] 7 Wiszniewska, A.; Hanus-Fajerska, E.; Muszy ´nska, E.; Ciarkowska, K. Natural Organic Amendments for Improved Phytoremediation of Polluted Soils: A Review of Recent Progress Pedosphere 2016 , 26 , 1–12 [ CrossRef ] 8 Heckenroth, A.; Rabier, J.; Dutoit, T.; Torre, F.; Prudent, P.; La ff ont-Schwob, I. Selection of Native Plants with Phytoremediation Potential for Highly Contaminated Mediterranean Soil Restoration: Tools for a Non-Destructive and Integrative Approach J. Environ. Manag 2016 , 183 , 850–863. [ CrossRef ] 9 Navarro-Cano, J.A.; Goberna, M.; Verd ú , M. Using Plant Functional Distances to Select Species for Restoration of Mining Sites J. Appl. Ecol 2019 , 56 , 2353–2362. [ CrossRef ] 10 Ilunga wa Ilunga, E.; Mahy, G.; Piqueray, J.; S é leck, M.; Shutcha, M.N.; Meerts, P.; Faucon, M.-P. Plant Functional Traits as a Promising Tool for the Ecological Restoration of Degraded Tropical Metal-Rich Habitats and Revegetation of Metal-Rich Bare Soils: A Case Study in Copper Vegetation of Katanga, DRC Ecol. Eng 2015 , 82 , 214–221. [ CrossRef ] 11 Salducci, M.-D.; Folzer, H.; Issartel, J.; Rabier, J.; Masotti, V.; Prudent, P.; A ff re, L.; Hardion, L.; Tatoni, T.; La ff ont-Schwob, I. How Can a Rare Protected Plant Cope with the Metal and Metalloid Soil Pollution Resulting from Past Industrial Activities? Phytometabolites, Antioxidant Activities and Root Symbiosis Involved in the Metal Tolerance of Astragalus tragacantha Chemosphere 2019 , 217 , 887–896. [ CrossRef ] [ PubMed ] 12 A ff holder, M.-C.; Pricop, A.-D.; La ff ont-Schwob, I.; Coulomb, B.; Rabier, J.; Borla, A.; Demelas, C.; Prudent, P As, Pb, Sb, and Zn Transfer from Soil to Root of Wild Rosemary: Do Native Symbionts Matter? Plant Soil 2014 , 382 , 219–236. [ CrossRef ] 13 Muszy ´nska, E.; Labudda, M.; Kral, A. Ecotype-Specific Pathways of Reactive Oxygen Species Deactivation in Facultative Metallophyte Silene Vulgaris (Moench) Garcke Treated with Heavy Metals Antioxidants 2020 , 9 , 102. [ CrossRef ] [ PubMed ] 14 Burns, J.H.; Anacker, B.L.; Strauss, S.Y.; Burke, D.J. Soil Microbial Community Variation Correlates Most Strongly with Plant Species Identity, Followed by Soil Chemistry, Spatial Location and Plant Genus AoB Plants 2015 , 7 , plv 030. [ CrossRef ] [ PubMed ] 15 Phillips, J.M.; Hayman, D.S. Improved Procedures for Clearing Roots and Staining Parasitic and Vesicular-Arbuscular Mycorrhizal Fungi for Rapid Assessment of Infection Trans. Br. Mycol. Soc 1970 , 55 , 158–161. [ CrossRef ] 16 Conesa, H.M.; Garc í a, G.; Faz, Á .; Arnaldos, R. Dynamics of Metal Tolerant Plant Communities’ Development in Mine Tailings from the Cartagena-La Uni ó n Mining District (SE Spain) and Their Interest for Further Revegetation Purposes Chemosphere 2007 , 68 , 1180–1185. [ CrossRef ] [ PubMed ] 17 Fern á ndez, S.; Poschenrieder, C.; Marcen ò , C.; Gallego, J.R.; Jim é nez-G á mez, D.; Bueno, A.; Afif, E Phytoremediation Capability of Native Plant Species Living on Pb-Zn and Hg-As Mining Wastes in the Cantabrian Range, North of Spain J. Geochem. Explor 2017 , 174 , 10–20. [ CrossRef ]

Int. J. Environ. Res. Public Health 2020 , 17 , 5506 20 of 22 18 Higueras, P.; Esbr í , J.M.; Garc í a-Ordiales, E.; Gonz á lez-Corrochano, B.; L ó pez-Berdonces, M.A.; Garc í a-Noguero, E.M.; Alonso-Azc á rate, J.; Mart í nez-Coronado, A. Potentially Harmful Elements in Soils and Holm-Oak Trees ( Quercus Ilex L.) Growing in Mining Sites at the Valle de Alcudia Pb-Zn District (Spain)—Some Clues on Plant Metal Uptake J. Geochem. Explor 2017 , 182 , 166–179. [ CrossRef ] 19 Pratas, J.; Favas, P.J.C.; D’Souza, R.; Varun, M.; Paul, M.S. Phytoremedial Assessment of Flora Tolerant to Heavy Metals in the Contaminated Soils of an Abandoned Pb Mine in Central Portugal Chemosphere 2013 , 90 , 2216–2225. [ CrossRef ] [ PubMed ] 20 Escarr é , J.; Lef è bvre, C.; Raboyeau, S.; Dossantos, A.; Gruber, W.; Cleyet Marel, J.C.; Fr é rot, H.; Noret, N.; Mahieu, S.; Collin, C.; et al. Heavy Metal Concentration Survey in Soils and Plants of the Les Malines Mining District (Southern France): Implications for Soil Restoration Water Air Soil Pollut 2011 , 216 , 485–504 [ CrossRef ] 21 Szarek-Łukaszewska, G. Vegetation of Reclaimed and Spontaneously Vegetated Zn-Pb Mine Wastes in Southern Poland Pol. J. Environ. Stud 2009 , 18 , 717–733 22 Pollock, L.J.; Morris, W.K.; Vesk, P.A. The Role of Functional Traits in Species Distributions Revealed through a Hierarchical Model Ecography 2012 , 35 , 716–725. [ CrossRef ] 23 Sardans, J.; Peñuelas, J. Plant-Soil Interactions in Mediterranean Forest and Shrublands: Impacts of Climatic Change Plant Soil 2013 , 365 , 1–33. [ CrossRef ] [ PubMed ] 24 Pierret, A.; Maeght, J.-L.; Cl é ment, C.; Montoroi, J.-P.; Hartmann, C.; Gonkhamdee, S. Understanding Deep Roots and Their Functions in Ecosystems: An Advocacy for More Unconventional Research Ann. Bot 2016 , 118 , 621–635. [ CrossRef ] [ PubMed ] 25 Azul, A.M.; Ramos, V.; Pato, A.; Azenha, M.; Freitas, H. Mycorrhizal Types in the Mediterranean Basin: Safety Teaching and Training J. Biol. Educ 2008 , 42 , 130–137. [ CrossRef ] 26 Wang, B.; Qiu, Y.-L. Phylogenetic Distribution and Evolution of Mycorrhizas in Land Plants Mycorrhiza 2006 , 16 , 299–363. [ CrossRef ] [ PubMed ] 27 Pawlowska, T.E.; Błaszkowski, J.; Rühling, Å. The Mycorrhizal Status of Plants Colonizing a Calamine Spoil Mound in Southern Poland Mycorrhiza 1997 , 6 , 499–505. [ CrossRef ] 28 Regvar, M.; Vogel, K.; Irgel, N.; Wraber, T.; Hildebrandt, U.; Wilde, P.; Bothe, H. Colonization of Pennycresses ( Thlaspi Spp.) of the Brassicaceae by Arbuscular Mycorrhizal Fungi J. Plant Physiol 2003 , 160 , 615–626 [ CrossRef ] 29 Roumet, C.; Lafont, F.; Sari, M.; Warembourg, F.; Garnier, E. Root Traits and Taxonomic A ffi liation of Nine Herbaceous Species Grown in Glasshouse Conditions Plant Soil 2008 , 312 , 69–83. [ CrossRef ] 30 Rajkumar, M.; Sandhya, S.; Prasad, M.N.V.; Freitas, H. Perspectives of Plant-Associated Microbes in Heavy Metal Phytoremediation Biotechnol. Adv 2012 , 30 , 1562–1574. [ CrossRef ] 31 Dietterich, L.H.; Gonneau, C.; Casper, B.B. Arbuscular Mycorrhizal Colonization Has Little Consequence for Plant Heavy Metal Uptake in Contaminated Field Soils Ecol. Appl 2017 , 27 , 1862–1875. [ CrossRef ] [ PubMed ] 32 Wang, F. Occurrence of Arbuscular Mycorrhizal Fungi in Mining-Impacted Sites and Their Contribution to Ecological Restoration: Mechanisms and Applications Crit. Rev. Environ. Sci. Technol 2017 , 47 , 1901–1957 [ CrossRef ] 33 Nadg ó rska-Socha, A.; Kandziora-Ciupa, M.; Ciepał, R. Element Accumulation, Distribution, and Phytoremediation Potential in Selected Metallophytes Growing in a Contaminated Area Environ. Monit. Assess 2015 , 187 , 441. [ CrossRef ] [ PubMed ] 34 Bothe, H. Plants in Heavy Metal Soils. In Detoxification of Heavy Metals ; Sherameti, I., Varma, A., Eds.; Springer: Berlin / Heidelberg, Germany, 2011; Volume 30, pp. 35–57 35 Marsili, S.; Roccotiello, E.; Carbone, C.; Marescotti, P.; Cornara, L.; Mariotti, M.G. Plant Colonization on a Contaminated Serpentine Site Northeast. Nat 2009 , 16 , 297–308. [ CrossRef ] 36 Szarek-Łukaszewska, G.; Grodzi ´nska, K. Grasslands of A Zn-Pb Post-Mining Area (Olkusz Ore-Bearing Region, S Poland) Pol. Bot. J 2011 , 56 , 245–260 37 Bergmeier, E.; Konstantinou, M.; Tsiripidis, I.; S ý kora, K.V. Plant Communities on Metalliferous Soils in Northern Greece Phytocoenologia 2009 , 39 , 411–438. [ CrossRef ] 38 Woch, M.W. Species Trait-Environment Relationships in Semi-Dry Brachypodium pinnatum Grasslands on Old Waste Heaps Left by Zn-Pb Mining in the Western Małopolska Region (S Poland) Tuexenia 2017 , 37 , 247–270.

Int. J. Environ. Res. Public Health 2020 , 17 , 5506 21 of 22 39 Turnau, K.; Gawro ´nski, S.; Ryszka, P.; Zook, D. Mycorrhizal-Based Phytostabilization of Zn–Pb Tailings: Lessons from the Trzebionka Mining Works (Southern Poland). In Bio-Geo Interactions in Metal-Contaminated Soils ; Kothe, E., Varma, A., Eds.; Springer: Berlin / Heidelberg, Germany, 2012; Volume 31, pp. 327–348 40 Hazard, C.; Johnson, D. Does Genotypic and Species Diversity of Mycorrhizal Plants and Fungi A ff ect Ecosystem Function? New Phytol 2018 , 220 , 1122–1128. [ CrossRef ] 41 Partida-Mart í nez, L.P.; Heil, M. The Microbe-Free Plant: Fact or Artifact? Front. Plant Sci 2011 , 2 . [ CrossRef ] 42 Raunkiær, C The Life Forms of Plants and Statistical Plant Geography ; Oxford University Press: Oxford, UK, 1934 43 Lombardo, E.; Bancheva, S.; Domina, G.; Venturella, G. Distribution, Ecological Role and Symbioses of Selected Shrubby Species in the Mediterranean Basin: A Review Plant Biosyst. Int. J. Deal. Asp. Plant Biol 2020 , 154 , 1–31. [ CrossRef ] 44 Bonis, A.; Grubb, P.J.; Coomes, A. Requirements of Gap-Demanding Species in Chalk Grassland: Reduction of Root Competition versus Nutrient Enrichment by Animals J. Ecol 1997 , 85 , 625–633. [ CrossRef ] 45 Li, H.-Y.; Li, D.-W.; He, C.-M.; Zhou, Z.-P.; Mei, T.; Xu, H.-M. Diversity and Heavy Metal Tolerance of Endophytic Fungi from Six Dominant Plant Species in a Pb-Zn Mine Wasteland in China Fungal Ecol 2012 , 5 , 309–315. [ CrossRef ] 46 Zhang, Y.; Li, T.; Zhao, Z.-W. Colonization Characteristics and Composition of Dark Septate Endophytes (DSE) in a Lead and Zinc Slag Heap in Southwest China Soil Sediment Contam. Int. J 2013 , 22 , 532–545 [ CrossRef ] 47 Oba, H.; Tawaray, K.; Wagatsuma, T. Arbuscular Mycorrhizal Colonization in Lupinus and Related Genera Soil Sci. Plant Nutr 2001 , 47 , 685–694. [ CrossRef ] 48 Baumel, A.; Auda, P.; Torre, F.; Medail, F. Morphological Polymorphism and RDNA Internal Transcribed Spacer (ITS) Sequence Variation in Armeria (Plumbaginaceae) from South-Eastern France Bot. J. Linn. Soc 2009 , 159 , 255–267. [ CrossRef ] 49 Kachenko, A.G. General Introduction & Background. In Ecophysiology and Phytoremediation Potential of Heavy Metal (Loid) Accumulating Plants ; The University of Sydney: New South Wales, Australia, 2008; pp. 1–52 50 Babst-Kostecka, A.A.; Waldmann, P.; Fr é rot, H.; Vollenweider, P. Plant Adaptation to Metal Polluted Environments—Physiological, Morphological, and Evolutionary Insights from Biscutella laevigata Environ. Exp. Bot 2016 , 127 , 1–13. [ CrossRef ] 51 Pastor, J.; Hern á ndez, A.J. Multi-Functional Role of Grassland Systems in the Ecological Restoration of Mines, Landfills, Roadside Slopes and Agroecosystems Options Mediterr 2008 , 79 , 6 52 Veselkin, D.V.; Konoplenko, M.A.; Betekhtina, A.A. Means for Soil Nutrient Uptake in Sedges with Di ff erent Ecological Strategies Russ. J. Ecol 2014 , 45 , 547–554. [ CrossRef ] 53 Tsiripidis, I.; Papaioannou, A.; Sapounidis, V.; Bergmeier, E. Approaching the Serpentine Factor at a Local Scale—A Study in an Ultramafic Area in Northern Greece Plant Soil 2010 , 329 , 35–50. [ CrossRef ] 54 Meerts, P.; Grommesch, C. Soil Seed Banks in a Heavy-Metal Polluted Grassland at Prayon (Belgium) Plant Ecol 2001 , 155 , 35–45. [ CrossRef ] 55 Colombo, R.P.; Mart í nez, A.E.; Pardo, A.F.; Di Bidondo, L.F.; Van Baren, C.; Lira, P.d.L.; Godeas, A.M Di ff erential E ff ects of Two Strains of Rhizophagus intraradices on Dry Biomass and Essential Oil Yield and Composition in Calamintha nepeta Rev. Argent. Microbiol 2013 , 45 , 114–118. [ CrossRef ] 56 Hüseyinova, R.; Kutbay, H.G.; Bilgin, A.; Kılıç, D.; Horuz, A.; Kırmano ˘glu, C. Sulphur and Some Heavy Metal Contents in Foliage of Corylus avellana and Some Roadside Native Plants in Ordu Province, Turkey Ekoloji 2009 , 18 , 10–16. [ CrossRef ] 57 Shallari, S.; Schwartza, C.; Haskob, A.; MorelaT, J.L. Heavy Metals in Soils and Plants of Serpentine and Industrial Sites of Albania Sci. Total Environ 1998 , 10. [ CrossRef ] 58 Turnau, K. Heavy Metal Content and Localization in Mycorrhizal Euphorbia cyparissias from Zinc Wastes in Southern Poland Acta Soc. Bot. Pol 1998 , 67 , 105–113. [ CrossRef ] 59 Brown, G.; Brinkmann, K. Heavy Metal Tolerance in Festuca ovina L. from Contaminated Sites in the Eifel Mountains, Germany Plant Soil 1992 , 143 , 239–247. [ CrossRef ] 60 Nworie, O.; Qin, J.; Lin, C. Trace Element Uptake by Herbaceous Plants from the Soils at a Multiple Trace Element-Contaminated Site Toxics 2019 , 7 , 3. [ CrossRef ] [ PubMed ] 61 Boularbah, A.; Schwartz, C.; Bitton, G.; Aboudrar, W.; Ouhammou, A.; Morel, J.L. Heavy Metal Contamination from Mining Sites in South Morocco: 2. Assessment of Metal Accumulation and Toxicity in Plants Chemosphere 2006 , 63 , 811–817. [ CrossRef ]

Int. J. Environ. Res. Public Health 2020 , 17 , 5506 22 of 22 62 Glavaˇc, N.K.; Djogo, S.; Raži´c, S.; Kreft, S.; Veber, M. Accumulation of Heavy Metals from Soil in Medicinal Plants Arch. Ind. Hyg. Toxicol 2017 , 68 , 236–244. [ CrossRef ] 63 Yildiz, D.; Kula, I.; Ay, G.; Baslar, S.; Dogan, Y. Determination of Trace Elements in the Plants of Mt. Bozdag, Izmir, Turkey Arch. Biol. Sci 2010 , 62 , 731–738. [ CrossRef ] 64 Gucwa-Przepi ó ra, E.; Chmura, D.; Sokołowska, K. AM and DSE Colonization of Invasive Plants in Urban Habitat: A Study of Upper Silesia (Southern Poland) J. Plant Res 2016 , 129 , 603–614. [ CrossRef ] [ PubMed ] 65 Suchkova, N.; Tsiripidis, I.; Alifragkis, D.; Ganoulis, J.; Darakas, E.; Sawidis, T. Assessment of Phytoremediation Potential of Native Plants during the Reclamation of an Area A ff ected by Sewage Sludge Ecol. Eng 2014 , 69 , 160–169. [ CrossRef ] 66 Hern á ndez, A.J.; Pastor, J. Incidencia De Metales Pesados En La Biodiversidad De Pastizales En El Emplazamiento De Una Explotaci ó n Abandonada De Baritina. In Los Sistemas Forrajeros: Entre La Producci ó n Y El Paisaje ; Reuni ó n Cient í fica XLVI, SEEP: Vitoria-Gasteiz, Spain, 2007; p. 8 67 Friede, M.; Unger, S.; Hellmann, C.; Beyschlag, W. Conditions Promoting Mycorrhizal Parasitism Are of Minor Importance for Competitive Interactions in Two Di ff erentially Mycotrophic Species Front. Plant Sci 2016 , 7 , 1465. [ CrossRef ] [ PubMed ] 68 Ban á sov á , V.; ˇ Durišov á , E.; Nadubinsk á , M.; Gurinov á , E.; ˇ Ciamporov á , M. Natural Vegetation, Metal Accumulation and Tolerance in Plants Growing on Heavy Metal Rich Soils. In Bio-Geo Interactions in Metal-Contaminated Soils ; Kothe, E., Varma, A., Eds.; Springer: Berlin / Heidelberg, Germany, 2012; Volume 31, pp. 233–250 69 Paj ˛ ak, M.; Halecki, W.; G ˛ asiorek, M. Accumulative Response of Scots Pine ( Pinus sylvestris L.) and Silver Birch ( Betula pendula Roth) to Heavy Metals Enhanced by Pb-Zn Ore Mining and Processing Plants: Explicitly Spatial Considerations of Ordinary Kriging Based on a GIS Approach Chemosphere 2017 , 168 , 851–859 70 Anawar, H.M.; Canha, N.; Santa-Regina, I.; Freitas, M.C. Adaptation, Tolerance, and Evolution of Plant Species in a Pyrite Mine in Response to Contamination Level and Properties of Mine Tailings: Sustainable Rehabilitation J. Soils Sediments 2013 , 13 , 730–741. [ CrossRef ] 71 Pedron, F.; Petruzzelli, G.; Barbafieri, M.; Tassi, E. Strategies to Use Phytoextraction in Very Acidic Soil Contaminated by Heavy Metals Chemosphere 2009 , 75 , 808–814. [ CrossRef ] 72 Newman, E.I.; Eason, W.R.; Eissenstat, D.M.; Ramos, M.I.R.F. Interactions between Plants: The Role of Mycorrhizae Mycorrhiza 1992 , 1 , 47–53. [ CrossRef ] 73 Kalinovic, J.V.; Serbula, S.M.; Radojevic, A.A.; Milosavljevic, J.S.; Kalinovic, T.S.; Steharnik, M.M. Assessment of As, Cd, Cu, Fe, Pb, and Zn Concentrations in Soil and Parts of Rosa spp. Sampled in Extremely Polluted Environment Environ. Monit. Assess 2019 , 191 , 15. [ CrossRef ] 74 Zekri, J.; Rached, O.; Sahli, L.; Yilmaz, A.; Temel, H.; Tahar, A. Assessment of Soil Contamination and Plant Stress Tolerance in an Antimony Mining Area: Case Study for Scabiosa atropurpurea L and Santolina chamaecyparissus Environ. Ecol 2019 , 37 , 747–757 75 Reeves, R.D.; Adigüzel, N.; Baker, A.J.M. Nickel Hyperaccumulation in Bornmuellera kiyakii Aytaç & Aksoy and Associated Plants of the Brassicaceae from Kızılda ˘g (Derebucak, Konya-Turkey) Turk. J. Bot 2009 , 33 , 33–40 76 Barni, E.; Siniscalco, C. Vegetation Dynamics and Arbuscular Mycorrhiza in Old-Field Successions of the Western Italian Alps Mycorrhiza 2000 , 10 , 63–72. [ CrossRef ] 77 Poschenrieder, C.; Llugany, M.; Lombini, A.; Dinelli, E.; Bech, J.; Barcel ó , J Smilax aspera L. an Evergreen Mediterranean Climber for Phytoremediation J. Geochem. Explor 2012 , 123 , 41–44. [ CrossRef ] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http: // creativecommons.org / licenses / by / 4.0 / ).