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Background and aims: Metallophytes are plants that can tolerate extreme metal concentrations in the soil in which they grow. The Dugald River zinc (Zn)-lead (Pb) gossan in Queensland (Australia) is one of the largest metal deposits in the world with a surface gossan formed after weathering over millions of years. It hosts a range of metallophytes which may have potential to be used in mine site rehabilitation. This study aimed to investigate the soil-plant relationships of metallophytes on the Dugald River gossan. Methods: Plant samples and associated rooting soil samples were collected across the gossan and then analysed for metal concentrations. Soil-plant metal relationships were subsequently explored to characterise the species in relation to metal uptake behaviour. Results: The metallophyte grass, Eriachne mucronata, dominated the gossan, yet there appeared to be no direct relationship between the occurrence of metallophytes and prevailing soil metal concentrations. Using transformation-based redundancy analysis (tb-RDA), two groups of metals, copper (Cu) and Zn-Cadmium (Cd), have been identified to be the primary metals driving species distribution. Crotalaria novae-hollandiae, was able to accumulate high concentrations of each of these metals in its leaves, with up to 16,200 mg Zn kg−1, 545 mg Cu kg−1 and 170 mg Cd kg−1. Conclusions: Soil metal concentrations alone are not suitable indications for metallophyte distribution or composition in a polymetallic environment. Crotalaria novae-hollandiae can tolerate high concentrations of metals and accumulate Zn-Cu-Cd above the respective hyperaccumulation thresholds; the species can be described for the first time as a strong polymetallic indicator-type metallophyte.

X-ray Fluorescence Spectroscopy (XRF) is a non-destructive analytical technique that has recently been developed as a tool for high throughput screening for the discovery of trace element hyperaccumulator species in herbarium collections. Herbarium XRF analysis was undertaken of representatives of the Brassicaceae family in the two herbaria in Belgrade (Serbia). This was coupled to field collection and chemical analysis of new samples of Cardamine waldsteinii. The data revealed a potentially new zinc hyperaccumulator species, C. waldsteinii, with 3050 mg kg−1 Zn in its leaves. This compares to 2210 mg kg−1 in the leaves of this species collected from the field. These values are below the notional hyperaccumulation threshold for zinc but are nevertheless unusual. The remarkably high accumulation factor for zinc are suggestive of the exceptional potential of C. waldsteinii for zinc uptake, even in non-metalliferous, zinc-deficient soils. We recommend following up this study with experimental investigations on C. waldsteinii to elucidate its ecophysiology and its zinc tolerance and accumulation characteristics.

Purposes To analyze the main edaphic factors that differentiate ultramafic from typical Cerrado environments and act as agents of the development of biochemical and morphological mechanisms of species adaptation to these harsh environments; and to determine the genetic diversity of three Cerrado?s shrubland native species (Justicia lanstyakii, Euploca salicoides, and Oxalis hirsutissima). Methods We conducted chemical analysis of metal contents in soils, as well as on elemental composition, and analysis of DNA extracted from leaf tissues of the three species found in both environments. In leaves of E. salicoides grown in both environments we evaluated the changes in the levels of non-structural carbohydrates (NSC), and total proteins. Results The accessions obtained in ultramafic soils were closer with each other, indicating genetic similarity and major differences in relation to the accessions collected in the Cerrado area. These differentiations probably are related with higher adaptation to soils rich in metals, mainly Ni in ultramafic, and Al in the Cerrado environments. The highest levels of NSC were observed in plants grown in ultramafic soils, including raffinose, which is related to responses to metal detoxification and drought. The allocation of Ni in the trichomes, which does not affect important processes of plant metabolism, is another mechanism developed by E. salicoides to overcome the hyperaccumulation of Ni in their tissues. Conclusions These findings can help select seed collection sites representative of the genetic diversity of native plant species for restoring degraded areas or for phytoremediation of metals. Made available in DSpace on 2022-06-15T10:20:00Z (GMT). No. of bitstreams: 1 Leide-Edaphic-factors-as-genetic-selection.pdf: 8532198 bytes, checksum: c217a3fdc7b391c15c06f63e01a18233 (MD5) Previous issue date: 2022

Background and aims: Phosphorus is one of the major nutrients that directly or indirectly affects all aspects of plant growth. Tropical nickel hyperaccumulators, including Phyllanthus rufuschaneyi and Rinorea cf. bengalensis from Borneo Island (in the Malaysian state of Sabah), have evolved to grow in extremely P-impoverished ultramafic soils. This study aimed to establish the response of the root and shoot ionome of these two agromining ‘metal crops’ to soil P availability. Methods: We undertook a soil P dosing trial on P. rufuschaneyi and R. cf. bengalensis in Sabah (Malaysia) over a period of 12 months. We measured the elemental concentrations in the soil solution and roots, as well as in the developed and developing stems and leaves. Results: The results show that root and shoot P accumulation increased markedly as soil P availability increased by 80-fold in P. rufuschaneyi, whereas R. cf. bengalensis did not increase as strongly to P supply, despite a 135-fold increase in soil solution P. The contrasting observations on the root and shoot P accumulation patterns in these species suggests distinct P acquisition strategies in these species. The non-responsiveness of R. cf. bengalensis to increasing soil P availability may be related to its possible association with mycorrhizal fungi. Conclusion: The findings of this study reveal the complexities of phosphate uptake, transport and accumulation in these ‘metal crops’. This information is essential to develop appropriate nutrient management in nickel agromining operations.

Nickel hyperaccumulation in Blepharidium guatemalense Standl. (Rubiaceae) was found in the tropical forests of south-eastern Mexico. This study aimed to document the geographic extent of nickel hyperaccumulation in this species, to understand its process of hyperaccumulation and to explore nickel distribution within the tissues of this plant. To accomplish these objectives, a complete non-destructive elemental screening of herbarium specimens was performed with a hand-held X-ray fluorescence spectrometer. Besides, rhizosphere soils and plant tissues were collected in Mexico and analyzed for physical–chemical parameters. Finally, elemental distribution maps of nickel and other elements in plant tissues were obtained by X-ray fluorescence spectroscopy and microscopy. This study revealed that Blepharidium guatemalense is distributed throughout Chiapas, Tabasco and Campeche, reaching the maximum nickel concentration in leaves (4.3 wt%) followed by roots and seeds (2.0 wt%) and bark (1.8 wt%). Simultaneous hyperaccumulation of cobalt and nickel was found in 15% of the herbarium specimens. Blepharidium guatemalense has uncommon re-distribution mechanisms via phloem since this tissue is the highest nickel-enriched from all parts of the plant (from roots to leaves). A high total nickel (mean of 610 µg g−1) was found in rhizosphere soils even though no evidence of ophiolite emplacement in that area has been reported. Blepharidium guatemalense represents the first hypernickelophore (> 1 wt% Ni) to be reported as growing in soils that are neither ultramafic nor enriched by anthropogenic pollutants.

Glass manufacturing operations lead to an increasing number of abandoned slag heaps contaminated with metallic trace elements (MTE). However, the relative influence of edaphic factors on the biodiversity of glasswork wastelands is still poorly understood although closely related to sustainable land management practices. Therefore, the objectives of this research were to provide new insights into glasswork wastelands through the investigation of (i) Orthoptera, diurnal Lepidoptera, plant communities, and (ii) abiotic parameters in the topsoils. To that end, biodiversity indices were computed from ecological inventories performed on the herbaceous layer. In addition, soil samples were taken from the topsoil layer (0-10 cm) to assess agronomic properties, actually (CEC-exchangeable) and potentially bioavailable MTE fractions (DTPA-extractable) and pseudo-total MTE contents. On the one hand, the studied site was able to support a substantially higher than excepted biodiversity with orthopteran assemblages similar to grasslands and a diurnal Lepidoptera diversity comparable to urban parks. We also noted a positive influence of plant richness on the diurnal Lepidoptera community structure. On the other hand, topsoil analysis revealed a severe Pb contamination (1800-3100 mg kg

Background The fern Dicranopteris linearis is a hyperaccumulator of rare earth elements (REEs), aluminium (Al) and silicon (Si). However, the physiological mechanisms of tissue-level tolerance of high concentrations of REE and Al, and possible interactions with Si, are currently incompletely known. Methods A particle-induced X-ray emission (μPIXE) microprobe with the Maia detector, scanning electron microscopy with energy-dispersive spectroscopy and chemical speciation modelling were used to decipher the localization and biochemistry of REEs, Al and Si in D. linearis during uptake, translocation and sequestration processes. Results In the roots >80 % of REEs and Al were in apoplastic fractions, among which the REEs were most significantly co-localized with Si and phosphorus (P) in the epidermis. In the xylem sap, REEs were nearly 100 % present as REEH3SiO42+, without significant differences between the REEs, while 24–45 % of Al was present as Al-citrate and only 1.7–16 % Al was present as AlH3SiO42+. In the pinnules, REEs were mainly concentrated in necrotic lesions and in the epidermis, and REEs and Al were possibly co-deposited within phytoliths (SiO2). Different REEs had similar spatial localizations in the epidermis and exodermis of roots, the necrosis, veins and epidermis of pinnae of D. linearis. Conclusions We posit that Si plays a critical role in REE and Al tolerance within the root apoplast, transport within the vascular bundle and sequestration within the blade of D. linearis.

International audience; Background and Aims: In tropical ultramafic soils, potassium (K) is typically the most growth limiting nutrient. However, tropical nickel (Ni) hyperaccumulator plants, including Phyllanthus rufuschaneyi and Rinorea cf. bengalensis (which are ‘metal crops’ used in agromining) from Malaysia, have unusually high K shoot accumulation compared to other species, despite naturally growing on severely K-impoverished ultramafic soils. This study aimed to establish the response to soil K availability in relation to uptake of K and other elements in the roots and shoots of P. rufuschaneyi and R. cf. bengalensis.Methodology: We undertook an experiment in which soluble K was dosed to ultramafic soil in pots with P. rufuschaneyi and R. cf. bengalensis in Sabah (Malaysia).Results: The results show that root K concentrations increased markedly as the soil K availability increased by 35-fold, whilst the corresponding effect on K accumulation in the shoots of P. rufuschaneyi and R. cf. bengalensis was not significantly different in relation to soil K dosing. Observed divergent responses between root and shoot K accumulation in these species suggests a separate genetic control of K uptake and xylem loading in P. rufuschaneyi and R. cf. bengalensis.Conclusion: The tight control of root-to-shoot K translocation and constrained K accumulation in shoots under a soil K gradient is likely an adaptive mechanism to the evolution of these species to grow in highly nutrient-impoverished ultramafic soils. This study provides information that will be useful for better nutrient management of tropical Ni metal farms that use K-efficient Ni ‘metal crops’.

Background and Aims The aim of this study was to test the frequency distributions of foliar elements from a large dataset from Kinabalu Park (Sabah, Malaysia) for departure from unimodality, indicative of a distinct ecophysiological response associated with hyperaccumulation. Methods We collected foliar samples (n = 1533) comprising 90 families, 198 genera and 495 plant species from ultramafic soils, further foliar samples (n = 177) comprising 45 families, 80 genera and 120 species from non-ultramafic soils and corresponding soil samples (n = 393 from ultramafic soils and n = 66 from non-ultramafic soils) from Kinabalu Park (Sabah, Malaysia). The data were geographically (Kinabalu Park) and edaphically (ultramafic soils) constrained. The inclusion of a relatively high proportion (approx. 14 %) of samples from hyperaccumulator species [with foliar concentrations of aluminium and nickel (Ni) >1000 μg g–1, cobalt, copper, chromium and zinc >300 μg g–1 or manganese (Mn) >10 mg g–1] allowed for hypothesis testing. Key Results Frequency distribution graphs for most elements [calcium (Ca), magnesium (Mg) and phosphorus (P)] were unimodal, although some were skewed left (Mg and Mn). The Ni frequency distribution was bimodal and the separation point for the two modes was between 250 and 850 μg g–1. Conclusions Accounting for statistical probability, the established empirical threshold value (>1000 μg g–1) remains appropriate. The two discrete modes for Ni indicate ecophysiologically distinct behaviour in plants growing in similar soils. This response is in contrast to Mn, which forms the tail of a continuous (approximately log-normal) distribution, suggestive of an extension of normal physiological processes.

Background: Phytoextraction is an in situ technique that can be applied to minerals and mining wastes using hyperaccumulator plants to purposely bio-concentrate high levels of metals or metalloids into their shoots in order to remove them from the substrate, while achieving monetary gain. Phytoextraction can be applied to a limited number of elements depending on the existence of hyperaccumulator plants with suitable characteristics. Although phytoextraction has been trialled in experimental settings, it requires testing at field scale to assess commercial broad-scale potential. Scope: The novelty and purported environmental benefits of phytoextraction have attracted substantial scientific inquiry. The main limitation of phytoextraction with hyperaccumulators is the number of suitable plants with a high accumulation capacity for a target element. We outline the main considerations for applying phytoextraction using selected elemental case studies in which key characteristics of the element, hyperaccumulation and economic considerations are evaluated. Conclusions: The metals cobalt, cadmium, thallium and rhenium and the metalloids arsenic and selenium are present in many types of minerals wastes, especially base metal mining tailings, at concentrations amenable for economic phytoextraction. Phytoextraction should focus on the most toxic elements (arsenic, cadmium, and thallium) or especially valuable elements (selenium, cobalt, and rhenium). The value proposition is in the clean-up of contaminated land in the case of toxic elements, whereas it is in the ‘bio-ore’ generated by the process in the case of valuable elements.

Globally, there is a major shift to electric vehicles to combat climate change and these vehicles are currently powered by lithium-ion batteries that contain nickel cobalt manganese oxide materials. This technological change from internal combustion engines means that demand for battery minerals will need to increase by factors of >20 for the critical metals required for batteries in the next three decades. If this scenario plays out, it will require a dramatic increase in the worldwide capacity to produce nickel, manganese, cobalt, and lithium raw materials of sufficient purity. This demand could partly be met by agromining technology, which is a ‘green technology’ that extracts valuable products, including high-purity metal salts useful for the battery industry, from selected plants known as ‘metal crops’. Farming for nickel, cobalt, and manganese is currently within reach, whereas lithium agromining has not yet been developed but has potential. Synopsis: Agromining offers a sustainable approach to economically produce battery-grade raw materials from unconventional sources, thus, producing ‘green technologies’ from ‘green sources’.

The molecular biology and genetics of the Ni–Cd–Zn hyperaccumulator Noccaea caerulescens has been extensively studied, but no information is yet available on Ni and Zn redistribution and mobilization during seed germination. Due to the different physiological functions of these elements, and their associated transporter pathways, we expected differential tissue distribution and different modes of translocation of Ni and Zn during germination. This study used synchrotron X-ray fluorescence tomography techniques as well as planar elemental X-ray imaging to elucidate elemental (re)distribution at various stages of the germination process in contrasting accessions of N. caerulescens. The results show that Ni and Zn are both located primarily in the cotyledons of the emerging seedlings and Ni is highest in the ultramafic accessions (up to 0.15 wt%), whereas Zn is highest in the calamine accession (up to 600 μg g–1). The distribution of Ni and Zn in seeds was very similar, and neither element was translocated during germination. The Fe maps were especially useful to obtain spatial reference within the seeds, as it clearly marked the vasculature. This study shows how a multimodal combination of synchrotron techniques can be used to obtain powerful insights about the metal distribution in physically intact seeds and seedlings.

The manganese (Mn) hyperaccumulator Gossia bidwillii is a tree species native to subtropical eastern Australia where it occurs on Mn-rich soils. Here, we conducted the first Mn accumulation and tissue-level distribution study on wild and experimentally grown G. bidwillii. Gossia bidwillii plants were subjected to different levels of Mn (250 µg g−1, 500 µg g−1, 1000 µg g−1) soil dosing treatments, whereas the wild G. bidwillii was sampled from growing on highly Mn-enriched natural soils. We used laboratory-based micro-X-ray Fluorescence (µXRF) elemental mapping to elucidate in situ distribution patterns of Mn and other elements in hydrated wild and Mn-dosed G. bidwillii leaves. The data from wild G. bidwillii revealed that it can be strongly Mn-hyperaccumulating with foliar Mn concentrations of 39 000 µg g−1 and 24 000 µg g−1 in old and young leaves, respectively. In the Mn dosing trial, G. bidwillii accumulated 24 400 µg g−1 in old leaves and 17 100 µg g−1 in young leaves in the highest treatment level. The laboratory based µXRF data revealed that Mn is uniformly enriched throughout the laminae and petioles of both young and old leaves in wild G. bidwillii; while in Mn-dosed G. bidwillii, the foliar Mn distribution was primarily concentrated at the leaf-tip and lamina. The approach employed by combining data from the field and controlled experiments was especially meaningful for investigating Mn accumulation in this species and gaining added insight into the phenomenon of Mn hyperaccumulation.

Plant and soil 475(1-2), 515 - 534 (2022). doi:10.1007/s11104-022-05388-3, PurposeThe Cunoniaceae are a major component of the New Caledonian flora with 91 endemic species that are highly unusual in that multiple metals are hyperaccumulated in different species. This makes it an ideal model system for studying the nature of the hyperaccumulation phenomenon.MethodsX-ray fluorescence spectroscopy (XRF) scanning of all herbarium collections of the Cunoniaceae was undertaken at the Herbarium of New Caledonia to reveal incidences of nickel (Ni) and manganese (Mn) accumulation. Following on, the Mn hyperaccumulating P. reticulata and the Ni hyperaccumulating P. xaragurensis were selected for detailed follow-up investigations using synchrotron-based X-ray fluorescence microscopy (XFM).ResultsThe systematic XRF screening of herbarium specimens showed that numerous species have high foliar Mn and Ni with species either accumulating Ni or Mn, but not both elements simultaneously. Soil ‘extractable’ Mn and Ni concentrations associated with Pancheria reticulata and P. xaragurensis greatly varies between the species. The XFM data shows that P. reticulata has a distinctive distribution pattern with Mn concentrated in large hypodermal cells. This contrasts with P. xaragurensis where Ni was mainly localized in and around the epidermis, and hypodermal cells were not observed., Published by Springer Science + Business Media B.V, Dordrecht [u.a.]

The extend of biogeochemical cycling of nickel (Ni) by tropical hyperaccumulator plants in their native habitat is largely unknown, although these unusual plants are suspected to play a major role in the recycling of this element in ultramafic ecosystems. In this study, we have assessed the biogeochemical cycling of Ni (and other elements, including mineral nutrients) by a tropical Ni hyperaccumulator plant, i.e., Phyllanthus rufuschaneyi, which is one of the most promising species for tropical Ni agromining. The study site was a young secondary forest in Sabah (Malaysia) where Phyllanthus rufuschaneyi occurs as the dominant species on an ultramafic Cambisol. For 2 years, we monitored a 100-m2 plot and collected information on weather, biomass increase, soil fertility, water fluxes to the soil and litter fluxes for a wide range of elements, including Ni. The Ni cycle is mainly driven by internal fluxes, notably the degradation and recycling of Ni-rich litter. Over the period of investigation, the Ni litter flux corresponded to the total Ni stock of the litter (5.2 g m−2 year−1). The results further show that Ni turnover varies significantly with the accumulation properties of the plant cover. This points to the major influence of Ni hyperaccumulator plants in building up Ni available stocks in the topsoils, as has also been shown in temperate ultramafic systems. Litterfall and throughfall contribute substantially to the cycling of phosphorus, sulphur and potassium in this ecosystem, with throughfall contributing 2-, 220- and 20-fold higher to the respective nutrient fluxes relative to litterfall. The magnesium:calcium ratio far exceeded 1 in the soil, but was < 1 in the leaves of Phyllanthus rufuschaneyi. The insights from this study should be taken into account when designing tropical agromining operations; as Ni stocks could be more labile than initially thought. The removal of Ni- and nutrients-rich biomass will likely affect available Ni (and major nutrients) for the next cropping seasons, and requires sustainable fertilisation, to be utilized to replenish depleted major nutrients. These findings also have major ecological implications.

International audience; Aims: The agronomy of tropical ‘metal crops’ had not yet been tested until a recent pot trial was undertaken to test the growth and nickel (Ni) yield of two promising Ni ‘metal crops’ (Phyllanthus rufuschaneyi and Rinorea cf. bengalensis) from Sabah, Malaysia. The present study investigates the growth and Ni yield in P. rufuschaneyi in response to inorganic and organic fertilisation under field conditions.Methods: A two-year field experiment was undertaken on an ultramafic Cambisol in Sabah. Two experiments were set up with different types of fertilisers, either organic or inorganic. Ten 9-m2 plots were amended with either NPK (370 kg ha−1) or N (120 kg ha−1) fertilisation and were monitored during the first year. Another experiment with 25-m2 plots was set up to test three treatments of organic amendment (0, 1 or 2 kg m−2 of shredded P. rufuschaneyi biomass).Results: The results show that despite considerable variability, Ni concentration was generally lower in plant shoots amended with N only (i.e. urea). However, treatments showed no statistical effect on plant biomass and Ni yield in the short-term, even though organic treatments had a strong positive effect on the availability of Ni.Conclusion: The short-term effects of inorganic and organic treatments on Ni yield of the tropical ‘metal crop’ Phyllanthus rufuschaneyi were not apparent (75 kg Ni ha−1 yr−1). The field trials are ongoing to assess long-term effects. The preliminary results suggest that with appropriate agronomic systems and plant breeding, the Ni yield could be substantially increased.

Aims: Nickel hyperaccumulator plants require highly evolved mechanisms to avoid cellular-level toxicity to cope with the high prevailing concentrations of nickel in their seeds and seedlings. This study aimed to investigate tissue-level distribution and redistribution of Ni and other physiologically relevant elements during the germination of hyperaccumulator plants. Methods : Berkheya coddii Roessler (Asteraceae) and Odontarrhena muralis sensu latu (Waldst. & Kit.) Endl. (O. chalcidica), formerly Alyssum murale (Waldst. & Kit.) (Brassicaceae), were germinated for several days at room temperature before using synchrotron micro X-ray Fluorescence Spectroscopy (µXRF) to obtain high-sensitivity and high-resolution elemental images of live/hydrated plants at various stages of seed germination and seedling growth. Results: The results show that fruits and seeds of both species were highly enriched in nickel. In Berkheya coddii, nickel was located in the cotyledons, the micropylar area, the seed coat, and the point of attachment of the pappus with the cypsela body. In Odontarrhena muralis seeds, nickel occurred in the cotyledons and hypocotyl. The emergence of true leaves initiates nickel and calcium redistribution within seedlings in both species. Conclusions: The diversity in physiological responses to nickel in Berkheya coddii and Odontarrhena muralis does not only occur at the mature stage, but is inherent to both species as seed elemental storage and tolerance mechanisms during seedling development differ.

International audience; Ultramafic soils are usually marginal in macronutrients (nitrogen (N), phosphorus (P), potassium (K) and calcium (Ca)) for growth of crop plants. Commercial nickel (Ni) agromining is dependent on attaining high yield and high Ni concentration in harvestable biomass of Ni hyperaccumulator species. We previously reported on the biomass responses of two promising tropical ‘metal crops’ (Phyllanthus rufuschaneyi and Rinorea cf. bengalensis) to rates of N, P, and K fertilisers. Calcium, sulphur (S) and organic matter amendments have varied effects on the biomass production and Ni uptake in temperate Ni hyperaccumulator species used in agromining, but the trends in tropical ‘metal crops’ are not reported to-date. We investigated the effects of these amendments on the growth performance and the Ni (and other elements) uptake in P. rufuschaneyi and R. cf. bengalensis The experiments consisted of a large 12-month randomised growth trial in large pots in Sabah (Malaysia) using ultramafic soils under different treatment levels of soluble Ca and S, and organic matter amendments. We found that Ca and S additions had no significant effects on the growth of P. rufuschaneyi and R. cf. bengalensis. Organic matter amendments had strong positive effect on the growth of R. cf. bengalensis (p

International audience; Background and aims: Pioneer plants may improve the ecological restoration of degraded ultramafic areas by plant-soil interaction processes. In this study, we assess the effect of the pioneer actinorhizal tree C. terminale (Casuarinaceae) on the recovery of plant communities and soil functions on degraded tropical ultramafic sites.Methods: Soil and plant samples were collected along a tree-age gradient in two degraded ultramafic sites in Sabah (Northern Borneo, Malaysia): a Technosol and a Leptosol. Chemical composition of plants and soils, and biological activity of soils were assessed at both sites. Plant colonisation was assessed by plot vegetation surveys.Results: An improvement in soil fertility parameters (pH reduction from 8.5 to 6.8, an increase in the concentrations of several nutrients and enhanced soil enzyme activities) was observed along the C. terminale age gradient. However, plant cover and diversity was only improved around mature trees at the site that was not impacted by mining.Conclusion: C. terminale promotes the recovery of several soil functions, mainly related to the storage and recycling of N, P, K, S. Besides plant-soil feedback, other environmental factors (i.e. exposition to sunlight, drought) may play an important role on revegetation of ultramafic soils.

Ultramafic (i.e. serpentine) soils are widespread in the Balkans and particularly in Albania. They account for a large part of plant endemism in that region and host several hyperaccumulator species, which are characterized by leaf nickel concentrations frequently above 1%. This rich nickel hyperaccumulating flora could serve as candidate to be used in phytoextraction and agromining. Despite recent interest in metal hyperaccumulating plants and agromining, very few studies have investigated the bacterial diversity and the influence of environmental factors on microbial gene profiles in the rhizosphere of hyperaccumulator plants growing on ultramafic soils. Because rhizospheric bacteria could be crucial to the success of phytoremediation, we studied a total of 48 nickel-hyperaccumulating plants which were sampled from four species that are widespread in Albania: Noccaea ochroleuca, Odontarrhena smolikana, O. rigida and O. chalcidica. All samples were taken from the ultramafic regions of Librazhd and Pogradec in eastern Albania in October 2015. Our study shows that Proteobacteria, Actinobacteria and Acidobacteria dominated the soil bacterial communities. Of these three phyla, only Proteobacteria was relatively abundant. This study underlines the influence of soil Cation Exchange Capacity on the bacterial community's diversity and structure. Based on the predicted metagenomes, the genes belonging to amino acid, lipid and carbohydrate metabolisms were identified as major gene families. Our study sheds some light on our understanding of how bacterial communities are structured within and affect the rhizosphere of hyperaccumulator plants from ultramafic soils in Albania.

Purposes To analyze the main edaphic factors that differentiate ultramafic from typical Cerrado environments and act as agents of the development of biochemical and morphological mechanisms of species adaptation to these harsh environments; and to determine the genetic diversity of three Cerrado’s shrubland native species (Justicia lanstyakii, Euploca salicoides, and Oxalis hirsutissima). Methods We conducted chemical analysis of metal contents in soils, as well as on elemental composition, and analysis of DNA extracted from leaf tissues of the three species found in both environments. In leaves of E. salicoides grown in both environments we evaluated the changes in the levels of non-structural carbohydrates (NSC), and total proteins. Results The accessions obtained in ultramafic soils were closer with each other, indicating genetic similarity and major differences in relation to the accessions collected in the Cerrado area. These differentiations probably are related with higher adaptation to soils rich in metals, mainly Ni in ultramafic, and Al in the Cerrado environments. The highest levels of NSC were observed in plants grown in ultramafic soils, including raffinose, which is related to responses to metal detoxification and drought. The allocation of Ni in the trichomes, which does not affect important processes of plant metabolism, is another mechanism developed by E. salicoides to overcome the hyperaccumulation of Ni in their tissues. Conclusions These findings can help select seed collection sites representative of the genetic diversity of native plant species for restoring degraded areas or for phytoremediation of metals.

This article has been accepted for publication in Metalomics Published by Oxford University Press. DOI: 10.1093/mtomcs/mfab045; International audience; Plants have developed a diversity of strategies to take up and store essential metals in order to colonize various types of soils including mineralized soils. Yet, our knowledge of the capacity of plant species to accumulate metals is still fragmentary across the plant kingdom. In this study, we have used the X-Ray Fluorescence technology to analyze metal concentration in a wide diversity of species of the Neotropical flora that was not extensively investigated so far. In total, we screened more than 11 000 specimens representing about 5000 species from herbaria in Paris and Cuba. Our study provides a large overview of the accumulation of metals such as manganese, zinc and nickel in the Neotropical flora. We report 30 new nickel hyperaccumulating species from Cuba, including the first records in the families Connaraceae, Melastomataceae, Polygonaceae, Santalaceae and Urticaceae. We also identified the first species from this region of the world that can be considered as manganese hyperaccumulators in the genera Lomatia (Proteaceae), Calycogonium (Melastomataceae), Ilex (Aquifoliaceae), Morella (Myricaceae) and Pimenta (Myrtaceae). Finally, we report the first zinc hyperaccumulator, Rinorea multivenosa (Violaceae), from the Amazonas region. The identification of species able to accumulate high amounts of metals will become instrumental to support the development of phytotechnologies in order to limit the impact of soil metal pollution in this region of the world.

Aims Agromining aims to improve the fertility of naturally metal-rich soils by extracting metals, such as nickel (Ni), using hyperaccumulator plants. Ultramafic soils are characterized by low fertility levels, limiting hyperaccumulator yields. Here, we characterize the potential benefits for phytoextraction efficiency of cocropping a Ni-hyperaccumulator (Odontarrhena chalcidica) and a legume (Vicia sativa), following a two-year field experiment. Methods A two-year field experiment was set up in an ultramafic zone in North-West Spain. Three treatments were tested: co-cropping, fertilized control with ammonium nitrate and non-fertilized control. Results Over the 2 years, co-cropping increased O. chalcidica’s biomass by 24% and 403% compared to fertilized and non-fertilized controls, respectively. Moreover, co-cropping had higher Ni-yields for both years, while fertilization had a negative effect on soil parameters. A non-metric multidimensional scaling analysis of the operational taxonomic units showed that the soil bacterial diversity changed over time. Soil exchangeable Ni and organic carbon influenced the phyla’s relative abundance. Metabolic genes were dominant and their relative abundances increased over time with co-cropping. Conclus i o n Pluriannua l co-cropping of a hyperaccumulator with a legume improved both hyperaccumulator and Ni yields. In contrast, mineral fertilization was shown to be detrimental to some soil microbial parameters. Thus, ameliorating agromining by replacing mineral fertilizers would combine an eco-efficient strategy with sustainable metal recovery., This work was supported by French National Research Agency through the national “Investissements d’avenir” program, reference ANR-10-LABX- 21 - LABEX RESSOURCES21, through the “Agromine” project, reference ANR-14-CE04-0005, through the European ERA-net FACCE_SURPLUS project “AGRONICKEL: Developing Ni agromining on ultramafic land in Europe”, reference ANR-15- SUSF-0003-05, through the “Application of Nickel hyperaccumulating plants in phytomining processes” program, reference I-LINK0900 and the LIFE Environment and Resource Efficiency Programme (Life Agromine; LIFE15 ENV/FR/ 000512).

The rare phenomenon of plant manganese (Mn) hyperaccumulation within the Australian flora has previously been detected in the field, which suggested that the tree genus Gossia (Myrtaceae) might contain new Mn hyperaccumulators. We conducted the first growth experiment on Gossia using a multi-factorial dosing trial to assess Mn, cobalt (Co), and zinc (Zn) (hyper)accumulation patterns in selected Gossia species (G. fragrantissima and G. punctata) after a systematic assessment of elemental profiles on all holdings of the genus Gossia at the Queensland Herbarium using handheld X-ray fluorescence spectroscopy. We then conducted detailed in situ analyses of the elemental distribution of Mn, Co, Zn and other elements at the macro (organ) and micro (cellular) levels with laboratory- and synchrotron-based X-ray fluorescence microscopy (XFM). Gossia pubiflora and Gossia hillii were newly discovered to be Mn hyperaccumulator plants. In the dosing trial, G. fragrantissima accumulated 17 400 µg g−1 Mn, 545 µg g−1 Co, and 13 000 µg g−1 Zn, without signs of toxicity. The laboratory-based XFM revealed distinct patterns of accumulation of Co, Mn, and Zn in G. fragrantissima, while the synchrotron XFM showed their localization in foliar epidermal cells, and in the cortex and phloem cells of roots. This study combined novel analytical approaches with controlled experimentation to examine metal hyperaccumulation in slow-growing tropical woody species, thereby enabling insight into the phenomenon not possible through field studies.

The aim of this study was to assess the potential of the woody nickel hyperaccumulator species

Aims: The tropical nickel (Ni) hyperaccumulator plants, Rinorea cf. bengalensis and Phyllanthus rufuschaneyi, are locally common on ultramafic soils in the Malaysian state of Sabah on the island of Borneo. The aim of this study was to determine whether the structure and diversity of R. cf. bengalensis rhizosphere microbial communities were dependant on the Ni concentrations in the different rhizosphere soils studied. Methods: Rinorea cf. bengalensis and Phyllanthus rufuschaneyi were subjected to a randomized block design experiment in which four Ni dose levels were applied (0, 60, 240, 600 mg Ni kg−1) × 5 replicates per level for 12 months cultivation. At the end of the trial foliar elemental concentrations were measured. In addition, rhizosphere soils were collected for elemental and microbial analyses. The microbial analysis consisted of measuring microbial enzymatic activities and a diversity analysis based on Illumina sequencing. Results: The Illumina sequencing analysis yielded 2,649,138 sequences grouped under 2430 different Operational Taxonomic Units (OTUs) belonging to 25 phyla. No impact of the Ni amendments was shown for the four microbial enzymatic activities tested, even at the highest Ni dose level (600 mg Ni kg−1). Higher microbial enzymatic activities were found in the rhizosphere of R. cf. bengalensis relative to P. rufuschaneyi. Nickel concentrations appeared to have a high impact on the P. rufuschaneyi rhizosphere soils, whereas no effect was found for the rhizosphere bacterial community diversity of R. cf. bengalensis. Conclusions: There may be a significant role of rhizodeposits associated with R. cf. bengalensis that protect and preserve the microbial communities in the rhizosphere, regardless of the prevailing nickel concentrations.

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

International audience

A large body of analytical data is available on the inorganic composition of many thousands of plant species, for which typical concentration ranges have been tabulated for major, minor, and trace elements. These elements include those that have been shown essential for plant growth, as well as others that lack this status, at least universally. Metalliferous soils, having abnormally high concentrations of some of the elements that are generally present only at minor (e.g. 200–2000 μg g−1) or trace (e.g. 0.1–200 μg g−1) levels, have attracted increasing attention during the last 50 years. The effects vary widely, depending on the species, the relevant elements, and soil characteristics that collectively influence the availability of metals to plants. Some of these soils are toxic to all or most higher plants. Others have hosted the development of specialized plant communities consisting of a restricted and locally characteristic range of metal-tolerant species. These typically show a slightly elevated concentration of the elements with which the soil is enriched, but in places a species may exhibit extreme accumulation of one or more of these elements, to a concentration level that can be hundreds or even thousands of times greater than that usually found in plants on the most common soils. These plants, now widely referred to as hyperaccumulators, are a remarkable resource for many types of fundamental scientific investigation (plant systematics, ecophysiology, biochemistry, genetics and molecular biology) and for applications such as phytoremediation and agromining. Systematic analysis of herbarium specimens by X-ray Fluorescence, combined with auxiliary collection data, can provide insights into phylogenetic patterns of hyperaccumulation, and has the potential to complement and add insights to biogeographical and phylogenetic studies.

Globally, the discovery of metal and metalloid hyperaccumulator plants has been hindered by systematic screening of plant species and is highly biased towards Ni hyperaccumulators due to the existence of a paper test that is only specific to Ni (based on dimethylglyoxime reagent). Consequently, more than 500 of the approximately 700 known hyperaccumulators species accumulate Ni. New technological advances now permit massive screening of herbarium specimens using non-destructive multi-element sensitive portable X-Ray Fluorescence Spectroscopy (pXRF) instead of via destructive techniques such as Atomic Absorption Spectrophotometry (AAS) or Inductively-coupled Plasma-Atomic Emission Spectroscopy (ICP-AES). This approach has already led to the discovery of numerous hyperaccumulator species for various elements new to science. This chapter introduces established and novel tools that can be used for discovering hyperaccumulator plant species in the field and in the herbarium.

Substantial unrealized opportunities exist for economic Ni agromining in the tropics. However, until recently this technology has remained relatively unexploited in this part of the world. In this chapter, we discuss the progress of tropical Ni agromining in two regions, namely Southeast Asia and the neotropical region. Significant advances have been made in Ni agromining operations in Southeast Asia, particularly Sabah (Malaysia), in the past few years: (i) exploring for suitable locations, (ii) screening for hyperaccumulator plants in native flora, (iii) selecting candidate hyperaccumulator species with high biomass production and shoot Ni concentrations (‘metal crops’), (iv) testing the agronomy of ‘metal crops’ to be used in viable agromining operations, and (v) demonstration of real-life agromining operations at field scale. The two most promising ‘metal crops’ in Sabah are Phyllanthus rufuschaneyi and Rinorea cf. bengalensis. However, Ni agromining developments in neotropical regions are still in their infant stages. Preliminary investigations have led to the discovery of a potential ‘metal crop’, Blepharidium guatemalense, having highly desirable attributes for possible Ni agromining. It is envisaged that tropical Ni agromining could be a productive alternative land use in these regions, and as such will significantly improve the livelihood of potential local ‘metal farmers’.

International audience; The hyperaccumulator Pycnandra acuminata is a New Caledonian rainforest tree known to have the highest concentration of nickel in any living organism with 25 wt% in its latex. All trees (with diameter >10 cm) and soil profiles in a 0.25-hectare permanent plot were sampled to assess the biogeochemical compartmentalisation of nickel in a dense stand of Pycnandra acuminata trees. Nickel stable isotope analysis permitted insights into the cycling of nickel in this ecosystem. The total tree biomass of the plot was ~281 tonnes ha-1, approximately 0.44 kg of cobalt, 49.1 kg of manganese, 257 kg of nickel and 6.76 kg of zinc. Nickel stable isotope analysis identified the biotic origin of the nickel in the soil upper layers with P. acuminata shoots enriched in lighter nickel isotopes. The δ60Ni latex signature suggests that long-distance transport, radial xylem and phloem loading are at play in Pycnandra acuminata.

Les herbiers permettent d’identifier efficacement de nouvelles espèces de plantes hyperaccumulatrices de métaux (e.g. nickel, manganèse, zinc) grâce à la spectrométrie de fluorescence des rayons X. Cribler l’Herbier du MNHN-Paris s’est révélé prometteur notamment pour des territoires peu étudiés comme les Amériques et Madagascar. Ces découvertes ouvrent des perspectives inédites pour la restauration écologique et la phytoextraction des métaux.

International audience; Background and aims: The genus Denhamia(Celastraceae) includes fifteen Australian species, many of which have a propensity for manganese (Mn) (hyper)accumulation. Among the key aims of this study were to: i) elucidate Mn accumulation in D. bilocularis, D. celastroides, D. pittosporoides and D. silvestris under controlled conditions; ii) examine the in situ distributions of Mn and other elements in tissues of i) above, and also in two other species growing in their natural habitat, i.e. D. silvestris and D. cunninghamii; iii) test numerous Denhamia herbarium specimens for Mn accumulation using portable X-ray fluorescence spectroscopy (XRF).Methods: Portable XRF and laboratory micro-XRF were used to examine Mn accumulation and foliar distribution patterns in several Australian Denhamia species. These techniques were variously applied to fresh field material and experimentally-raised plants treated with Mn (250 μg g−1, 500 μg g−1 and 1000 μg g−1) and dry herbarium material.Results: The findings revealed D. bilocularis as a new Mn hyperaccumulator, with foliar Mn concentrations of up to 15,300 μg g−1 in herbarium samples, and 13,700 μg g−1 in experimentally grown plants. Laboratory μXRF maps consistently showed that foliar Mn accumulation was localized at the extremities of wild D. cunninghamii and D. silvestris leaves; whereas it was found in the vasculature of experimentally grown D. bilocularis and D. pittosporoides leaves.Conclusions: This study presents evidence that Mn accumulation across the Australian Denhamia species is taxonomically variable, however further work is required.

Initial experiments using Mediterranean Ni-hyperaccumulator plants for the purpose of phytomining were carried out in the 1990s. In order to meet commercial phytoextraction requirements, a technology has been developed using hyperaccumulator species with adapted intensive agronomic practices on naturally Ni-rich soils. Ultramafic soils in the Balkans and other parts of Europe display a great variability in Ni concentrations and available Ni levels. In Albania, Vertisols are currently being used for low-productivity agriculture (pasture or arable land) on which phytomining could be included in cropping practices. In northwestern Greece (Pindus and Vourinos mountain regions), agricultural soils may occur on ultramafic Cambisols. In Spain and Austria, these soils are much more erratically distributed and are seldom used for crops and pastures. In the Balkans, Odontarrhena chalcidica (synonym Alyssum murale) occurs widely on these ultramafic soils and is a spontaneous weed that grows among other crops. Field studies across Europe have now been carried out outside Mediterranean areas and have evaluated the Ni-hyperaccumulator O. chalcidica, as well as two other species native to northwestern Greece (Bornmuellera emarginata and B. tymphaea). At each site, local hyperaccumulator plants were tested for comparison (Noccaea goesingense in Austria and Odontarrhena serpyllifolia s.l. in Spain), in the context of two recent EUfunded projects (by Agronickel and LIFEAgromine). Soil and crop management practices are being developed in order to optimize the Ni agromining process. Field studies have evaluated the potential benefits of fertilization regimes, crop selection, and cropping patterns (introducing agroecological practices), and bioaugmentation using plant-associated microorganisms.

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

International audience; 17 Aims. Rinorea cf. bengalensis is a Ni hyperaccumulator which occurs in Sabah (Malaysia), on Borneo Island, 18 that is able to accumulate considerable amounts of Ni and influences the Ni cycle in surface soil layers, both in 19 terms of Ni concentration and Ni isotopic composition. In this study, the biogeochemical processes underpinning 20 Ni isotopic fractionation in the soil-plant system and the mechanisms regulating Ni homeostasis in R. cf. 21 bengalensis plants were elucidated. 22 Methods. Two specimens of R. cf. bengalensis of different ages and associated surface soils were collected from 23 ultramafic soils in Sabah. Soil mineralogy, Ni concentrations, speciation and isotopic signatures were 24 subsequently determined in plant and soil samples. 25 Results. Nickel in R. cf. bengalensis leaves is mainly complexed with citrate. Soil Ni available fractions have 26 different δ 60 Ni values depending on the Ni bearing phases. Rinorea cf. bengalensis specimens take up lighter Ni 27 isotopes and a pronounced isotopic fractionation within the plant is observed, especially in the young specimen. 28 Conclusions. The results suggest that the observed fractionation in the young plant can be attributable to kinetic 29 effects (lighter isotopes move faster), which become less evident in the older specimen, as Ni is redistributed and 30 homogenized through phloem loading and unloading processes. 31 2

International audience; In Albania, ultramafic outcrops cover 11% of the surface and have the potential to support nickel phytomining. In a large-scale in-situ experiment on an ultramafic Vertisols in Pojske we are studying the influence of agronomical practices on Ni phytoextraction yield of Odontarrhena chalcidica (syn. Alyssum murale). Three cropping systems were compared in three plots in 2016-2017; POJ-1 Plot (0.3 ha) was established with plants that had germinated spontaneously without any treatments; POJ-2 plot (0.3 ha) was covered by plants that had germinated spontaneously and was treated with mineral fertilizer (N50P50K50 kg ha(-1)); and POJ-3 Plot (400 m(2)) was divided in four sub plots, where O. chalcidica was planted at a density of 4 plants m(-2) on which, we neither applied fertilizer, nor NPK fertilizer (N65P65K65), pig (FPM; N260:P105:K260 + 15 kg ha(-1)N, P, K) or chicken manure (FCHM; N260:P390:K260 +15 kg ha(-1) N, P, K. Irrigation and mechanical control of weeds was done on POJ-3. After 8 months, shoot Ni concentration, biomass, and Ni yields were higher in O. chalcidica treated with manure and the cost of biomass production was smaller. Nickel yield was more promising (145 kg ha(-1)) than in previous field trials. This study highlights that, using manure, the Ni yield increases Ni phytomining net values, thus agromining can become an economically justifiable agricultural cropping system.

International audience; Aim The identification and use of hyperaccumulator plants in mining projects has been recognized as an important component of mine planning at several sites around the world. The objective of this research was to provide information on relevant plant tissue chemistry and an indicative assessment of the potential for phytomining at Weda Bay Nickel (WBN), Halmahera. Methods The first stage was the identification of native nickel hyperaccumulator plants. In total, 280 plant tissue samples from 10 nickel accumulator species and 46 matching rhizosphere soil samples were collected. Chemical analyses of plant tissue samples were performed and physico-chemical parameters of the rhizosphere soils were also measured. Results A total of three species were considered as metal crops: Rinorea aff. bengalensis (up to 22,200 mg kg−1 dry weight at 2 m above ground level), Ficus trachypison (1060 mg kg−1) and Trichospermum morotaiense (5180 mg kg−1), but only R. aff. bengalensis has sufficiently high Ni concentrations in biomass to warrant field trials. Conclusions Utilising a successional planting strategy, F. trachypison and T. morotaiense could be used to facilitate site conditions, followed by the metal crop R. aff. bengalensis. Using this design, a nickel yield of 330 kg per hectare would be possible every 4 years. In addition to allowing the recovery of nickel, this approach could be an integrated mine site rehabilitation strategy to mitigate environmental impacts, improve soil quality and facilitate transition to other land-uses such as native forest.

Selenium (Se), a trace element essential for human and animal biological processes, is deficient in many agricultural soils. Some extremely rare plants can naturally accumulate extraordinarily high concentrations of Se. The native legume Neptunia amplexicaulis, endemic to a small area near Richmond and Hughenden in Central Queensland, Australia, is one of the strongest Se hyperaccumulators known on Earth, with foliar concentrations in excess of 4000 μg Se g−1 previously recorded. Here, we report on the Se distribution at a whole plant level using laboratory micro X-ray Fluorescence Microscopy (μXRF) and scanning electron microscopy (SEM-EDS), as well as on chemical forms of Se in various tissues using liquid chromatography-mass spectrometry (LC-MS) and synchrotron X-ray absorption spectroscopy (XAS). The results show that Se occurs in the forms of methyl-selenocysteine and seleno-methionine in the foliar tissues, with up to 13 600 μg Se g−1 total in young leaves. Selenium was found to accumulate primarily in the young leaves, flowers, pods and taproot, with lower concentrations present in the fine-roots and stem and the lowest present in the oldest leaves. Trichomes were not found to accumulate Se. We postulate that Se is (re)distributed in this plant via the phloem from older leaves to newer leaves, using the taproot as the main storage organ. High concentrations of Se in the nodes (pulvini) indicate this structure may play an important a role in Se (re)distribution. The overall pattern of Se distribution was similar in a non-Se tolerant closely related species (Neptunia gracilis), although the prevailing Se concentrations were substantially lower than in N. amplexicaulis.

Background and Aims Hybanthus austrocaledonicus (Violaceae) is a nickel (Ni) hyperaccumulator endemic to New Caledonia. One of the specimens stored at the local herbarium had a strip of bark with a remarkably green phloem tissue attached to the sheet containing over 4 wt% Ni. This study aimed to collect field samples from the original H. austrocaledonicus locality to confirm the nature of the green ‘nickel-rich phloem’ in this taxon and to systematically assess the occurrence of Ni hyperaccumulation in H. austrocaledonicus and Hybanthus caledonicus populations. Methods X-ray fluorescence spectroscopy scanning of all collections of the genus Hybanthus (236 specimens) was undertaken at the Herbarium of New Caledonia to reveal incidences of Ni accumulation in populations of H. austrocaledonicus and H. caledonicus. In parallel, micro-analytical investigations were performed via synchrotron X-ray fluorescence microscopy (XFM) and scanning electron microscopy with X-ray microanalysis (SEM-EDS). Key Results The extensive scanning demonstrated that Ni hyperaccumulation is not a characteristic common to all populations in the endemic Hybanthus species. Synchrotron XFM revealed that Ni was exclusively concentrated in the epidermal cells of the leaf blade and petiole, conforming with the majority of (tropical) Ni hyperaccumulator plants studied to date. SEM-EDS of freeze-dried and frozen-hydrated samples revealed the presence of dense solid deposits in the phloem bundles that contained >8 wt% nickel. Conclusions The occurrence of extremely Ni-rich green phloem tissues appears to be a characteristic feature of tropical Ni hyperaccumulator plants.