Vidiro Gei

Guillaume Echevarria

Peter D Erskine

Sandrine Isnard

Bruno Fogliani

Emmanuelle Montargès-Pelletier

Tanguy Jaffré

Kathryn M Spiers

Jan Garrevoet

Antony Van

Antony Van Der Ent
email: a.vanderent@uq.edu.au

Soil chemistry, elemental profiles and elemental distribution 1 in nickel hyperaccumulator species from New Caledonia

Keywords: Hyperaccumulator, Elemental distribution, Latex, X-ray Fluorescence Microscopy

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Hyperaccumulators are unusual plants that accumulate particular metals or metalloids in their living tissues to levels that are 2-3 orders of magnitude greater than is normal for most plants growing on similar soils [START_REF] Van Der Ent | Hyperaccumulators of metal and metalloid trace elements: facts and fiction[END_REF]Reeves et al. 2018a,b). Nickel hyperaccumulator plants are defined as plants with Ni concentrations in leaves >1000 µg g -1 dry weight [START_REF] Jaffré | Sebertia acuminata: A hyperaccumulator of nickel from New Caledonia[END_REF][START_REF] Reeves | Tropical hyperaccumulators of metals and their potential for phytoextraction[END_REF][START_REF] Van Der Ent | Ultramafic nickel laterites in Indonesia (Sulawesi, Halmahera): mining, nickel hyperaccumulators and opportunities for phytomining[END_REF]. Discovering hyperaccumulators and understanding their ecology could lead to identifying potential species to be utilized in novel phytotechnologies such as agromining for phytoextraction of valuable metals. They could be used in accumulating trace metals in naturally-mineralised agricultural soils, low grade ores, mine tailings, or mineral processing wastes [START_REF] Chaney | Potential effects of waste constituents on the food chain[END_REF]van der Ent et al. 2015a); and then harvested and processed to extract high-grade bio-ore or 'eco-catalysts' (Brooks et al. 1998;Brooks and Robinson 1998;[START_REF] Chaney | Improved understanding of hyperaccumulation yields commercial phytoextraction and phytomining technologies[END_REF]Losfeld et al. 2015b). Nickel phytomining is envisaged to become transformative in the rehabilitation of tropical laterite mining operations as part of rehabilitation strategies [START_REF] Van Der Ent | Ultramafic nickel laterites in Indonesia (Sulawesi, Halmahera): mining, nickel hyperaccumulators and opportunities for phytomining[END_REF]2015a;Losfeld et al. 2015a,b;[START_REF] Erskine | Incorporating Hyperaccumulator Plants into Mine Rehabilitation in the Asia-Pacific Region[END_REF].

Currently, there are approximately 500 documented Ni hyperaccumulators worldwide (Reeves et al. 2018a). The majority of the Ni hyperaccumulators have been recorded in Brazil (130) [START_REF] Reeves | The flora and biogeochemistry of the ultramafic soils of Goiás state, Brazil[END_REF], Southern Europe and Minor Asia (80-90) [START_REF] Brooks | Hyperaccumulation of nickel by Alyssum Linnaeus (Cruciferae)[END_REF][START_REF] Adıgüzel | Important serpentine areas of Turkey and distribution patterns of serpentine endemics and nickel accumulators[END_REF], New Caledonia (65) [START_REF] Jaffré | The metal hyperaccumulators from New Caledonia can broaden our understanding of nickel accumulation in plants[END_REF]van der Ent et al. 2015c), and Malaysia (24) (van der Ent et al. 2015a,b;2018c). Recent screening of herbarium samples using a portable XRF instrument in New Caledonia has revealed the existence of 99 Ni hyperaccumulators and 74 Mn hyperaccumulators (11 known previously), meaning that more hyperaccumulators are to be discovered with such fast and non-destructive methods [START_REF] Gei | Tools for discovery of hyperaccumulator plant species and understanding their ecophysiology[END_REF]2020). One of the most unusual Ni hyperaccumulators is the tree Pycnandra (formerly Sebertia) acuminata, endemic to New Caledonia, whose latex contains up to 257 mg Ni g -1 dry latex [START_REF] Jaffré | Sebertia acuminata: A hyperaccumulator of nickel from New Caledonia[END_REF][START_REF] Jaffré | The discovery of nickel hyperaccumulation in the New Caledonian tree Pycnandra acuminata: 40 years on[END_REF]. Hyperaccumulation is an extreme phenotypic expression of transition element metabolic regulation in plants, which involves specific transporters and sequestration of metal ions and chelates in specific sink tissues [START_REF] Krämer | Transition metal transport[END_REF][START_REF] Andresen | Trace metal metabolism in plants[END_REF]. Hence, knowledge of the specific sequestering and transport mechanisms within these hyperaccumulating plants is crucial to better understand this phenomenon (van der Ent et al. 2017a,b). The frequent occurrence of hyperaccumulation in a number of different clades suggests that Ni hyperaccumulation evolved repeatedly in different lineages [START_REF] Pollard | The genetic basis of metal hyperaccumulation in plants[END_REF][START_REF] Krämer | Metal Hyperaccumulation in Plants[END_REF]), but it is unknown whether physiological mechanisms of hyperaccumulation differ between the phylogenetic groups (i.e. whether non-related hyperaccumulating species have similar eco-physiological processes -inferred from cellular and tissue-level elemental distribution -associated with hyperaccumulation).

Our understanding of the diversity of Ni hyperaccumulation mechanisms is limited because studies on the spatial elemental distribution in tissues and cells have only been performed for less than 10% of the >500 Ni hyperaccumulating plant species known globally [START_REF] Mesjasz-Przybyłowicz | Extreme nickel hyperaccumulation in the vascular tracts of the tree Phyllanthus balgooyi from Borneo[END_REF]. Most of the studies initially conducted concerned small, herbaceous plants from the families Brassicaceae, Asteraceae, and Celastraceae (e.g. Alyssum (now Odontarrhena), Noccaea (formerly Thlaspi), Berkheya, Senecio, and Stackhousia) and the leaves were typically the main analysed parts, while stems and roots were less often investigated [START_REF] Mesjasz-Przybyłowicz | Proton microprobe and X-ray fluorescence investigations of nickel distribution in serpentine flora from South Africa[END_REF]Mesjasz-Przybyłowicz et al. 1996a,b, 1997a,b,c, 2001a[START_REF] Berazaín | Nickel localization in tissues of different hyperaccumulator species of Euphorbiaceae from ultramafic areas of Cuba[END_REF][START_REF] Küpper | Cellular compartmentation of nickel in the hyperaccumulators Alyssum lesbiacum, Alyssum bertolonii and Thlaspi goesingense[END_REF][START_REF] Bhatia | Elemental mapping using PIXE shows the main pathway of nickel movement is principally symplastic within the fruit of the hyperaccumulator Stackhousia tryonii[END_REF][START_REF] Bhatia | Studies on spatial distribution of nickel in leaves and stems of the metal hyperaccumulator Stackhousia tryonii Bailey using nuclear microprobe (micro-PIXE) and EDXS techniques[END_REF][START_REF] Broadhurst | Nickel localization and response to increasing Ni soil levels in leaves of the Ni hyperaccumulator Alyssum murale[END_REF][START_REF] Broadhurst | Interaction of nickel and manganese in accumulation and localization in leaves of the Ni hyperaccumulators Alyssum murale and Alyssum corsicum[END_REF][START_REF] Mcnear | Application of quantitative fluorescence and absorption-edge computed microtomography to image metal compartmentalization in Alyssum murale[END_REF][START_REF] Tappero | Hyperaccumulator Alyssum murale relies on a different metal storage mechanism for cobalt than for nickel[END_REF]). The only tropical woody shrub analysed was Hybanthus floribundus (Violaceae) for which Ni was mainly reported in the leaf epidermis [START_REF] Bidwell | Subcellular localization of Ni in the hyperaccumulator, Hybanthus floribundus (Lindley) F. Muell[END_REF][START_REF] Kachenko | Quantitative elemental localisation in leaves and stems of nickel hyperaccumulating shrub Hybanthus floribundus var. floribundus using micro-PIXE spectroscopy[END_REF]. There was also a short report on species from the genera Phyllanthus (Phyllanthaceae), Euphorbia and Leucocroton (Euphorbiaceae) from Cuba which reported that Ni concentration was highest in laticifers of all the species, which all had latex [START_REF] Berazaín | Nickel localization in tissues of different hyperaccumulator species of Euphorbiaceae from ultramafic areas of Cuba[END_REF]), but it confirmed that leaf epidermis was the storage location for all species studied. For the majority of investigated Ni hyperaccumulator plant species, Ni is a priori preferentially accumulated in foliar epidermal cells [START_REF] Mesjasz-Przybyłowicz | Proton microprobe and X-ray fluorescence investigations of nickel distribution in serpentine flora from South Africa[END_REF]Mesjasz-Przybyłowicz et al. 1996a,b, 1997b,c, 2001b;[START_REF] Küpper | Cellular compartmentation of nickel in the hyperaccumulators Alyssum lesbiacum, Alyssum bertolonii and Thlaspi goesingense[END_REF][START_REF] Bidwell | Subcellular localization of Ni in the hyperaccumulator, Hybanthus floribundus (Lindley) F. Muell[END_REF][START_REF] Bhatia | Studies on spatial distribution of nickel in leaves and stems of the metal hyperaccumulator Stackhousia tryonii Bailey using nuclear microprobe (micro-PIXE) and EDXS techniques[END_REF][START_REF] Broadhurst | Nickel localization and response to increasing Ni soil levels in leaves of the Ni hyperaccumulator Alyssum murale[END_REF]) with the exception of Berkheya coddii, where it occurs at high concentrations in the palisade parenchyma [START_REF] Groeber | Fate of nickel and calcium in seedlings of the hyperaccumulator Berkheya coddii during germination[END_REF]. The accumulation of Ni in leaf vacuoles as complexes with organic acids is often interpreted as a mechanism of metal detoxification and maintaining homeostasis [START_REF] Seregin | Physiological role of nickel and its toxic effects on higher plants[END_REF]. Recently, several detailed studies on tropical woody shrubs and trees of the genera Phyllanthus, Glochidion (Phyllanthaceae), and Rinorea (Violaceae) shed more light on cellular and sub-cellular distribution by using X-ray Fluorescence Microscopy (µXRF) and Proton Induced X-ray Emission (PIXE) techniques, which provided highly resolved mapping and low limits of detection (van der Ent et al. 2017a(van der Ent et al. ,b, 2018b)). These studies have shown that in phylogenetically distant species there is remarkably similar chemical form and distribution of Ni within plant organs and tissues.

This study is a comparative assessment of elemental concentrations and distribution in distantly related species from New Caledonia to establish whether similar patterns of Ni hyperaccumulation mechanisms, observed so far globally, hold true for woody New Caledonian hyperaccumulator plants. Therefore, synchrotron µXRF was used to produce highly resolved elemental mapping across different tissues in an array of species that occur in New Caledonia. The model plants analysed in this study were chosen to be as representative as possible of New Caledonian hyperaccumulator lineages (Fig. 1). The five selected species represent five families and four plant orders (Ericales, Gentianales, Malpighiales, Oxalidales) known to contain hyperaccumulator species worldwide. We aimed to investigate whether distribution patterns of Ni at the cellular and tissue level in the roots, stem, and leaves, and those of other physiologically relevant elements (e.g. Ca, K, Mn, Co, Zn), differ among these species.

MATERIALS AND METHODS

2.1 Species ecology -All five taxa (Geissois pruinosa var. pruinosa, Homalium francii, Hybanthus austrocaledonicus, Psychotria gabriellae, and Pycnandra acuminata) (Fig. 1) are endemic to New Caledonia and grow on soils derived from ultramafic bedrock and outcrops.

All taxa have a wide distribution in New Caledonia (Fig. 2). Homalium francii, H. austrocaledonicus, P. gabriellae, and P. acuminata occur in dense humid forest, the first three species being common understory shrubs, whereas the latter is an uncommon upper mid-story to canopy tree [START_REF] Swenson | Revision of Pycnandra subgenus Sebertia (Sapotaceae) and a generic key to the family in New Caledonia[END_REF]. Geissois pruinosa is an ecologically ubiquitous species that occurs in maquis, most often in forest edges and sometimes in dense humid forests, however the var. pruinosa is restricted to the southern part of New Caledonia [START_REF] Jaffré | Hyperaccumulation of nickel by Geissois species[END_REF][START_REF] Hopkins | Flore de la Nouvelle-Calédonie[END_REF]). These taxa are ultramafic obligates, except for Homalium francii [START_REF] Jaffré | Etude floristique et structurale de deux forêts denses humides sur roches ultrabasiques en Nouvelle-Calédonie[END_REF] and Hybanthus austrocaledonicus [START_REF] Paul | Exceptional phloem nickel in Hybanthus austrocaledonicus (Violaceae) from New Caledonia[END_REF]) which also occur on other types of soils.

Collection of plant samples and preparation -All species were collected in New

Caledonia (Fig. 2). All samples were collected from natural populations in the Parc Provincial de la Rivière Bleue, except for Geissois pruinosa var. pruinosa which was collected at Monts des Koghis. Branch segments were collected from shrubs or obtained from the canopy of mature trees with a pole pruner, packed in moist paper and kept in sealed plastic bags, then brought to the PETRA III Synchrotron at DESY (Deutsches Elektronen-Synchrotron, Hamburg, Germany) in a fresh state. In addition, tissue samples were excised with a razor blade in the field and immediately shock-frozen using a metal mirror technique in which the samples were pressed between a block of copper (Cu)-metal cooled by liquid N 2 and a second cooled Cu-metal block attached to a Teflon holder. This ensured extremely fast freezing of the plant tissue samples to prevent cellular damage by ice crystal formation. The frozen samples were then wrapped in aluminium (Al) foil, transported and subsequently stored in a cryogenic container (at least -190°C) until further processing (e.g. sectioning and mounting for µXRF analysis as described below).

Freeze-dried specimens were prepared by sealing frozen samples in Ultralene film held onto XRF sample cups (polypropylene, 6 µm) with small holes punctured in the film for sublimed water to escape. The freeze-drying process started at -196°C by inserting the framed frozen samples onto a large steel block cooled in liquid nitrogen. The cooled block with samples was then loaded into the freeze-drying machine and vacuum-pumped and set to -85°C.

Freeze-drying then progressed very slowly (by changing the set temperature with 5°C increments) over the course of four days until room temperature was reached.

2.3

Light microscopy and histochemistry -Plant tissue samples were fixed in 3% glutaraldehyde upon collection. Sections of 50-100 µm thickness were obtained with a vibratome (Leica VTS1000) after embedding in agarose and stained with 1% toluidine blue to reveal anatomical features to assist in the interpretation of the element maps (Supporting Information Figure S1 andS2).

Soil sampling and determination of chemical properties -Soil samples were taken

from near the base of each plant from which the tissue samples were also collected in the Parc Provincial de la Rivière Bleue (Table 1). In total three soil samples were collected for G. pruinosa var. pruinosa, three for P. gabriellae and one for H. austrocaledonicus and one for Pycnandra acuminata. Homalium francii was collected very close to the P. acuminata so the habitat elemental concentration values would be similar, hence no sample was collected. The soil samples were taken with a plastic scoop from a depth of 10-20 cm. These soil samples were air-dried and sieved (<2 mm). Sub-samples (~300 mg) were digested using 9 mL 70% HNO 3 and 3 mL 37% HCl per sample in a microwave digester (Milestone Start D) for 1.5 h and diluted to 40 mL with ultrapure water before analysis to obtain pseudo-total elemental concentrations. Soil pH was measured in a 1:2.5 soil:water mixture after 2 h of shaking.

Exchangeable trace elements were extracted in 0.1 M Sr(NO 3 ) 2 at a soil:solution ratio of 1:4 (10 g soil with 40 ml solution) and 2 h shaking time (adapted from [START_REF] Kukier | Amelioration of nickel phytotoxicity in muck and mineral soils[END_REF].

As a means of estimating potentially phytoavailable trace elements, diethylenetriamine pentaacetic acid (DTPA-extractant) was used according to the method of [START_REF] Becquer | Disponibilité du nickel le long d'une toposéquence de sols développés sur roches ultramafiques de Nouvelle-Calédonie[END_REF] which was adapted from the original method by [START_REF] Lindsay | Development of a DTPA soil test for zinc, iron, manganese, and copper[END_REF], with the following modifications: exclusion of triethanolamine (TEA), pH adjusted to 5.3, 5 g soil used with 25 mL extractant to prevent saturation of the DTPA, and an extraction time of 1 h.

Chemical analysis of bulk tissue samples and soil extracts -Plant tissue samples

(leaves, bark, branches, roots, fruits, flowers) for bulk chemical analysis were collected in the field from the same plants. Latex was collected by cutting the bark of P. acuminata.

These samples were dried at 70°C for five days in a drying oven. The dried plant tissue samples were subsequently ground and digested using 4 ml HNO 3 (70%) in a microwave oven (Milestone Start D) for a 45-minute programme and diluted to 30 mL with ultrapure water (Millipore 18.2 MΩ•cm at 25°C) before analysis with ICP-AES (Varian Vista Pro II) for Ni, Co, Cr, Cu, Zn, Mn, Fe, Mg, Ca, Na, K, S, and P. Soil extracts were also analysed using ICP-AES for the same elements. In-line internal addition standardization using yttrium was used to compensate for matrix-based interferences.

X-ray fluorescence microscopy (µXRF) -

The X-ray fluorescence microscopy experiment was undertaken at Beamline P06 at the PETRA III (Deutsches Elektronen-Synchrotron; DESY, Hamburg, Germany), a 6 GeV 3 rd Generation Synchrotron Radiation Source. The undulator beam was monochromatised with a cryogenically cooled Si(111) channel-cut monochromator to an energy of 12 keV with a flux of ~1.1 × 10 10 photon/s. A Kirkpatrick-Baez mirror pair was used to focus the incident beam to 700 × 530 nm (hor × ver). The Maia detector uses a large detector array in backscatter geometry to maximize detected signal and count-rates for efficient imaging. Maia enables high overall count-rates and uses an annular detector geometry, where the beam passes though the detector and strikes the sample at normal incidence [START_REF] Kirkham | The Maia spectroscopy detector system: engineering for integrated pulse capture, low latency scanning and real time processing[END_REF][START_REF] Siddons | Maia X-ray microprobe detector array system[END_REF]. This enables a large solid-angle (~1.2 steradian) to be achieved in order to either maximize detected signal or to reduce the dose and potential damage to a specimen (Ryan et al. 2010;2014). We first conducted a quick 'survey scan' (50-100 µm with a dwell time of 1-2 ms, taking 5-10 min. in total) to select the precise scan area. Then a 'detailed scan' was conducted (resolution of 2-10 µm and a dwell time of 8-20 ms, taking 60-180 min. in total).

The live samples were prepared on-site immediately prior to scanning at beamline P06. The samples were hand cut with a stainless-steel razor blade ('dry knife') and analysed within 10 minutes after excision. The fresh and freeze-dried samples were mounted between two sheets of Ultralene thin film (4 µm) stretched over a Perspex frame magnetically attached to the horizontal-vertical motion stage at atmospheric temperature (~20°C). The possibility of radiation-induced damage in µXRF analysis (especially in fresh/hydrated samples) is an important consideration that may limit the information sought from the analysis (van der Ent et al. 2017b). In a recent study, radiation dose limits for µXRF analysis were assessed, and in hydrated plant tissue dose-limits are 4.1 kGy before detectable damage occurs [START_REF] Jones | Assessing radiation dose limits for X-ray fluorescence microscopy analysis of plant specimens[END_REF]. To limit radiation damage, we used a fast scanning (per-pixel dwell time is less than 10 ms, and hence effective radiation dose is low).

Data processing and statistics -

The XRF event stream was analysed using the Dynamic Analysis method [START_REF] Ryan | Dynamic analysis: on-line quantitative PIXE microanalysis and its use in overlap-resolved elemental mapping[END_REF][START_REF] Ryan | Quantitative trace element imaging using PIXE and the nuclear microprobe[END_REF] as implemented in GeoPIXE [START_REF] Ryan | Quantitative PIXE microanalysis of geological material; using the CSIRO proton microprobe[END_REF][START_REF] Brearley | Nutrient limitation in a Malaysian ultramafic soil[END_REF]. The matrix file used for the spectra fitting was an assumed freeze-dried plant material composition of C 31 O 15 H 51 N 2 S 0.8 with a density of 0.75 g cm -3 , and for live/fresh samples the composition was C 7.3 O 33 H 59 N 0.7 S 0.8 with a density of 0.90 g cm -3 and considering two layers of Ultralene (4 µm).

RESULTS

Soil chemistry in the habitat of the five species -Most of the Soils originated from

non-(Rivière-Bleue) or only moderately serpentinised (Monts des Koghis) peridotite and were moderately weathered (i.e. they have full or partial ferralitic properties). The range of soil pH values was quite narrow around pH 6.0 (pH 5.8-6.2) except for the soils hosting Psychotria gabriellae, which were more acidic (< pH 5.5) than the other soils (Table 1).

Total Fe ranged from 128-388 mg Fe g -1 with most around 300 mg Fe g -1 and total Al from 12.2-42.5 mg Al g -1 . Soils had high Mg concentrations from 5.2-20.1 mg Mg g -1 and depleted concentrations of Ca. All soils were extremely depleted in total K with most values around 200 µg g -1 and none exceeded 1000 µg g -1 . Furthermore, P total concentrations were very low (from 280-780 µg P g -1 ), as were S total concentrations (590-1200 µg S g -1 ). Total Co, Cr, Mn, and Ni concentrations reflected typical values from ferralic ultramafic soils: 340-1080 µg Co g -1 , 339-1080 µg Cr g -1 , 2880-7280 µg Mn g -1 , and 2.7-10.5 mg Ni g -1 (Table 1).

Metal availability was assessed by both Sr(NO 3 ) 2 and DTPA extractions (Table 1). The former provides a good picture of exchangeable ions retained by the soil Cation Exchange Capacity (CEC), whereas DTPA extracts the isotopically-exchangeable pool, i.e. the Ni pool that supplies hyperaccumulators and non-hyperaccumulators [START_REF] Echevarria | Assessment of phytoavailability of nickel in soils[END_REF][START_REF] Echevarria | Assessment and control of the bioavailability of nickel in soils[END_REF][START_REF] Massoura | Response of excluder, indicator, and hyperaccumulator plants to nickel availability in soils[END_REF][START_REF] Estrade | Weathering and vegetation controls on nickel isotope fractionation in surface ultramafic environments (Albania)[END_REF]. The Sr(NO 3 ) 2 exchangeable Ni in all soils ranged from 6.4-38 µg Ni g -1 . Moreover, DTPA-extractable Ni was very high in all sampled soils. It varied from 300-627 µg Ni g -1 with no apparent difference between plant species. One soil (P. gabriellae from Monts des Koghis) had exchangeable Co that was remarkably high (i.e. 8 µg Co g -1 ) and exchangeable Mn was also highly available (i.e. 285 µg Mn g -1 ). In the case of DTPA-extractable Co, it was noticeable that most of the soils had similar concentrations, limited between 49-78 µg Co g -1 ; which appears higher than in typical ultramafic soils [START_REF] Echevarria | Genesis and behaviour of ultramafic soils and consequences for nickel biogeochemistry[END_REF]van der Ent et al. 2018a). For other metals, exchangeable concentrations were limited in all soils and within expected values.

Elemental profiles of the five species

Bulk elemental concentrations of major trace elements were obtained for different parts of the plant for the five taxa (Tables 2 &3) and described below.

Roots -It is not practically possible to entirely clean roots from soil particle contamination.

Soil particles, and in particular nanometric or submicrometric particles, may be embedded and cannot be entirely removed by rigorous washing or brushing. However, some stable elements which are very unlikely to be taken up in large quantities by roots (e.g. Cr, Fe) help to detect soil contamination. Typically, K was low in the roots (<2460 µg K g -1 ) and preferentially transferred in the leaves: all three species contained more than 1 Wt.% K in leaves. A similar pattern was seen for Ni with highly enhanced translocation to the leaves: (<3.41 mg Ni g -1 in roots of all three species whereas it reached 3.2 mg Ni g -1 in leaves).

Bark -In the bark samples of G. pruinosa var. pruinosa and H. francii, Ca was extremely enriched (16 and 25 mg Ca g -1 respectively), whereas for H. austrocaledonicus and P. acuminata, Ca content was in the same range as in all other parts. The bark (including phloem) had very high concentrations of Ni in H. austrocaledonicus (up to 48.5 mg Ni g -1 ), and in P. acuminata (up to 10 mg Ni g -1 ). On the contrary, G. pruinosa var. pruinosa and H. Leaf tissues (whole leaf) -Foliar Ca concentrations (Table 2) were typically five-fold higher than in the soils, in contrast to the extremely high soil Mg concentrations that had been effectively limited from uptake. All species except G. pruinosa var. pruinosa reached extremely high K concentrations in leaves (sometimes >10 mg K g -1 ) in comparison to soil concentrations ranging from 224-864 µg K g -1 (Table 1). The maximum foliar K concentration for G. pruinosa var. pruinosa remained in the lower range compared to other species (Table 2), but it was still five times more than the total K in the soil. Even though soil S concentrations were low (<1200 µg S g -1 ), foliar concentrations were remarkably high reaching up to 8320 µg S g -1 in the leaves. All five species are hypernickelophores (although the maximum value in this study for Homalium francii was 9480 µg Ni g -1 ) (Table 3, Fig. 3), and Ni concentration values were consistent with earlier reports for these species [START_REF] Jaffré | Ecophysiologie -Accumulation du nickel par une Rubiacée de Nouvelle-Calédonie, Psychotria douarrei (G. Beauvisage) Däniker[END_REF][START_REF] Reeves | Tropical hyperaccumulators of metals and their potential for phytoextraction[END_REF][START_REF] Jaffré | The metal hyperaccumulators from New Caledonia can broaden our understanding of nickel accumulation in plants[END_REF]. Although Co foliar concentrations were highly variable for a given species, H. francii and H. austrocaledonicus reached values ≥120 µg Co g -1 . Manganese concentrations of the five species in leaves were comparatively low (28-120 µg Mn g -1 ), given the high available Mn concentrations in the soils (571-1090 µg Mn g -1 DTPA-extractable Mn).

Reproductive organs (flowers, fruits and seeds) -Flowers of P. gabriellae were highly enriched in Ni, contrary to those of G. pruinosa var. pruinosa (Table 3). The fruits (i.e. pericarp, except for P. gabriellae as the endocarp surround the seed) were also strongly enriched in Ni in both P. acuminata (mean of 6130 µg Ni g -1 ) and P. gabriellae (mean of 9810 µg Ni g -1 ) but relatively low in G. pruinosa var. pruinosa (1510 µg Ni g -1 ).

Concentrations of other transition metals (i.e. Co, Cr, Mn, Zn) in fruits were remarkably low in comparison with those of Ni. Seeds of P. acuminata (seed stricto sensu) and P. gabriellae (seed stricto sensu surrounded by the endocarp i.e. pyrene) contained less Ni than the fruits (or flowers in case of P. gabriellae), with high range values (4110 and 5420 µg Ni g -1 Ni respectively). The mesocarp of P. acuminata exuded green latex when sliced, suggesting high Ni concentration in this seed.

Latex of Pycnandra acuminata -Samples of latex collected from the trunks of P. acuminata in Rivière Bleue had a bright turquoise colour (Fig. 1f). The latex contained noticeable concentrations of Ca that exceeded 10 mg Ca g -1 and were always more than 10 times higher than Mg concentrations (Table 2). It contained some K (1140-3800 µg K g -1 ) and Na (1560-3320 µg Na g -1 ), with all other major elements showing concentrations below 1200 µg g -1 . It contained surprisingly high concentrations of Al (mean concentration of 810 µg Al g -1 with one value around 1500 µg Al g -1 ) and Zn (mean concentration of 800 µg Zn g -1 ). As expected [START_REF] Sagner | Hyperaccumulation, complexation and distribution of nickel in Sebertia acuminata[END_REF][START_REF] Perrier | Nickel speciation in Sebertia acuminata, a plant growing on a lateritic soil of New Caledonia[END_REF][START_REF] Schaumlöffel | Speciation analysis of nickel in the latex of a hyperaccumulating tree Sebertia acuminata by HPLC and CZE with ICP MS and electrospray MS-MS detection[END_REF][START_REF] Callahan | LC-MS and GC-MS metabolite profiling of nickel(II) complexes in the latex of the nickel-hyperaccumulating tree Sebertia acuminata and identification of methylated aldaric acid as a new nickel(II) ligand[END_REF] Elemental distribution in whole leaf fragments -Potassium was highly concentrated in the midrib of all species except P. gabriellae (Fig. 4). It was also localised in the secondary veins for H. austrocaledonicus and G. pruinosa var. pruinosa and had a high background concentration throughout the leaves of H. austrocaledonicus but not G. pruinosa var.

pruinosa. In P. gabriellae there was a strong enrichment of K in minor veins whilst in H. francii, K was enriched in the lamina surrounding the secondary veins. Alternatively, K was depleted in the area surrounding the midrib of P. acuminata but was somewhat enriched at the base of the secondary veins and depleted towards the outer ends.

There was no notable enrichment of Ca in the leaves of P. gabriellae (Fig. 4). In the other species Ca was mainly concentrated in major veins and appears as small Ca-oxalate crystals (small dots). In G. pruinosa var. pruinosa and P. acuminata these crystals underlined the structure of minor veins order; whereas in H. austrocaledonicus and H. francii they were more scattered throughout the leaf blade. Ca was totally absent in the veins of H. austrocaledonicus but homogenously distributed throughout the leaf blade.

In all species, Ni was strongly concentrated in the midveins and secondary veins except P. acuminata, where Ni was concentrated in laticifers (Fig. 5). In H. austrocaledonicus and in G. pruinosa var. pruinosa Ni was also highly concentrated in the minor veins order. That was not the case for H. francii and P. gabriellae, for the latter there was also a notable enrichment of Ni in the immediate surroundings of the main veins. In P. acuminata, Ni had a peculiar distribution. It was mainly distributed along a network of elongated cellular structures (laticifers) that were not part of the vascular system, unlike Ca or K that clearly highlighted the vascular system. Manganese was co-localized with Ni in the veins of H. francii, but it had a different distribution than Ni in G. pruinosa var. pruinosa (Fig. 5) as it was mainly localised in the midrib and absent from all secondary and minor veins in this species. It was, however, highly enriched in the surrounding areas of the midrib and the secondary vein.

Cobalt in H. austrocaledonicus and P. acuminata is co-localized with Ni throughout the leaf, although at much lower concentrations.

Images and anatomical sections of the actual scanned freeze-dried samples are provided in Suppl. Info (Fig. 4,5). It should be noted that the concentrations of all elements appeared higher in the midvein and leaf margins (that are curved inwards in H. austrocaledonicus) due to greater thickness of these regions (hence generating more fluorescence X-rays). To a lesser extent, this was also true for the secondary veins that are slightly thicker than the rest of the leaf blade. While Ni concentrations appeared to be very high in the midribs of P. gabriellae and P. acuminata, the cross sections (Fig. 7) revealed that Ni is in fact equally distributed in the epidermis of midrib and lamina.

Comparison of fresh/live leaves and freeze-dried leaf fragments

There appeared to be differences in the leaf K distribution of G. pruinosa var. pruinosa between freeze-dried and fresh material (Fig. 4 and6). In freeze-dried material, K was mainly distributed in the vascular system of the leaf: in the mid-rib and secondary veins. In the fresh leaf, it seemed to be absent from all vascular tissues and concentrated in cell clusters in the leaf lamina. With P. gabriellae, K in fresh samples was higher in the midrib, whereas it was mostly located in secondary veins and almost absent from the mid-rib in freeze-dried leaves.

The distribution of Ni was generally similar (higher concentration in the mid-rib) in fresh and freeze-dried material for both species.

Elemental distribution in leaf midribs -As the concentrations of all elements were apparently higher in the leaf midrib (but refer to earlier notes about the effect of sample thickness on XRF signal), a more detailed scan of these tissues was undertaken on crosssectioned fresh material for two species (P. gabriellae and P. acuminata, Fig. 7). In P. gabriellae, K was present at high concentrations in all the tissues except in the epidermis.

Potassium appeared to be located also in the vacuoles of most of the cells. In P. acuminata, K was also present in high concentration throughout the midrib, and was particularly rich in the parenchyma, K concentrations were much lower in the vascular system and almost depleted in the sclerenchyma surrounding vascular bundle.

Calcium was much less concentrated in midrib of both species. In P. gabriellae it appeared as small localised patches principally distributed in the parenchyma. These patches were Caoxalate crystals. In P. acuminata, Ca seemed to be only present in the adaxial epidermis of the midrib and not anywhere else. Nickel and Mn seemed to be clearly co-localised in the epidermis of both species. In P. gabriellae, both abaxial and adaxial epidermal cell layers contained the highest concentrations of both elements with Mn concentrations higher in the leaf blade epidermal tissues. However, in P. acuminata, only the adaxial epidermis had the highest Ni concentrations while Mn concentrations were intermediate to high in both epidermal tissues. In this species, very few hotspots with very high Ni concentrations also appeared in parenchyma and collenchyma (Fig. 7).

Elemental distribution in leaf petioles -Petiole sections were examined as fresh samples for G. pruinosa var. pruinosa, P. gabriellae and P. acuminata and as freeze-dried samples for H. francii (Fig. 8 and9). In all species K concentration was high in the cortex and phloem but occurred in much lower concentration in the xylem (Fig. 8). However, in P. gabriellae and P. acuminata, high K concentrations were notable in xylem rays. K concentration in the epidermis varied between species, with particularly high concentrations in G. pruinosa var. pruinosa. Calcium distribution was similar to K, but with lower concentrations in addition to low concentrations of Ca in epidermal cells. Note that Ca appeared as small dots in the form of Ca-oxalate.

Nickel in G. pruinosa var. pruinosa and P. gabriellae was located in the epidermis of the petiole with lower, but non-negligible, concentrations in the outer cortex of P. gabriellae (Fig. 9). In H. francii, the reverse was observed, and Ni was mostly concentrated in the inner part (parenchyma) and absent from the epidermis and the vascular bundle. In P. acuminata, a few large patches of high Ni concentration occurred randomly in the parenchyma, and small, but more regular, patches were observed in the adaxial epidermis of the petiole (the flatter part in the two sections). These large Ni patches were interpreted as leaking laticifers after sectioning of the fresh petiole. In H. francii, Zn was located in similar areas as Ni but at lower concentrations. Manganese in G. pruinosa var. pruinosa and P. gabriellae was only concentrated in the epidermis of the petiole.

Elemental distribution in fresh stem sections of Psychotria gabriellae -In stem sections, the K concentrations were highest in the epidermis and progressively decreased towards the vascular tissues (Fig. 10). High concentrations, though much lower than in the epidermis, were also found in phloem tissues, pith and in the inner part of xylem tissues. Calcium was ubiquitous in the stem as localised Ca-oxalate particles, except in the xylem tissues. The distribution pattern of Ni was clearly similar to that of K in stems. Nickel was even more pronouncedly enriched in the epidermal cell layer than K.

Cell-level elemental distribution in Psychotria gabriellae -High-resolution scans (0.5 µm) of a fresh/live hand-cut leaf blade of P. gabriellae were obtained (Fig. 11). The abaxial epidermis of this species contains two layers of large cells whereas the adaxial epidermis had only one layer (Fig. 11). Potassium was enriched in the mesophyll and highly enriched in the vacuoles of the abaxial epidermal cells. In contrast, Ca was strongly depleted in the vacuoles of these cells and only detectable in epidermal cell walls, but it formed localized Ca fibres (raphides of Ca-oxalate precipitates) in the spongy mesophyll. The distribution of Mn and Co was rather similar to each other. Both elements were concentrated in the vacuoles of the adaxial epidermal cells. Zinc was slightly enriched in the spongy mesophyll. Nickel was extremely enriched in the vacuoles of the adaxial epidermal cells and to a lesser extent in the vacuoles of the outer layer of abaxial epidermal cells. It also appeared to be present in the apoplastic space between the inner layer cells of the abaxial epidermis. It was notably low in the palisade mesophyll.

DISCUSSION

Soil-plant interactions and elemental uptake

While most Ni hyperaccumulators worldwide have been described from serpentinite soils (i.e. soils derived from serpentinite minerals characterised by very high Mg/Ca, very low K and high pH, see [START_REF] Echevarria | Genesis and behaviour of ultramafic soils and consequences for nickel biogeochemistry[END_REF], in New Caledonia hyperaccumulation also occurs on nonserpentinised peridotites. The high diversity of soils derived from ultramafic rocks is an important pedological feature of New Caledonia, ranging from magnesium-rich brown soils (e.g. Cambisols) to impoverished Fe-rich soils (e.g. Ferralsols) [START_REF] Jaffré | Composition chimique et conditions de l'alimentation minérale des plantes sur roches ultrabasiques (Nouvelle Calédonie)[END_REF][START_REF] Isnard | How did the ultramafic soils shape the flora of the New Caledonian hotspot?[END_REF]. All of the studied species reach the status of hypernickelophore (plant with >10 mg Ni g -1 ); which may be partly explained by the high availability of Ni in all of the soil samples. This is mainly due to two processes; in the case of Monts des Koghis, soil erosion rejuvenates ultramafic properties of the ferralic material in the soils (van der Ent et al. 2018a), thus keeping exchangeable Mg and Ni at high concentrations with some contribution of moderate laterization. In the case of Rivière Bleue, the alluvial conditions and temporary waterlogging likely contribute to the dissolution of Ni-bearing Fe-oxyhydroxides, thus releasing substantial amounts of available Ni [START_REF] Antić-Mladenović | Impact of controlled redox conditions on nickel in a serpentine soil[END_REF]. Forest areas over peridotite humusrich alluvium are Ni-rich and host the famous P. acuminata [START_REF] Jaffré | Sebertia acuminata: A hyperaccumulator of nickel from New Caledonia[END_REF]) and other hyperaccumulators (H. austrocaledonicus, H. guillainii, P. gabriellae) [START_REF] Jaffré | Ecophysiologie -Accumulation du nickel par une Rubiacée de Nouvelle-Calédonie, Psychotria douarrei (G. Beauvisage) Däniker[END_REF].

For major elements, four species out of five had notably high K concentrations reaching almost 10 mg K g -1 in leaves except in G. pruinosa var. pruinosa (typically 2000 µg K g -1 ).

These concentrations are high for K-impoverished ultramafic soils (mean for K for 146 species in forest on ultramafic soils is 8.6 mg K g -1 ), compared to plants growing on nonultramafic soils [START_REF] Jaffré | Étude écologique du peuplement végétal des sol dérivés de roches ultrabasiques en Nouvelle Calédonie[END_REF]. The low Ca concentration in the soil and low Ca/Mg quotient contributes to the edaphic stresses on ultramafic soils [START_REF] Proctor | Vegetation and soil and plant chemistry on ultramafic rocks in the tropical Far East[END_REF]. In New Caledonia, exchangeable Ca 2+ are very low while Mg concentration are extremely high leading to very low Ca/Mg quotient on serpentinite [START_REF] Jaffré | Composition chimique et conditions de l'alimentation minérale des plantes sur roches ultrabasiques (Nouvelle Calédonie)[END_REF]) and also on peridotite in this study. The Ca/Mg quotient in leaves of all five species is high and closer to 1 (with the exception of H. austrocaledonicus). The ability for preferential Ca over Mg uptake is characteristic for many ultramafic species [START_REF] Brady | Evolutionary ecology of plant adaptation to serpentine soils[END_REF][START_REF] Brearley | Nutrient limitation in a Malaysian ultramafic soil[END_REF]), including Ni hyperaccumulators [START_REF] Bani | In-situ phytoextraction of Ni by a native population of Alyssum murale on an ultramafic site (Albania)[END_REF]van der Ent et al. 2018a).

Potassium and phosphorus are the most limiting nutrients in tropical ultramafic soils [START_REF] Proctor | Vegetation and soil and plant chemistry on ultramafic rocks in the tropical Far East[END_REF], where they have been leached or strongly bound to Fe oxides. Nickel hyperaccumulators efficiently take up these elements and significantly contribute to their biogeochemical cycling in ultramafic soils [START_REF] Echevarria | Genesis and behaviour of ultramafic soils and consequences for nickel biogeochemistry[END_REF]. The association between Ni and nutrient assimilation remain unknown, though it has been suggested that extensive uptake of Ni is the result of root foraging and assimilation of essential nutrients that are depleted in the soils. Another explanation would be that Ni accumulation slows down plant development leading to stunted habit and greater uptake/accumulation of nutrients [START_REF] Brooks | Some New Zealand and New Caledonia plant accumulators of nickel[END_REF].

Sulphate is typically the dominant anion in ferralic ultramafic soils; therefore, it is not surprising that it is co-accumulated where cations are highly accumulated within the plant to ensure ionic balance.

In the case of Co, the foliar concentrations are sometimes high and associated with a high DTPA-Co/DTPA-Ni quotient in the soil. The Co availability is probably also enhanced during temporary reduction events in alluvial soils from Rivière Bleue (where four out the five studied species were sampled). Similar results have been reported for a tropical Ni-Co hyperaccumulator plant (Glochidion cf. sericeum) from Borneo where unusually high Co uptake is linked to temporary waterlogged ultramafic soils (van der Ent et al. 2018c).

Latex of Pycnandra acuminata

Citrate is the main chelating agent of Ni in the latex of P. acuminata [START_REF] Lee | The relation between nickel and citric acid in some nickel-accumulating plants[END_REF][START_REF] Schaumlöffel | Speciation analysis of nickel in the latex of a hyperaccumulating tree Sebertia acuminata by HPLC and CZE with ICP MS and electrospray MS-MS detection[END_REF][START_REF] Callahan | LC-MS and GC-MS metabolite profiling of nickel(II) complexes in the latex of the nickel-hyperaccumulating tree Sebertia acuminata and identification of methylated aldaric acid as a new nickel(II) ligand[END_REF]). However, citrate complexes have much higher stability constants (log K 1 values) with trivalent ions such as Al than with Ni, typical values for 1:1 complexes are reported to be 10.2, 4.8 and 4.5 for Al, Ni and Zn respectively (more than 10 5 times more stable than the Ni-citrate complex) [START_REF] Martell | NIST Critically Selected Stability Constants of Metal Complexes Database[END_REF][START_REF] Cardiano | Sequestration of aluminium(III) by different natural and synthetic organic and inorganic ligands in aqueous solution[END_REF]. This may explain why we observed such high Al concentrations in the latex despite the low availability of soil Al. Zinc, which is more available than Al, was also high in the latex because it has a stability constant similar to Ni. High Al uptake and transfer by laticifers, while in low concentration in branches, fruits and seeds, is possibly an indication that this network loads metals including Ni from the root up to all other plant parts independently from the vascular system (van der Ent et al. unpublished).

Effects of freeze-drying on elemental distribution

Scattering of X-rays by water reduces XRF signal, meaning that less signal from hydrated samples (live samples) is detected. Furthermore, this effect is more pronounced with low fluorescence energy (e.g. K). Quite the opposite, freeze-drying increases the apparent concentration of an element by removing the solute. Calcium is present as Ca-rich deposits, presumably Ca-oxalate crystals [START_REF] Franceschi | Calcium oxalate in plants: formation and function[END_REF], and as a consequence is not impacted by the water loss during freeze-drying. Given that K is the most mobile cation in plant tissues, it is likely to be more affected by the movement of water upon freeze-drying.

There are several lines of evidence that show that freeze-drying does modify the distribution of elements at micro-scale [START_REF] Tylko | X-ray microanalysis of biological material in the frozen-hydrated state by PIXE[END_REF][START_REF] Wang | Micro-PIXE analyses of frozen-hydrated semi-thick biological sections[END_REF]). This study is the first to report extensive µXRF observations of fresh plant samples allowing the analysis of larger surfaces than for frozen-hydrated samples which need to be constantly maintained under a cold stream of N 2 gas (typically able to keep a sample area < 2 mm diameter wide frozen).

Patterns of Ni hyperaccumulation and other elements

The tissue-level and cellular-level distribution of Ni in the epidermal cells in P. gabriellae, H. austrocaledonicus, and H. francii conforms with the majority of studied Ni hyperaccumulator plant species globally, including (temperate) herbaceous species (see e.g. [START_REF] Broadhurst | Nickel localization and response to increasing Ni soil levels in leaves of the Ni hyperaccumulator Alyssum murale[END_REF]) and (tropical) woody plants (see van der Ent et al. 2017a,b). However, two of the studied species (G. pruinosa var. pruinosa and P. acuminata) have distinctive patterns in their Ni tissue-level distribution from what has been observed so far in other species. Geissois pruinosa var. pruinosa accumulates high Ni but much less potassium than all the other species (K hyperaccumulation is a general feature among all Ni hyperaccumulators globally).

In fact, K and Ni appear to have inverse foliar distribution in G. pruinosa var. pruinosa whereas they typically co-accumulate in other species (e.g. P. gabriellae).

Nickel is associated in tissues adjacent to vascular bundles in the epidermal tissues (and/or cortex); within vacuoles and apoplastic space in cell walls. When there is a lower Ni concentration it is only seen in the cell walls, but if there is a higher concentration it is observed in both the cell walls and in the vacuoles. That could mean that the preferred localisation for Ni storage is apoplastic space, only the extreme levels of hyperaccumulation would require active uploading into the vacuoles. The local distribution of Mn and Co are usually identical to that of Ni, inferring that co-accumulation could be due to the same transport pathways. However, the distribution of Zn is dissimilar to Ni, except for in P. gabriellae, and this mirrors the findings in the tropical Ni-Zn hyperaccumulator Dichapetalum gelonioides [START_REF] Nkrumah | Contrasting nickel and zinc hyperaccumulation in subspecies of Dichapetalum gelonioides from Southeast Asia[END_REF].

CONCLUSIONS

Many tropical hyperaccumulator species have extraordinarily high Ca concentrations compared to prevailing soils concentrations [START_REF] Van Der Ent | Multi-element concentrations in plants parts and fluids of Malaysian nickel hyperaccumulator plants and some economic and ecological considerations[END_REF]. Correlations between Ca and Ni have been reported in the leaves of Hybanthus floribundus from Australia [START_REF] Farago | Plants that accumulate metals (Part VI): Further studies of an Australian nickel accumulating plant[END_REF]. Here we report high Ca concentration in leaves, branches and bark, probably associated with calcium oxalate crystals deposited in epidermis and cortex (and pith in P. gabriellae). Calcium oxalate formation is a general mechanism for regulating bulk-free Ca 2+ level in tissues and organs. It can also have a support function when located in the sclerenchyma. For P. acuminata, the high Ca contents in the abaxial cortex of petioles and midribs suggests that it has a role in reinforcing the structure of these long petioles. In P. acuminata, Ca is present in vascular bundles, also probably as Ca-oxalate crystals, and acts as a component of their structures. Nickel and Ca are proxies for two different and disconnected networks in this plant, the former for laticifers and the latter for the vascular system.

The laticifers constitute an independent network of cells that operate in parallel to the vascular systems (xylem and phloem) and is the main sink for Ni. Although the epidermal layer of P. acuminata leaves has been reported to be rich in Ni [START_REF] Perrier | Nickel speciation in Sebertia acuminata, a plant growing on a lateritic soil of New Caledonia[END_REF]), SEM-EDS used in that study only probed to a maximum depth of 5 µm and hence was unable to reveal laticifers. Because it has a greater penetration depth (>1000 µm for hydrated biological samples) synchrotron µXRF using hard X-rays (incident energy >8 keV) allows direct observation of the unique distribution of Ni in P. acuminata. Nickel is mostly present in laticifers and transported throughout the whole tree from roots to leaves. The occurrence of these Ni-rich laticifers is a specific phenomenon that has not been visualized with µXRF in other species to date. Laticifers are impossible to section without their destruction because they are pressurized [START_REF] Pickard | Laticifers and secretory ducts: two other tube systems in plants[END_REF]. Consequently, physical sectioning of laticifers causes them to expel their contents, resulting in Ni spills. In order to visualise laticifers, nondestructive techniques such as synchrotron X-ray fluorescence tomography (µXRF-CT) are required (de Jonge and Vogt 2010; [START_REF] Lombi | Fast X-Ray fluorescence microtomography of hydrated biological samples[END_REF]van der Ent et al. 2017b), and intact laticifers can be readily observed in large maps of intact tissues. pruinosa and Pycnandra acuminata. The maps measure 2.28 × 4.49 mm at 5 µm resolution with 10 ms dwell (Homalium), 5.5 × 3.08 mm at 8 µm resolution with 8 ms dwell (Psychotria), 6.3 × 5.68 mm at 7 µm resolution with 8 ms dwell (Geissois), and 3.9 × 2.35 mm at 5 µm resolution with 8 ms dwell (Pycnandra). Maps were cropped to fit figure panels. Table 1. Habitat soil elemental concentrations in the natural habitats of Geissois pruinosa var. pruinosa, Hybanthus austrocaledonicus, Psychotria gabriellae and Pycnandra acuminata. The concentration values are ranges and means in µg g -1 or in mg g -1 when indicated with an asterisk* expressed on dry weight basis. The digests and extracts were analysed with ICP-AES. The 'total' is extractable with HCL/HNO 3 digest, 'DTPA' is extractable with Diethylenetriamine pentaacetic acid solution, and 'Sr(NO 3 ) 2 ' is extractable with strontium nitrate solution.

The Homalium francii samples were collected in close proximity of Pycnandra acuminata, therefore the soil habitat chemistry will be similar.

Species

  francii had very low Ni concentrations (410 and 920 µg Ni g -1 respectively) in the bark. Stem tissues (without bark) -Similarly to the bark, Ni concentrations were low in the branches of both G. pruinosa var. pruinosa and H. francii. They were intermediate in H. austrocaledonicus and P. acuminata and high in P. gabriellae (>15 mg Ni g -1 ) (data not shown).

  , Ni concentrations were extremely high and ranged from 110-150 mg Ni g -1 . However, Co concentrations in the latex were relatively low (mean of 41 µg Co g -1 ) compared to Ni concentrations. The Ni/Co ratio in latex was approximately 325, whereas it was approximately 10 in the soil DTPA extract.

Figure 1 .Figure 2 .

 12 FIGURE CAPTIONS

Figure 3 .

 3 Figure 3. Boxplots of the foliar concentrations of K, Mg, Ca and Mn, Co*, Ni in Geissois pruinosa var. pruinosa (n = 12), Psychotria gabriellae (n = 12, *n = 10), Homalium francii (n = 12), Hybanthus austrocaledonicus (n = 10; *n = 9) and Pycnandra acuminata (n=12).Boxplots show ranges, medians, percentiles and outliers. All values in µg g -1 dry weight.

Figure 4 .

 4 Figure 4. Synchrotron µXRF maps of K and Ca of freeze-dried leaf portions of Geissois pruinosa var. pruinosa, Psychotria gabriellae, Homalium francii, Hybanthus austrocaledonicus and Pycnandra acuminata. The maps measure 21.65 × 2.65 mm at 11 µm resolution with 10 ms dwell (Hybanthus), 17.85 × 10.29 mm at 17 µm resolution with 10 ms dwell (Homalium), 23.27 × 7.39 mm at 10 µm resolution with 8 ms dwell (Psychotria), 16.63 × 11.91 mm at 10 µm resolution with 8 ms dwell (Geissois), and 17.52 × 11.91 mm at 10 µm resolution with 8 ms dwell (Pycnandra). Maps were cropped to fit figure panels.

Figure 5 .

 5 Figure 5. Synchrotron µXRF maps of Ni, Co and/or Mn of freeze-dried leaf portions of Geissois pruinosa var. pruinosa, Psychotria gabriellae, Homalium francii, Hybanthus austrocaledonicus and Pycnandra acuminata. The maps measure 21.65 × 2.65 mm at 11 µm resolution with 10 ms dwell (Hybanthus), 17.85 × 10.29 mm at 17 µm resolution with 10 ms dwell (Homalium), 23.27 × 7.39 mm at 10 µm resolution with 8 ms dwell (Psychotria), 16.63 × 11.91 mm at 10 µm resolution with 8 ms dwell (Geissois), and 17.52 × 11.91 mm at 10 µm resolution with 8 ms dwell (Pycnandra). Maps were cropped to fit figure panels.

Figure 6 .

 6 Figure 6. Synchrotron µXRF maps of K, Ca, Ni and Mn of fresh leaf portions of Geissois pruinosa var. pruinosa, and Psychotria gabriellae. The maps measure 29.39 × 9.15 mm at 8 µm resolution with 17 ms dwell (Geissois) and 16.2 × 12.3 mm at 15 µm resolution with 10 ms dwell (Psychotria). Maps were cropped to fit figure panels.

Figure 7 .

 7 Figure 7. Synchrotron µXRF maps of K, Ca, Ni and Mn of fresh leaf midrib portions of Psychotria gabriellae and Pycnandra acuminata. The maps measure 3.49 × 2.12 mm at 8 µm resolution with 10 ms dwell (Psychotria) and 7.4 × 2.8 mm at 6 µm resolution with 8 ms dwell (Pycnandra). Maps were cropped to fit figure panels.

Figure 8 .

 8 Figure 8. Synchrotron µXRF maps of K and Ca of freeze-dried petiole portions of Homalium francii; and of fresh petioles portions of Psychotria gabriellae, Geissois pruinosa var.

Figure 9 .

 9 Figure 9. Synchrotron µXRF maps of Ni and Zn, of freeze-dried petiole portions of Homalium francii; and Ni and Mn or Co of fresh petioles portions of Psychotria gabriellae, Geissois pruinosa var. pruinosa and Pycnandra acuminata. The maps measure 2.28 × 4.485mm at 5 µm resolution with 10 ms dwell (Homalium), 5.5 × 3.08 mm at 8 µm resolution with 8 ms dwell (Psychotria), 6.3 × 5.68 mm at 7 µm resolution with 8 ms dwell (Geissois), and 3.9 × 2.35 mm at 5 µm resolution with 8 ms dwell (Pycnandra). Maps were cropped to fit figure panels.

Figure 10 .

 10 Figure 10. Synchrotron µXRF maps of K, Ca, Ni and Zn, of fresh stem portions of Psychotria gabriellae. The maps measure 4.7 × 4.58 mm at 8 µm resolution with 8 ms dwell. Maps were cropped to fit figure panels.

Figure 11 .

 11 Figure 11. Synchrotron µXRF maps of K, Ca, Mn, Co, Ni and Zn, of fresh leaf blade cross section of Psychotria gabriellae. The maps measure 0.16 × 0.27 mm at 0.5 µm resolution with 15 ms dwell (Psychotria). Maps were cropped to fit figure panels.

Sr(NO 3 ) 2 ) Fe (total)* Fe (Sr(NO 3 ) 2 ) Co (total) Co (DTPA) Co (Sr(NO 3 ) 2 )

  Only one sample, * ranges and means in mg g -1 , n = number of analysed samples

	Psychotria gabriellae	3	2.7-5.7 [3.7]	300-443 [352]	8.4-26.3 [18.9] 204-322 [250]	7.5-67.5 [46.1]	0.4-4.1 [2.8]
	Pycnandra acuminata	1	8.3	599	38	321	9.8	0.9
		ᵅ
		n	pH	Mg (total)*	Habitat soil chemistry Al (total)* P (total)	S (total)	K (total)
	Geissois pruinosa var. pruinosa 3	6.1-6.2 [6.2]	15.2-20.1 [18.4] 12.2-14.6 [13.2] 278-305 [296]	585-959 [712]	233ᵅ
	Hybanthus austrocaledonicus	1	5.9	7.2	34.9	295	648	-
	Psychotria gabriellae	3	5.1-5.9 [5.4]	8.8-14.1 [11.9] 22.3-31.9 [28.1] 751-779 [765] 887-1200 [1080]	231-864 [627]
	Pycnandra acuminata	1	5.8	5.2	42.5	373	716	224
	Species	n	Ca (total)	Cr (total)*	Cr (DTPA)	Cr (Sr(NO 3 ) 2 )	Mn (total)	Mn (DTPA)
	Geissois pruinosa var. pruinosa 3 1270-1550 [1370]	6.7-8.0 [7.3]	0.3-0.5 [0.4]	0.1-0.3 [0.2] 4160-4320 [4230] 571-614 [594]
	Hybanthus austrocaledonicus	1	1020	6.6	0.2	0.2	7080	842
	Psychotria gabriellae	3 1370-3250 [2510]	1.8-12 [5.4]	0.5-1 [0.9]	0.2-0.7 [0.6] 2880-4670 [3540] 780-1090 [891]
	Pycnandra acuminata	1	459	8.6	0.5	0.1	7280	875
	Species Mn (Geissois pruinosa var. pruinosa 3 16.1-20.5 [18.1] n	354-388 [366]	0.04-0.3 [0.2]	845-933 [875] 55.6-58.7 [57.1]	0.7-0.9 [0.8]
	Hybanthus austrocaledonicus	1	30.6	289	0.1	952	48.6	0.4
	Psychotria gabriellae	3	32.9-285 [181]	128-375 [217]	0.1-16 [9.7]	339-638 [445] 71.3-77.8 [75.4]	0.7-8 [5]
	Pycnandra acuminata	1	105	346	0.2	1080	65.5	1.3
	Species	n	Ni (total)*	Ni (DTPA)	Ni (Sr(NO 3 ) 2 )	Zn (total)	Zn (DTPA)	Zn (Sr(NO 3 ) 2 )
	Geissois pruinosa var. pruinosa 3	9.9-10.5 [10.2]	320-426 [367]	6.4-8.2 [7.1]	324-345 [336] 13.1-15.5 [13.9]	0.3-0.4 [0.3]
	Hybanthus austrocaledonicus	1	8	627	18.2	287	10.5	0.3

Table 2 .

 2 Bulk macro-elemental concentrations of various plant parts of Geissois pruinosa var. pruinosa, Homalium francii, Hybanthus austrocaledonicus, Psychotria gabriellae, and Pycnandra acuminata. The concentration values are ranges and means in µg g -1 or in mg g -1 when indicated with an asterisk* expressed on dry weight basis. Hash# indicates sample size if different from the n. Acid digests and were analysed with ICP-AES.

	Species	n	Ca	Fe	K	Mg	Na	P	S
					Leaves
	Geissois pruinosa var. pruinosa 12	2.97-4.7 [3.91]	10-46 [29]	1.09-1.83 [1.35]	1.83-5.17 [4.16]	220-740 [400]	92-490 [270]; 11#	0.51-8.32 [1.87]
	Homalium francii	12	1.24-3.61 [2.02]	37-300 [140]	1.17-10.4 [5.84]	1.2-3.33 [2.27]	130-3090 [1810]	160-1460 [580]	0.85-3.48 [2.02]
	Hybanthus austrocaledonicus	10	1.16-2.29 [1.71]	31-71 [47]	1.83-10.200 [6.28]	2.76-4.9 [3.4]	730-6000 [2400]	7-680 [360]; 8#	1.14-3.82 [2.75]
	Psychotria gabriellae	12	1.40-5.17 [2.82]	19-1510 [170]	0.48-19.0 [3.09]	0.91-2.93 [1.6]	390-2180 [950]	140-860 [480]; 10# 0.75-5.27 [2.94]
	Pycnandra acuminata	12	0.66-5.62 [1.23]	26-340 [81]	3.3-7.74 [5.20]	0.51-1.85 [0.98]	1130-2590 [1580]	55-810 [290]	0.51-1.93 [1.17]
					Branches
	Geissois pruinosa var. pruinosa 3	0.84-5.26 [3.47]	7.7-12 [9.2]	0.44-1.89 [1.05]	0.51-1.8 [1.04]	140-330 [230]	110-600 [330]	0.33-0.75 [0.480]
	Homalium francii	3	3.67-5.46 [4.36]	63-76 [71]	1.3-2.56 [1.74]	0.55-1.13 [0.91]	160-190 [170]	87-220 [140]	0.36-0.71 [0.52]
	Hybanthus austrocaledonicus	2	1.66-1.700 [1.68]	7-15 [11]	2.19-2.97 [2.58]	0.44-0.8 [0.62]	610-670 [640]	130-170 [150]	0.47-0.73 [0.6]
	Psychotria gabriellae	4	2.26-4.68 [3.01]	15-45 [26]	2.65-4.47 [3.39]	0.23-0.61 [0.35]	270-2710 [1150]	69-160 [110]	0.52-0.7 [0.59]
	Pycnandra acuminata	2	1.55-4.26 [2.90]	180-1030 [610]	1.77-4.38 [3.07]	0.43-2.44 [1.44]	1600-1850 [1720]	340-740 [540]	0.72-1.04 [0.88]
					Bark
	Geissois pruinosa var. pruinosa 1	16.3	9.1	0.61	2.11	75	100	0.81
	Homalium francii	1	24.8	15	2.56	0.24	37	170	0.74
	Hybanthus austrocaledonicus	4	0.93-1.44 [1.17]	15-240 [82]	7.69-8.88 [8.38]	0.37-0.74 [0.52]	21-350 [150]	93-220 [170]	0.76-2.47 [1.38]
	Pycnandra acuminata	2	0.6-5.28 [2.94]	21-22 [22]	0.43-0.49 [0.46]	0.07-0.0199 [0.09] 840-3780 [2310]	51-82 [66]	0.3-1.0 [0.65]
					Roots
	Homalium francii	1	2.13	5710	1950	1.92	1920	240	0.71
	Hybanthus austrocaledonicus	2	0.77-5.13 [2.95]	870-1620 [1250]	2.41-2.46 [2.44]	0.53-1.73 [1.13]	53-390 [220]	140-220 [180]	0.74-0.97 [0.85]
	Psychotria gabriellae	2	0.26-6.43 [3.35]	1080-2870 [1970]	1.51-1.73 [1.62]	0.34-1.17 [0.75]	930-1430 [1180]	160-2310 [1240]	0.76-0.81 [0.79]
					Flowers
	Geissois pruinosa var. pruinosa 2	3.56-5.18 [4.37]	15-15 [15]	5.09-7.35 [6.22]	2.58-3.43 [3.0]	210-1040 [620]	530-860 [700]	1.14-1.75 [1.44]
	Psychotria gabriellae	2	3.76-3.88 [3.82]	28-38 [33]	9.57-9.58 [9.57]	1.94-2.48 [2.21]	180-640 [410]	740-760 [750]	0.59-2.04 [1.31]
				Fruits (pericarp except for P. gabriellae)
	Geissois pruinosa var. pruinosa 1	2.47	11	4.14	1.64	940	730	0.87
	Psychotria gabriellae	3	2.05-5.92 [3.94]	12-17 [15]	7.15-8.5 [7.82]	1.9-2.15 [2.03]	240-390 [310]	610-950 [760]	1.47-1.66 [1.59]
	Pycnandra acuminata	2	0.91-2.48 [1.7]	14-20 [17]	4.39-4.81 [4.600]	0.63-2.18 [1.41]	680-1180 [930]	200-680 [440]	0.67-0.91 [0.79]
				Seeds (

with endocarp in P. gabriellae)

	Psychotria gabriellae	2.1	13	2.44	1.5	54	1000	1.39
	Pycnandra acuminata	0.78	9.8	2.69	0.74	830	560	0.43
				Latex
	Pycnandra acuminata	7.46-17.04 [12.25]	61-200 [130]	1.14-3.8 [2.47]	0.48-1.36 [0.92]	1560-3320 [2440]	310-570 [440]	0.7-1.19 [0.94]

Table 3 .

 3 Bulk trace elemental concentrations of various plant parts of Geissois pruinosa var. pruinosa, Homalium francii, Hybanthus austrocaledonicus, Psychotria gabriellae, and Pycnandra acuminata. The concentration values are ranges and means in µg g -1 or in mg g -1 when indicated with an asterisk* expressed on dry weight basis. Hash# indicates sample size if different from the n. Acid digests and were analysed with ICP-AES.

	Species	n	Al	Co	Cr	Cu	Mn	Ni	Zn
					Leaves
	Geissois pruinosa var. pruinosa	12	9.3-62 [42]	0.5-55 [39]	3.4-78 [56]	1.9-7.4 [2.3]	56-180 [93]	3.21-10.6 [8.99]	25-57 [39]
	Homalium francii	12	11-230 [100]	0.1-120 [28]	4-120 [39]	5.1-29 [12]	24-76 [45]	2.65-9.48 [5.4]	26-220 [110]
	Hybanthus austrocaledonicus	10	57-97 [75]	1.8-130 [30]; 9# 5.9-81 [38]; 7#	1.3-9.8 [6.6]	77-180 [120]	9.22-15.6 [12.7]	48-260 [100]
	Psychotria gabriellae	12	20-160 [90]	3.2-90 [37]; 10# 1.2-51 [27]; 9# 3-36 [12]; 11#	22-140 [72]	10.2-32.200 [21.4]	14-200 [95]
	Pycnandra acuminata	12	16-170 [69]	0.3-73 [18]	2.2-41 [19]; 9#	0.1-9.7 [2.4]	21-51 [28]	6.71-17.9 [13.3]	23-94 [50]
					Branches
	Geissois pruinosa var. pruinosa	3	5.6-7.7 [6.5]	2.3-12 [5.7]	0.7-1.8 [1.3]	3.1-21 [9.9]	9.7-22 [15]	0.47-1.07 [0.72]	24-56 [39]
	Homalium francii	3	16-19 [18]	0.2-1.7 [0.9]	2.7-2.9 [1.2]	1-8.4 [1.4]	8.8-17 [14]	0.87-0.95 [0.92]	20-25 [22]
	Hybanthus austrocaledonicus	2	4.9-6.1 [2.3]	0.1-0.9 [0.5]	0.5-1.7 [1.1]	1.3-1.6 [1.5]	21-25 [23]	2.03-2.34 [2.18]	13-14 [14]
	Psychotria gabriellae	4	6-22 [12]	0.5-2.8 [2]	1.1-3.3 [2]	2.6-4.7 [3.9]	14-73 [35]	4.61-14.6 [8.12]	22-53 [33]
	Pycnandra acuminata	2	29-130 [82]	0.3-0.4 [0.3]	0.9-31 [16]	3.2-9.4 [6.3]	23-64 [44]	2.82-6.86 [4.84]	25-32 [28]
					Bark
	Geissois pruinosa var. pruinosa 1	11	6.3	1.3	2.6	25	0.41	24
	Homalium francii	1	21	2	0.6	0.5	20	0.92	45
	Hybanthus austrocaledonicus	4	8.8-17 [13]	0.5-6.2 [3.3]	5.7-69 [30]	0.3-3 [1.5]	55-170 [130]	7.59-48.5 [26 400]	39-170 [97]
	Pycnandra acuminata	2	7.9-13 [10]	0.03-0.3 [0.2]	1.4-2.7 [1.3]	1.8-2.3 [2.1]	5.2-65 [35]	7.99-10.6 [9290]	35-50 [42]
					Roots
	Homalium francii	1	740	0.2	140	9.1	110	1.24	36
	Hybanthus austrocaledonicus	2	68-250 [160]	0.04-2.2 [1.1]	16-43 [30]	1.6-1.9 [1.7]	120-130 [120]	2.42-3.41 [2.91]	19-22 [21]
	Psychotria gabriellae	2	120-160 [140]	0.6-3 [1.8]	25-35 [30]	4-7.2 [5.6]	58-170 [120]	2.2-2.83 [2.51]	14-16 [15]
					Flowers
	Geissois pruinosa var. pruinosa	2	7-9.2 [8.1]	7-16 [12]	2.1-3.9 [1.3]	11-17 [14]	28-65 [47]	2.71-3.4 [3.06]	18-32 [25]
	Psychotria gabriellae	2	27-60 [44]	0.4-11 [5.8]	0.1-16 [8.1]	4.6-6.3 [5.5]	34-150 [91]	14.51-18. 0 [16.3]	43-61 [52]
				Fruits (pericarp except for P. gabriellae)
	Geissois pruinosa var. pruinosa	1	5.4	5.2	1.9	11	24	1.51	12
	Psychotria gabriellae	3	6.7-19 [12]	0.9-1.6 [1.1]	1.5-7.6 [2.2]	5.2-6 [5.6]	20-25 [22]	8.66-11.3 [9.81]	29-34 [31]
	Pycnandra acuminata	2	5.5-6.8 [6.2]	0.03-1 [0.5]	0.4-1.8 [1.1]	3.6-5.8 [2.6]	10-30 [20]	3.68-8.59 [6.13]	13-35 [24]
				Seeds (with endocarp in P. gabriellae)
	Psychotria gabriellae	1	7.2	0.2	1.4	6.1	54	5.42	20
	Pycnandra acuminata	1	7.1	-	4.7	1.3	22	4.11	14

Acknowledgements

The authors thank the Province Sud de Nouvelle-Calédonie for permission to collect the plant material samples (permits 1503-2016/ARR/DENV and 1206-2018/ARR/DENV)), and S.

Palermo for access to Monts des Koghis. This research was undertaken at P06 at DESY, a member of the Helmholtz Association (HGF). The research leading to this result has been supported by the project CALIPSOplus under the Grant Agreement 730872 from the EU Framework Programme for Research and Innovation HORIZON 2020. A. van der Ent was the recipient of a Discovery Early Career Researcher Award (DE160100429) from the Australian Research Council. V. Gei was the recipient of an Australia Awards PhD Scholarship from the Australian Federal Government.

SUPPLEMENTARY INFORMATION

Soil chemistry, elemental profiles and elemental distribution in nickel hyperaccumulator species from New Caledonia Vidiro Gei 1 , Guillaume Echevarria 1,2 , Peter D. Erskine 1 , Sandrine Isnard 3,4 , Bruno Fogliani 5 , Emmanuelle Montargès-Pelletier 6 , Tanguy Jaffré 3,4 , Kathryn Spiers 7 , Jan Garrevoet 7 , Antony van der Ent 1,2* 1 Centre for Mined Land Rehabilitation, Sustainable Minerals Institute, The University of Queensland, Australia.

2 Université de Lorraine -INRAE, Laboratoire Sols et Environnement, UMR 1120, France.