Sarah Louise Robin
email: sarah.robin@etudiant.unc.nc

Cyril Marchand

Brian Ham

France Pattier

Christine Laporte- Magoni

Arnaud Serres

Influences of species and watersheds inputs on trace metal accumulation in mangrove roots

Keywords: Coastal wetlands, Elements cycling, Ultrabasic, Bioconcentration, Iron plaque, New Caledonia

Mangrove forest is a key ecosystem between land and sea, and provides many services such as trapping sediments and contaminants. These contaminants include trace metals (TM) that can accumulate in mangroves soil and biota. This paper innovates by the comparative study of the effects of the watershed inputs on TM distribution in mangrove soil, on roots bioconcentration factors of two species (Avicennia marina and Rhizophora stylosa), and on Fe plaque formation and immobilization of these TM. Two mangrove forests in New Caledonia were chosen as study sites. One mangrove is located downstream ultramafic rocks and a Ni mine (ultrabasic site), whereas the second mangrove ends a volcano-sedimentary watershed (non-ultrabasic site). TM concentrations (Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Zn) were measured in soil, porewaters, and roots of both species via ICP-OES or Hg analyzer. Analyzed TM were significantly more concentrated in soils at the ultrabasic site with Fe, Cr, and Ni the most abundant. Iron, Mn, and Ni were the most concentrated in the roots with mean values of 9,651, 192, and 133 mg kg -1 respectively. However, the bioconcentration factors (BCF) of Fe (0.16) and Ni (0.11) were low due to a lack of ions in the dissolved phase and potential uptake regulation. The uptake of TM by mangrove trees was influenced by concentrations in soil, but more importantly by their potential bioavailability and the physiological characteristics of each species. TM concentrations and BCF were lower for R. stylosa probably due to less permeable root system. A. marina limits TM absorption through Fe plaque formation on its pneumatophores with a capacity to retain TM up to 94 % for Mn. Mean Fe plaque formation is potentially correlated to Fe concentration in soil. Eventually, framboids of pyrite were observed within root tissues in the epidermis of A. marina's pneumatophores.

Introduction

Mangrove forest is an intertidal ecosystem developing along subtropical and tropical coastlines. Even though mangrove accounts for less than 0.5% of the world's forest surface area, it is a remarkable sink for atmospheric CO2 and plays a crucial role in the flow of energy, nutrients, and in the carbon cycle [START_REF] Kristensen | Organic carbon dynamics in mangrove ecosystems: A review[END_REF][START_REF] Donato | Mangroves among the most carbon-rich forests in the tropics[END_REF]. Mangroves provide a physical protection to the coastline against storms and wave actions and act as a buffer between the land and the sea by accumulating sediments and associated contaminants [START_REF] Massel | Surface wave propagation in mangrove forests[END_REF][START_REF] Mcivor | Reduction of wind and swell waves by mangroves[END_REF][START_REF] Lee | Ecological role and services of tropical mangrove ecosystems: a reassessment[END_REF]. Nevertheless, this ecosystem is particularly endangered, notably due to urbanization and the release of anthropogenic effluents, some countries using mangroves as filters. In the 80s and the 90s, about 35% of mangrove forests area were lost [START_REF] Valiela | Mangrove forests: One of the world's threatened major tropical environments[END_REF][START_REF] Alongi | Present state and future of the world's mangrove forests[END_REF], Duke et al., 2007). In a few emerging countries, the estimation reached up to 8% per year [START_REF] Polidoro | The loss of species: mangrove extinction risk and geographic areas of global concern[END_REF]. Between 2000 and 2012, the annual loss rates decreased significantly in some regions, ranging from 0.16% to 0.39%, which demonstrates global awareness on the significance of the conservation of mangroves [START_REF] Hamilton | Creation of a high spatio-temporal resolution global database of continuous mangrove forest cover for the 21st century (CGMFC-21): CGMFC-21[END_REF].

Trace metals (TM) are metallic elements naturally present in limited concentrations in the environment due to their occurrence in the Earth's crust [START_REF] Turekian | Distribution of the elements in some major units of the Earth's crust[END_REF]. However, anthropogenic activities have considerably increased their concentrations in many ecosystems [START_REF] Shtiza | Chromium and nickel distribution in soils, active river, overbank sediments and dust around the Burrel chromium smelter (Albania)[END_REF][START_REF] Cao | Temporal variation and ecological risk assessment of metals in soil nearby a Pb-Zn mine in southern China[END_REF]. As TM are not biologically or chemically degraded, they can accumulate in the environment or be transported over long distances [START_REF] Marx | Long-distance transport of urban and industrial metals and their incorporation into the environment: sources, transport pathways and historical trends[END_REF]. They can be transferred to mangrove forests via atmospheric routes (wind, rain) or aquatic routes (rivers, sea) [START_REF] Marx | Long-distance transport of urban and industrial metals and their incorporation into the environment: sources, transport pathways and historical trends[END_REF]McGowan, 2010, Chowdhury et al., 2017). Mangrove soils are mainly characterized by low oxygen levels (anoxic conditions) and high sulfur and organic matter (OM) contents [START_REF] Holmer | Biogeochemical cycling of sulfur and iron in sediments of a South-East Asian mangrove, Phuket island, Thailand[END_REF][START_REF] Dittmar | Mangroves, a major source of dissolved organic carbon to the oceans[END_REF]. Strong biogeochemical activity, OM richness, and high sedimentation rate are three factors that make mangrove ecosystem a sink for contaminants [START_REF] Harbison | Mangrove muds--A sink and a source for trace metals[END_REF], Wu et al., 2014[START_REF] Cennerazzo | Dynamique des HAP et des composés organiques issus de leur transformation dans les compartiments du sol et de la rhizosphère[END_REF]. TM are quickly immobilized in soil by precipitation with pyrite (FeS2) or by adsorption onto OM for example [START_REF] Marchand | Relationships between heavy metals distribution and organic matter cycling in mangrove sediments (Conception Bay, New Caledonia)[END_REF][START_REF] Chakraborty | Partitioning of metals in different binding phases of tropical estuarine sediments: importance of metal chemistry[END_REF][START_REF] Cennerazzo | Dynamique des HAP et des composés organiques issus de leur transformation dans les compartiments du sol et de la rhizosphère[END_REF]. The dynamics of TM within mangrove soil are well studied worldwide with their partitioning mainly impacted by physico-chemical parameters (redox potential, pH) and bonding phases (carbonates, OM, sulfides, oxides, …) [START_REF] Jayachandran | Effect of pH on transport and transformation of Cu-sediment complexes in mangrove systems[END_REF][START_REF] Duan | Neutral monosaccharides and their relationship to metal contamination in mangrove sediments[END_REF][START_REF] Huang | Study of mercury transport and transformation in mangrove forests using stable mercury isotopes[END_REF].

Mangroves flora adapted to the habitat specificities such as poor O2 availability and high salinity (Duke et al., 1998, Kathiresan and[START_REF] Kathiresan | Biology of mangroves and mangrove ecosystems[END_REF]. Metallic stress is another challenge for mangrove species since TM can inhibit development processes and reduce photosynthetic activity [START_REF] Prasad | Metal fractionation studies in surfacial and core sediments in the Achankovil river basin in India[END_REF][START_REF] Nagajyoti | Heavy metals, occurrence and toxicity for plants: a review[END_REF]. Many authors studied the mechanisms of mangrove plants against metallic stress [START_REF] Zheng | Accumulation and biological cycling of heavy metal elements in Rhizophora stylosa mangroves in Yingluo Bay, China[END_REF][START_REF] Cheng | Metal (Pb, Zn and Cu) uptake and tolerance by mangroves in relation to root anatomy and lignification/suberization[END_REF][START_REF] Nath | Assessment of biotic response to heavy metal contamination in Avicennia marina mangrove ecosystems in Sydney Estuary, Australia[END_REF]. Multiple studies showed that essential metals (i.e. Cu and Zn) are regulated with their transfer to shoots limited [START_REF] Machado | Trace metals in mangrove seedlings: role of iron plaque formation[END_REF][START_REF] Macfarlane | Accumulation and partitioning of heavy metals in mangroves: A synthesis of field-based studies[END_REF]. Non-essential metals (i.e. Cr and Pb) are rather excluded from the root system or accumulated on the surface of the roots, and are not transferred to the highest organs (MacFarlane andBurchett, 2000, Chowdhury et al., 2017).

In some mangrove species such as the Avicennia, the formation of an iron plaque was observed at the surface of their root system [START_REF] Machado | Trace metals in mangrove seedlings: role of iron plaque formation[END_REF][START_REF] Pi | Effects of wastewater discharge on formation of Fe plaque on root surface and radial oxygen loss of mangrove roots[END_REF][START_REF] Lin | Seedlings influenced by sulfur and iron amendments[END_REF]. This plaque results from the precipitation of Fe (III) via the oxidation of free Fe (II) from the diffusion of O2 by the plant in its rhizosphere [START_REF] Taylor | Use of the DCB technique for extraction of hydrous iron oxides from roots of wetland plants[END_REF]Crowder, 1983, Williams et al., 2014). Studies have also exposed the role of bacteria at the root surface for biological oxidation of Fe 2+ into Fe plaque, but to a lesser extent [START_REF] Maisch | Iron lung: how rice roots induce iron redox changes in the rhizosphere and create niches for microaerophilic Fe(II)-oxidizing bacteria[END_REF]. This Fe plaque helps prevent the absorption of excessive TM and other pollutants by the mangrove through the root system [START_REF] Otte | Iron plaque on roots of Aster tripolium L.: interaction with zinc uptake[END_REF][START_REF] Pi | Formation of iron plaque on mangrove roots receiving wastewater and its role in immobilization of wastewater-borne pollutants[END_REF][START_REF] Yamaguchi | Arsenic distribution and speciation near rice roots influenced by iron plaques and redox conditions of the soil matrix[END_REF]. Iron plaque can only be formed under waterlogged conditions since Fe 2+ must be soluble, brought to the surface by transpiration pull, oxidized automatically by O2 diffusion, and precipitated [START_REF] St-Cyr | Microscopic observations of the iron plaque of a submerged aquatic plant (Vallisneria americana Michx)[END_REF]. The formation of the Fe plaque is driven by many abiotic and biotic factors [START_REF] Mendelssohn | Factors controlling the formation of oxidized root channels: A review[END_REF]. Higher Fe 2+ solubility leading to higher Fe plaque formation is observed at lower pH and redox potential. However, pH is higher in the rhizosphere of more efficient O2-diffusing species that also lead to higher Fe plaque formation [START_REF] Tripathi | Roles for root iron plaque in sequestration and uptake of heavy metals and metalloids in aquatic and wetland plants[END_REF]. There is still a debate on how the Fe fraction in the soil influence Fe plaque formation. [START_REF] St-Cyr | Iron oxide deposits on the roots of phragmites australis related to the iron bound to carbonates in the soil[END_REF] showed positive correlation between Fe plaque formation and the Fe fraction bound to carbonates while other studies exposed a positive correlation with the exchangeable fraction only [START_REF] Tripathi | Roles for root iron plaque in sequestration and uptake of heavy metals and metalloids in aquatic and wetland plants[END_REF]. Consequently, it is difficult to predict the magnitude of Fe plaque formation at a specific site due to the number of factors involved but it has been extensively confirmed that Fe 2+ availability and O2 diffusion capacity of the species are the two predominant factors affecting Fe plaque development [START_REF] Tripathi | Roles for root iron plaque in sequestration and uptake of heavy metals and metalloids in aquatic and wetland plants[END_REF].

In New Caledonia (NC), some mangrove forests develop between ultramafic soils, including Ni mining sites, and the largest lagoon in the world. Massive exploitation of ultramafic laterites intensifies erosion and favors TM transfer towards rivers and the lagoon, as laterites display high concentrations of some TM [START_REF] Bird | The impacts of opencast mining on the rivers and coasts of New Caledonia[END_REF][START_REF] Fernandez | A combined modelling and geochemical study of the fate of terrigenous inputs from mixed natural and mining sources in a coral reef lagoon (New Caledonia)[END_REF]. Multiple studies have been conducted on the dynamic of TM in New Caledonian mangroves with different stress factors (Bourgeois et al., 2019a[START_REF] Marchand | The partitioning of transitional metals (Fe, Mn, Ni, Cr) in mangrove sediments downstream of a ferralitized ultramafic watershed (New Caledonia)[END_REF][START_REF] Noël | EXAFS analysis of iron cycling in mangrove sediments downstream a lateritized ultramafic watershed (Vavouto Bay, New Caledonia)[END_REF]. It has been shown that the distribution and partitioning of TM is partially affected by the initial concentrations and speciation of TM from the effluent. Iron, Ni, and Cr, characteristic metals of the ultramafic rocks, have been found in concentrations much higher than world average in mangroves soil downstream those rocks, and those elements are mostly detected in oxide or oxy/hydroxide forms [START_REF] Marchand | The partitioning of transitional metals (Fe, Mn, Ni, Cr) in mangrove sediments downstream of a ferralitized ultramafic watershed (New Caledonia)[END_REF][START_REF] Noël | Ni cycling in mangrove sediments from New Caledonia[END_REF]. The mobility and bioavailability of these TM are mostly influenced by the vegetation [START_REF] Marchand | Structuration écologique et bilan des processus biogéochimiques au sein d'une mangrove 'atelier[END_REF], the OM content [START_REF] Marchand | Relationships between heavy metals distribution and organic matter cycling in mangrove sediments (Conception Bay, New Caledonia)[END_REF], and the redox conditions (Bourgeois et al., 2019a), which will affect the biogeochemical processes that take place in the soil and will consequently impact the speciation of the TM. The two predominant species are Rhizophora stylosa seaward and Avicennia marina landward [START_REF] Marchand | Typologies et biodiversité des mangroves de Nouvelle-Calédonie[END_REF]. The root systems of these two species are broadly different with distinct particularities. R. stylosa has extensive aerial stilt support roots [START_REF] Kathiresan | Biology of mangroves and mangrove ecosystems[END_REF] while A. marina possesses pneumatophores (roots growing upward) equipped of lenticels that allow the passive diffusion of oxygen [START_REF] Purnobasuki | Morphology of four root types and anatomy of rootroot junction in relation gas pathway of Avicennia marina (Forsk) Vierh roots[END_REF]. To our knowledge, only one study looked at the bioaccumulation of TM from soil to roots and shoots of mangroves in NC and was not interested in the functions of the Fe plaque in TM absorption [START_REF] Marchand | Trace metal geochemistry in mangrove sediments and their transfer to mangrove plants (New Caledonia)[END_REF]. Globally, the processes of TM exchange at the soil-vegetation interface are understudied worldwide, especially in situ.

In the context of mangrove developing downstream lateritic soils enriched in TM, our main objective was to assess the influence of watershed inputs on the accumulation of TM in the root systems of the two main mangrove species developing along the semi-arid coastline of New Caledonia. In addition, we were interested in the influence of elemental inputs and soil redox conditions on the formation of Fe plaque at the root surface of A. marina. Eventually, a potential relationship between Fe plaque concentrations and the amount of TM retained within the plaque was investigated. To achieve those objectives, concentrations of TM in the rhizosphere and root system of the two species as well as in the Fe plaque of A. marina were quantified by Optical Emission Spectrometry by Inductive Current Plasma (ICP-OES), and the roots were analyzed by Scanning Electron Microscopy (SEM). Two study sites, one ultrabasic and one non-ultrabasic, were chosen to evaluate the impact of TM-rich watersheds on TM distribution in mangroves downstream.

We hypothesize that due to the capacity of A. marina to diffuse O2 to its rhizosphere, thanks to a more permeable root system, the root bioconcentration of TM is higher in this species than in R. stylosa. We also expect that the accumulation of TM is not subject to a threshold and therefore, TM are found in higher concentrations in the roots when it is also the case in the soil.

Material & method

Study sites

New Caledonia is a French archipelago, located in the South Pacific Ocean, 1,500 km away from the East coast of Australia and enclosed by the largest lagoon in the world, registered at UNESCO since 2008 (UNESCO, 2020). The main island is about 500 km long and 50 km wide between 20°S and 23°S. Mangrove forest represents more than 35,000 hectares and 80% of the western coast of the main island is covered by this ecosystem [START_REF] Marchand | Typologies et biodiversité des mangroves de Nouvelle-Calédonie[END_REF]. The western coastline is subject to a semi-arid climate and to semidiurnal tidal cycle [START_REF] Douillet | A numerical model for fine suspended sediment transport in the southwest lagoon of New Caledonia[END_REF]. The intertidal zone of NC is the habitat of more than 20 mangrove species. Floristic diversity of mangrove forests is divided into monospecific stands depending on multiple factors such as frequency of tidal immersion and porewater salinity [START_REF] Duke | Factors influencing biodiversity and distributional gradients in mangroves[END_REF]. On the West coast of the main island, three distinct monospecific stands are observed depending on the elevation.

On the seashore, Rhizophora spp. cover 50% of mangrove area with porewater salinity ranging from 5 to 40 g L -1 . A. marina covers >15% of mangrove area and develops higher in the intertidal zone with salinity between 35 and 70 g L -1 . Eventually, salt-flat is the highest topographical stand of vegetation composed mainly of Salicornia, and flourishes on soil with salinity ranging from 60 to 100 g L -1 [START_REF] Marchand | Typologies et biodiversité des mangroves de Nouvelle-Calédonie[END_REF][START_REF] Marchand | Trace metal geochemistry in mangrove sediments and their transfer to mangrove plants (New Caledonia)[END_REF].

On the main island, ultramafic soil represents a third of the territory with alteration profiles particularly rich in Ni [START_REF] Dalvi | The past and the future of nickel laterites[END_REF][START_REF] Nicholson | Geochemistry and age of the Nouméa Basin lavas, New Caledonia: Evidence for Cretaceous subduction beneath the eastern Gondwana margin[END_REF]. Open-casts Ni mines are exploited all along the central mountain chain. Ultramafic soils, including those open-cast mines, are eroded during rainfall events, and part of it flows into nearby rivers. Two study sites were therefore chosen. The first one for lateritic inputs from its watershed and another one for the absence of known lateritic inputs.

The first study site is a mangrove located at the mouth of the Dumbéa River (22°172.797'S, 166°429.493'E), in the Gadji Bay (Fig. 1). 9 km upstream the river is an old Ni open-cast mine (22˚143.293'S, 166˚491.718'E) (Fig. 1) exploited from the 19 th to mid-20 th century but deserted since then [START_REF] Marchand | The partitioning of transitional metals (Fe, Mn, Ni, Cr) in mangrove sediments downstream of a ferralitized ultramafic watershed (New Caledonia)[END_REF]. The first site is named "ultrabasic site" thereafter. The second site is a mangrove located in the Apogoti bay, 3.5 km south of the mouth of the Dumbéa River (22°202.072'S, 166°439.939'E) (Fig. 1). It is named thereafter "non-ultrabasic site" since it is a volcano-sedimentary soil mainly made of quartz and plagioclase and its watershed does not include the Dumbéa River or ultramafic soils (Service de la Géologie de Nouvelle-Calédonie, 2016). The hydrography of the Gadji Bay is rather south to north and therefore the effluent from the Dumbéa River does not flow into the Apogoti Bay [START_REF] Douillet | A numerical model for fine suspended sediment transport in the southwest lagoon of New Caledonia[END_REF]. At both sites, the monospecific stands identified are homogeneous in terms of tree density and height.

Sampling and processing

Samples were collected in June 2020, during the short dry season. On both sites, stands of A. marina and R. stylosa stands were identified (Fig. 1). For each species, triplicates of 30 cm long soil cores were collected with an Eijkelkamp gouge auger about 20 m apart from each other (Fig. 1). Soil cores were cut along depth with a wooden knife into 5 sections of 6 cm each. This sampling strategy was chosen because we are studying the transfer of TM to the root system that develops in the 30 first centimeters and smaller steps in sections probably would not give further information [START_REF] Kathiresan | Biology of mangroves and mangrove ecosystems[END_REF]. The pH and redox potential (Eh) were immediately measured using a pH meter (pH3110 -WTW). A glass electrode (SENTIX -Xylem Analytics) was used to measure the pH, calibrated with standards prior to measurements, while a combined Pt and Ag/AgCl electrode (SENTIX -Xylem Analytics) was used to measure the Eh, calibrated with standards prior to measurements. The soil sections were then placed into tightly closed plastic bags. To extract porewater from the soil, a rhizon (Rhizon SMS -10 cm, OD 2.5 mm -Rhizosphere) was inserted into each soil section to which a 12 mL syringe was connected. The syringe was kept fully pulled with an adapted wooden block. The plastic bags with the soil and the attached syringes were immediately placed into a cooler (~4 °C) until processed at the laboratory less than 6 h after sampling. Within the same area where the soil cores were collected, coarse roots of R. stylosa were chopped with a saw with a segment out of the soil and a segment immersed in the soil. For A. marina, pneumatophores were gently teared from the main roots. All biotic samples were immediately placed into plastic bags and kept in a cooler (~4 °C) until processed at the laboratory less than 6 h after sampling.

Upon arrival at the laboratory, the porewaters were collected and salinity was measured using a refractometer (ATC). Porewater samples were filtered at 0.45 µm and 2 drops of 70% HNO3 were added before storage at 4 °C. The soil samples were tightly closed and kept in the freezer at -20 °C. Triplicate of pneumatophores at both sites were kept fresh for Fe plaque extraction (see 2.3.3) but the rest of the biotic samples were also placed in the freezer. Frozen soil samples were lyophilized 72 h (FreeZone -LABCONCO) before sieving at 2 mm and then ground with a ball mill (FRITSCH). The frozen roots and pneumatophores were washed with distilled water. Transversal sections were obtained using a proxxon (Dremmel 3000, SpeedClic SC544) and dried in a heat chamber at 40 °C until reaching constant mass. One dried section per root and per pneumatophore were kept intact in a desiccator for scanning electron microscopy (SEM). The other dried sections were ground using a cutting mill (POLYMIX -px-mfc90d) for TM analysis.

Analysis

Reagents

All reagents were analytical grade or better. Sodium bicarbonate and ammonium fluoride were obtained from Sigma Aldrich. Tri-sodium citrate dihydrate was obtained from AnalaR NORMAPUR, VWR. Sodium dithionite was obtained from Panreac. Concentrated nitric acid (70%) was obtained from Ajax Finechem.

Multi-element (Ca, Co, Cr, Cu, Fe, K, Mg, Mn, Ni, Na, Zn) standard solution for ICP (1000 mg L -1 in HNO3 2%) was obtained from CPAChem and Pb standard solution for ICP (1000 mg L -1 in HNO3 2%) from Perkin-Elmer. Certified reference materials (ISE 1729 sample ID 910 "Clay Soil" and IPE 1804 sample ID 198 "Banana") were obtained from the Wageningen Evaluating Programmes for Analytical Laboratories.

Iron plaque extraction

Triplicate of fresh pneumatophores for both sites were washed with distilled water and immerged sections were obtained using a proxxon. In order to extract the Fe plaque from the root surface, a treatment with a solution of dithionite-citrate-bicarbonate (DCB) established by [START_REF] Taylor | Use of the DCB technique for extraction of hydrous iron oxides from roots of wetland plants[END_REF] and modified by [START_REF] Lin | Seedlings influenced by sulfur and iron amendments[END_REF] was used. Briefly, 0.5 g of pneumatophore was immersed in 20 mL of the DCB solution (0.03 M Na3C6H5O7 •2H2O, 0.125 M NaHCO3, and 0.144 M Na2S2O4 in MilliQ water) for 3 h at room temperature. Samples were then rinsed with MilliQ water to obtain 25 mL of final volume. The extract was filtered at 0.45 µm and kept at 4 °C until analysis. Treated samples were dried in a heat chamber at 40 °C until constant mass, then ground using a cutting mill.

Trace metals extraction

Acid extraction elaborated by Bourgeois et al. (2019a) was used to extract TM from soil and biota.

Briefly, 100 mg of dried sample were weighed in a 15 mL polypropylene tube and 1 mL of 10% FNH4:HNO3 concentrated (w/v) for the soil samples and 1% FNH4:HNO3 concentrated (w/v) for biotic samples was added. Sample was vortexed and let at room temperature overnight. The sample was then heated in a sand bath for 6 h at 100 °C. Once cooled down, the volume was adjusted to 10 mL with MilliQ water and the sample was vortexed. After centrifugation at 3,000 rpm for 5 min, the supernatant was transferred to a new tube and kept at 4 °C until analysis. For quality control, certified materials (ISE sample ID 910 and IPE sample ID 198) were also extracted and analyzed. Results can be exploited but questionable. In biotic extracts, the z-score's absolute value is inferior to 2 for all TM except for Co that has a score between 2 and 3 (SM 1). Due to matrix interferences, Cu and Pb were not measured in the DCB extracts.

Trace metals analysis

Total organic carbon

The percentage of total organic carbon (TOC) in the soil was obtained via a TOC analyzer (TOC-LCPH-SSM500A -Shimadzu Corporation).

Scanning electron microscopy SEM

Semi-quantitative analysis of dried transversal sections of roots and pneumatophores was performed by Scanning Electron Microscopy (SEM) using a JEOL JSM-IT 300 LV apparatus coupled with an Energy Dispersive Spectroscopy (EDX) Oxford CAM 80 device at the Electronic Microscopy Platform (P2M) at the University of New Caledonia. Before analysis, because of the electric insulator nature of the samples, they were coated with a 4 nm Pt layer using a Leica EM ac600 vacuum evaporator to avoid electrons accumulation on the surface. Coated biotic sections and dried ground soil samples were then glued to aluminum stubs with conductive carbon tape before observation.

Mineralogical analysis

Mineralogical composition of soil samples was determined by X-ray powder diffraction (XRD) (PANalytical -AERIS XRD Diffractometer) with a Co source at the ISEA laboratory. A rock sample originated from the watershed of the non-ultrabasic site and sampled nearby the mangrove site was also analyzed by XRD. Scans were taken between 5 and 80 °2θ with a generator power of 600 W. Spectra were treated and analyzed with the High Score software.

Data analysis

Iron plaque calculations

Iron plaque concentration was calculated using the following equation [START_REF] Lin | Seedlings influenced by sulfur and iron amendments[END_REF]:

[Fe plaque]= 0.1591 × (mg) (kg) (1)
To obtain the mass percentages of TM retained in the Fe plaque, the following equation was used:

% = mg mg + mg ×100
(2)

Bioconcentration factor

The bioconcentration factor (BCF) was calculated by dividing the concentration of TM in roots by the concentration of TM in the soil in the section between 6 and 12 cm deep that corresponds to the depth at which roots were collected. The collection of roots between 6 and 12 cm validates our sampling strategy of 30 cm soil cores.

b). Concentration of dissolved Fe along the vertical gradient is positively correlated to the pH (0.66) and negatively correlated to the Eh (-0.91) under R. stylosa at the ultrabasic site (SM 2).

For both mangrove sites and in the watershed rock of the non-ultrabasic site, the main identified mineralogical phase quartz (SiO2) (Fig. 3). Plagioclase (NaAlSi3O8 / CaAl2Si2O8) and illite (KAl4Si2O12)

were identified for the non-ultrabasic site and the watershed rock of this site (Fig. 3c-e). For this rock sample, vermiculite ((Mg,Ca)0,7(Mg,Fe,Al)6(Al,Si)8O22(OH)4) and enstatite (Mg,Fe)SiO3, two Fe-bearing phases, were named (Fig. 3e) while talc (Mg3Si4O10(OH)2) and chlorite ((Fe,Mg,Al)6(Si,Al)4O10(OH)8) were identified in the mangrove soil of the non-ultrabasic site (Fig. 3c,d). The neoformed pyrite crystals (FeS2)

were detected at both mangrove sites (Fig. 3a-d). For the ultrabasic site, three Fe-bearing mineralogical phases were identified: the oxyhydroxide goethite (α-FeOOH) and the phyllosilicates antigorite ((Mg,Fe)3Si2O5(OH)4) and clinochlore ((Mg,Fe)5Al(Si3Al)O10(OH)8).

For the non-ultrabasic site, all identified mineralogical phases are present along the cores (Fig. 3c,d).

However, for the ultrabasic site, goethite and clinochlore are mainly detected close to the surface and tend to vanish as depth increases. Pyrite is present in the sections lacking those two Fe-bearing phases (Fig. 3a,b).

Trace metals distribution in soil

For each soil core, all TM except for Pb and Zn are significantly more concentrated in the soil at the ultrabasic site (SM 3). As shown in the supplementary materials (SM 4), no significant differences in TM concentrations in porewaters between species have been detected.

Sections of roots and pneumatophores were collected between 6 and 12 cm deep and the concentrations of TM in the soil for this section are presented in Table 1. In the soil between 6 and 12 cm deep, all TM are significantly more concentrated at the ultrabasic site. Iron, Ni, and Cr are the three most concentrated TM in the soil at both sites with mean concentrations of 176,991 mg kg -1 , 3,499 mg kg -1 , and 2,294 mg kg -1 , respectively, at the ultrabasic site, and 38,828 mg kg -1 , 560 mg kg -1 , and 460 mg kg -1 , respectively, at the non-ultrabasic site. Under both species and at both sites, Hg is the least concentrated TM with a mean values of 0.063 mg kg -1 at the ultrabasic site and to 0.046 mg kg -1 at the non-ultrabasic site followed by Pb with mean values of 20 mg kg -1 at the ultrabasic site and 11 mg kg -1 at the non-ultrabasic site.

Bioaccumulation of trace metals from soil to roots

Iron is the most concentrated TM in the roots of both species (16,375 mg kg -1 for A. marina and 2,928 mg kg -1 for R. stylosa) with Mn (301 mg kg -1 for A. marina and 82 mg kg -1 for R. stylosa) and Ni (164 mg kg -1 for A. marina and 102 mg kg -1 for R. stylosa) the 2 nd and 3 rd most concentrated TM (Table 1).

However, Fe and Ni, as well as Co, Cr, and Hg have low BCF (0.16, 0.11, 0.16, 0.07, and 0.11, respectively) (Fig. 4a). Manganese, Cu, and Zn have higher BCF (0.93, 0.42, 0.26), and Pb is the TM with the highest BCF (1.60) (Fig. 4a).

The ratio of TM concentrations in porewaters over concentrations in soil samples (dissolved/solid ratio) are low for Fe (0.14), Ni (0.27), and Cr (0.08), while high for Mn (3.34), Cu (1.86), and Zn (3.03) (Fig. 4b).

The dissolved/solid ratio of Co is high (2.81) while that of Pb is low (0.40) (Fig. 4b).

Chromium, Cu, Fe, Pb, and Zn are significantly more concentrated in the pneumatophores of A. marina than in the roots of R. stylosa (Table 1). Cobalt, Hg, Ni, and Mn are also more concentrated in pneumatophores but not significantly (p-value > 0.05) (Table 1). The BCF of A. marina is always greater than that of R. stylosa (Fig. 4a), which is not always the case regarding TM dissolved/solid ratios (Fig. 4b).

Iron plaque and associated immobilized trace metals

Variabilities are high but the concentration of the Fe plaque at the surface of A. marina's pneumatophores is higher at the ultrabasic site (4,132 mg kg -1 ) compared to the non-ultrabasic site (1,863 mg kg -1 ) (Fig. 5a). However, the percentages of TM retained in the plaque is not site-dependent with slight differences between the ultrabasic site and the non-ultrabasic site (Fig. 5b). Mass percentages of TM contained in the Fe plaque vary between 47% and 94% (Fig. 5b). Nickel is the TM the least immobilized by the Fe plaque with mean mass concentration of 51% while Co and Mn are the most immobilized with mean values of 85% and 92%, respectively (Fig. 5b).

Scanning electron microscopy -energy dispersive X-ray spectroscopy

Roots of R. stylosa and pneumatophores of A. marina were analyzed by SEM. Figure 6 shows a cross section of a pneumatophore of A. marina, cut into two images, which allows scanning of the root from the vascular bundle to the epidermis. EDX elemental spectra associated to the four different parts of the root are also displayed. From all roots observed, Fe is the only TM that has been detected by the EDX apparatus on biotic samples, and seem to be localized in the epidermis. The vascular bundle, inner, and outer cortical cells seem to be free of TM according to SEM-EDX analysis. Interestingly, aggregates of framboidal pyrite like that can be seen in the soil were observed in the epidermis of some pneumatophores of A. marina (Fig. 7a)

and were identified via elemental mapping using EDX (Fig. 7b).

Discussion

Influence of watersheds inputs on mangrove soil conditions

At the non-ultrabasic site, the highly negative Eh values associated to the low Fe concentrations in the dissolved phase indicate that the soil is anoxic. The presence of pyrite under both species but not in the watershed rock implies that sulfate reduction processes occur in the soil. Conversely, at the ultrabasic site, less negative Eh values and higher concentrations of dissolved Fe in the upper soil layers (Fig. 2a,b) suggest suboxic conditions, which most probably result from continuous goethite inputs from upstream lateritic soils allowing Fe reduction. The latter process and goethite dissolution led to the release of Fe in porewaters, which can be further precipitated as pyrite during sulfate-reduction processes in the deeper anoxic layers [START_REF] Marchand | Distribution and characteristics of dissolved organic matter in mangrove sediment pore waters along the coastline of French Guiana[END_REF][START_REF] Kristensen | Biogeochemical cycles: global approaches and perspectives[END_REF].

Larger organic carbon accumulation was measured at the non-ultrabasic site. Considering that mangrove characteristics (zonation, tree density, height) were similar between the two sites, we suggest that different mineralization processes and rates may explain this trend. In fact, the suboxic conditions that prevail at the ultrabasic site due to the presence of large amount of goethite may result in more efficient decay processes than the anoxic non-ultrabasic site.

At both sites, pH is significantly more acidic under R. stylosa than under A. marina (Fig. 2). In mangrove soil, OM mineralization and sulfide oxidation are the two main processes that result in H + release and thus in soil acidification [START_REF] Clark | Redox stratification and heavy metal partitioning in Avicennia-dominated mangrove sediments: a geochemical model[END_REF]. R. stylosa is closer to the shore and more influenced by daily tidal fluctuations. Inputs of electron acceptors is greater for OM mineralization or sulfides oxidation under this species leading to a more acidic soil (Bourgeois et al., 2019a). The pH is not significantly different between sites under A. marina and is significantly more acidic at the non-ultrabasic site under R. stylosa even though we suggest mineralization rates are slower at this latter site (Fig. 2). The soil anoxia at the nonultrabasic site may indicate a greater amount of reduced sulfide in its soil compared to the suboxic ultrabasic-site. This leads to more oxidation reactions of sulfides under R. stylosa, which contributes to the soil acidification [START_REF] Noël | EXAFS analysis of iron cycling in mangrove sediments downstream a lateritized ultramafic watershed (Vavouto Bay, New Caledonia)[END_REF].

The geology of the watershed affects the mineralogy of mangrove soil and thus the conditions (TOC, Eh, pH…) as well as the elemental composition. Chemical interactions between different ligands in the soil and TM determine the speciation they take in the system. Those forms control the mobility, the bioavailability, and the toxicity of these TM [START_REF] Chakraborty | Partitioning of metals in different binding phases of tropical estuarine sediments: importance of metal chemistry[END_REF].

Concentrations of trace metals in soil driven by watersheds inputs

At the ultrabasic site, the higher mean concentrations of the analyzed TM in the soil can be explained by the difference in the geology of the watersheds (Table 1). The ultrabasic-site's watershed is composed mostly of laterites rich in goethite and other phases that contain TM such as antigorite (Fig. 3a,b) [START_REF] Baltzer | La sédimentation et la diagenèse précoce sur les côtes à mangrove en aval des massifs ultrabasiques en Nouvelle-Calédonie[END_REF][START_REF] Vithanage | Occurrence and cycling of trace elements in ultramafic soils and their impacts on human health: A critical review[END_REF]. Furthermore, the erosion and leaching of the old Ni mine located upstream the Dumbéa River contributes to the input of Ni and other TM such as Co, Cr, and Mn [START_REF] Fandeur | XANES Evidence for oxidation of Cr(III) to Cr(VI) by Mn-oxides in a lateritic regolith developed on serpentinized ultramafic rocks of New Caledonia[END_REF][START_REF] Metian | Trace element bioaccumulation in reef fish from New Caledonia: Influence of trophic groups and risk assessment for consumers[END_REF][START_REF] Gwenzi | Occurrence, behaviour, and human exposure pathways and health risks of toxic geogenic contaminants in serpentinitic ultramafic geological environments (SUGEs): A medical geology perspective[END_REF]. At the non-ultrabasic site, the volcano-sedimentary soil mainly composed of quartz and plagioclase (Fig. 3c,d) contributes less importantly to analyzed TM inputs to the mangrove soil. Nevertheless, two Fe-bearing phases were identified in the watershed rock: vermiculite and enstatite (Fig. 3e). Enstatite was previously identified in the Dumbéa watershed and can also be a Ni-bearing phase [START_REF] Trescases | L'évolution géochimique supergène des roches ultrabasiques en zone tropicale: formation des gisements nickélifères de Nouvelle-Calédonie[END_REF][START_REF] Baltzer | La sédimentation et la diagenèse précoce sur les côtes à mangrove en aval des massifs ultrabasiques en Nouvelle-Calédonie[END_REF]. In the conditions that prevail in our study sites, medium to important rainfall and slightly acidic soil with daily basic sea water influx, enstatite is not stable [START_REF] Oelkers | An experimental study of enstatite dissolution rates as a function of pH, temperature, and aqueous Mg and Si concentration, and the mechanism of pyroxene/pyroxenoid dissolution[END_REF]Schott, 2001, Halder and[START_REF] Halder | Far from equilibrium enstatite dissolution rates in alkaline solutions at earth surface conditions[END_REF]. The alteration products of enstatite are talc and chlorite, which were identified in the mangrove soils, with the latter potentially bearing Fe and Ni as well (Fig. 3c,d) [START_REF] Trescases | L'évolution géochimique supergène des roches ultrabasiques en zone tropicale: formation des gisements nickélifères de Nouvelle-Calédonie[END_REF]. Illite can also potentially contribute to Fe input or storage [START_REF] Xie | Soluble Fe release from iron-bearing clay mineral particles in acid environment and their oxidative potential[END_REF].

TM concentrations in the soil of the ultrabasic site are close to those measured by Marchand et al.

(2016) on the same site in 2012 with Fe, Ni, and Cr the three most concentrated in descending order. Iron and Ni concentrations in soil are much higher than world average. Here, Fe represents as high as 18% in mass of the soil, whereas average values in mangroves vary between 0.02 and 6% with a mean value around 1-2% [START_REF] Alagarsamy | Distribution and seasonal variation of trace metals in surface sediments of the Mandovi estuary, west coast of India[END_REF][START_REF] Chatterjee | Distribution and possible source of trace elements in the sediment cores of a tropical macrotidal estuary and their ecotoxicological significance[END_REF][START_REF] Kruitwagen | Status of pollution in mangrove ecosystems along the coast of Tanzania[END_REF]. In the present study, Ni values as high as 4,000 mg kg -1 were measured (Table 1) downstream the Dumbéa River while literature review exposed maximum values of Ni in mangroves soil of 200 mg kg -1 [START_REF] Bayen | Occurrence, bioavailability and toxic effects of trace metals and organic contaminants in mangrove ecosystems: A review[END_REF]. Even though, the concentrations of TM in the soil at the non-ultrabasic are lower than those of the ultrabasic site, relatively high values of Fe and Ni were still measured when compared to world average: 680 mg kg -1 for Ni and 4% in mass of the soil for Fe (Table 1).This can be explained by the high background noise of TM in New-Caledonian soils and marine sediments, mainly Fe, Ni, Cr, and Mn [START_REF] Beliaeff | Guide pour le suivi de la qualité du milieu marin en Nouvelle-Calédonie[END_REF]. Natural or anthropogenic-driven erosion of nearby soils in addition to wild and man-made effluents driven by the currents contribute to TM accumulation in the soil of the non-ultrabasic site. This mangrove is located in an urbanized area and models of current show possible inputs from urban discharged as well as an influence from a containment site located 2.5 km south-east of the studied mangrove [START_REF] Douillet | Atlas hydrodynamique du lagon Sud-Ouest de Nouvelle-Calédonie[END_REF].

TM concentrations in the soil are therefore dependent on the watershed inputs with the leaching of laterized soil the major factor causing the ultrabasic site to be richer in TM than the other site. We hypothesize that the bioaccumulation of TM in the roots of both studied mangrove species is partly dependent on other factors than TM concentrations in the soil such as the nature of the TM (essential or nonessential).

Bioaccumulation factors differ between trace metals and are independent of the watershed inputs

Iron and Ni concentrations in mangrove roots at both sites are relatively high (Table 1). Iron concentrations obtained in tissues for the ultrabasic site are as high as values reported in Marchand et al.

(2016) in the same mangrove around 10,000 mg kg -1 . Measured Fe concentrations at both sites are much higher than those measured in roots of mangroves in India with a maximum value of 938 mg kg -1 [START_REF] Chowdhury | Accumulation of trace metals by mangrove plants in Indian sundarban wetland: Prospects for phytoremediation[END_REF]. We measured Ni mean values of 164 mg kg -1 in A. marina's pneumatophores, which is greater than the maximum values reported in literature of 100 mg kg -1 [START_REF] Lewis | Fate and effects of anthropogenic chemicals in mangrove ecosystems: A review[END_REF][START_REF] Bayen | Occurrence, bioavailability and toxic effects of trace metals and organic contaminants in mangrove ecosystems: A review[END_REF]. However, the BCF of these two TM are low (Fig. 4a) with the dissolved fractions minor in comparison to the solid fractions in the soil (Fig. 4b). [START_REF] Chowdhury | Bioremoval of trace metals from rhizosediment by mangrove plants in Indian Sundarban Wetland[END_REF] estimated that 95% of Fe present in soil of mangrove forests is not bioavailable for the biota since most of it is associated to sulfides.

Therefore, even though Fe and Ni are essential metals, their absorption by the plant may be limited due to a lack of ions in the dissolved phase, which is also the case for the non-essential metal Cr that is usually one of the least bioavailable TM in the soil as it was observed here (Fig. 4) [START_REF] Zhu | Factors influencing the heavy metal bioaccessibility in soils were site dependent from different geographical locations[END_REF]. In the previous study done at the ultrabasic site, Fe and Ni were the most concentrated TM in roots and leaves of A. marina and R.

stylosa but with low bioconcentration and translocation factors as well, partly due to biological barriers that were not explored in the study [START_REF] Marchand | Trace metal geochemistry in mangrove sediments and their transfer to mangrove plants (New Caledonia)[END_REF]. The high concentrations measured in the roots, especially Fe and Ni, despite low BCF, are explained by the very high concentrations of those TM in the soil at both sites as exposed previously in the discussion.

A low BCF was also calculated for Co at both sites but with a high dissolved/solid ratio in soil (Fig. 4), confirming the observation made by [START_REF] Marchand | Trace metal geochemistry in mangrove sediments and their transfer to mangrove plants (New Caledonia)[END_REF] at the ultrabasic site. A higher dissolved fraction potentially indicates more ions in the bioavailable form. This result indicates that the uptake of Co by mangroves must be regulated at the biological level rather than at the physico-chemical level. Cobalt is an essential metal since it is a component of many enzymes and co-enzymes and particularly participates to plant growth and CO2 assimilation [START_REF] Nagajyoti | Heavy metals, occurrence and toxicity for plants: a review[END_REF][START_REF] Reece | Plant structure, growth, and development[END_REF]. However, some publications acknowledge Co as a non-essential metal for mangroves (Bourgeois et al., 2019b[START_REF] Nath | Assessment of biotic response to heavy metal contamination in Avicennia marina mangrove ecosystems in Sydney Estuary, Australia[END_REF]. It is therefore possible that only small quantities of Co are required by the plant, and for the rest of it, mangroves regulate Co uptake through biological mechanisms such as regulation at the root surface with the epidermis acting as a barrier [START_REF] Macfarlane | Accumulation and partitioning of heavy metals in mangroves: A synthesis of field-based studies[END_REF].

The non-essential metal Pb, weakly present in the dissolved phase compared to the solid phase in the soil (Fig. 4b), is highly bioaccumulated in the root systems at both sites (Fig. 4a). The maximum value of 39 mg kg -1 is still much less than the alarming values obtained in roots of the same species worldwide reaching up to more than 200 mg kg -1 [START_REF] Macfarlane | Accumulation and partitioning of heavy metals in mangroves: A synthesis of field-based studies[END_REF][START_REF] Lewis | Fate and effects of anthropogenic chemicals in mangrove ecosystems: A review[END_REF][START_REF] Bayen | Occurrence, bioavailability and toxic effects of trace metals and organic contaminants in mangrove ecosystems: A review[END_REF]. Lead is usually easily mobilized in the soil [START_REF] Zhu | Factors influencing the heavy metal bioaccessibility in soils were site dependent from different geographical locations[END_REF][START_REF] Yan | Accumulation and tolerance of mangroves to heavy metals: a review[END_REF], and has a high chelation affinity with roots cell walls (associated to carbohydrates) [START_REF] Turner | The accumulation of zinc by subcellular fractions of roots of Agrostis Tenuis sibth. in relation to zinc tolerance[END_REF]Marshall, 1972, Verkleij and[START_REF] Verkleij | Mechanisms of metal tolerance in higher plants, in, Heavy metal tolerance in plants -evolutionary aspects[END_REF]. In A.

marina, it was observed that Pb is largely accumulated as immobile, bound to cell walls, and sequestered in the roots mainly in the epidermal layers (MacFarlane andBurchett, 2000, 2002). This implies that even though not much Pb is present in the dissolved phase, the plant absorbs a large proportion of it. For A.

marina, BCF values greater than 1 were also described for Pb in literature [START_REF] Nath | Assessment of biotic response to heavy metal contamination in Avicennia marina mangrove ecosystems in Sydney Estuary, Australia[END_REF].

Regarding the essential metals Cu, Mn, and Zn, their uptakes by mangroves may not be regulated.

Relatively high concentrations of those TM in the dissolved phase compared to the solid phase (Fig. 4b) imply that they are potentially more bioavailable and therefore are better absorbed by the roots of both species (high to moderate BCF) (Fig. 4a). The BCF values obtained for Mn in A. marina are closed to those exposed in literature at both sites [START_REF] Nath | Assessment of biotic response to heavy metal contamination in Avicennia marina mangrove ecosystems in Sydney Estuary, Australia[END_REF][START_REF] Yadav | Effect of heavy metals on the carbon and nitrogen ratio in Avicennia marina from polluted and unpolluted regions[END_REF]. [START_REF] Marchand | Trace metal geochemistry in mangrove sediments and their transfer to mangrove plants (New Caledonia)[END_REF] also exposed a greater mobility and bioavailability of Zn and Cu compared to Fe and Ni at the ultrabasic site. Copper is important in ATP synthesis and plays an essential role in the respiratory electron transport chain [START_REF] Demirevska-Kepova | Biochemical changes in barley plants after excessive supply of copper and manganese[END_REF]. Zinc is the second most abundant TM in plants after Fe and the only metal present in the 6 enzymes classes [START_REF] Broadley | Zinc in plants[END_REF]. Copper and Zn also have a high chelation affinity with the cell walls of mangroves roots with Cu preferentially chelated with amino acids and Zn with organic acids [START_REF] Verkleij | Mechanisms of metal tolerance in higher plants, in, Heavy metal tolerance in plants -evolutionary aspects[END_REF]Schat, 1990, MacFarlane et al., 2007).

High values of some TM in roots, particularly Fe and Ni, are therefore due to high concentrations of TM in the soil at both sites. TM are not similarly bioconcentrated in the roots, with Pb the most transferred from soil to roots and Cr the least. The BCF of TM is not site-dependent indicating that the watershed inputs do not influence the uptake of TM by the plants. TM mobility and biological and/or physiological factors do affect TM transfer from soil to roots.

Trace metals bioaccumulation in roots is species-dependent

In this study, results evidenced the greater TM uptake in roots of A. marina compared to R. stylosa (Table 1). One explanation is that roots of R. stylosa are weakly permeable due to a thickening mechanism and exodermis cell lignification facing metallic stress that act as a physical barrier to TM uptake [START_REF] Cheng | Interactions among Fe2+, S2-, and Zn2+ tolerance, root anatomy, and radial oxygen loss in mangrove plants[END_REF][START_REF] Cheng | Metal (Pb, Zn and Cu) uptake and tolerance by mangroves in relation to root anatomy and lignification/suberization[END_REF]. A. marina, with more permeable pneumatophores, is more subject to TM absorption through the root system. We focused on pneumatophores rather than on main roots. One hypothesis is that TM are accumulated in pneumatophores as a mechanism to limit metallic stress in the central root system [START_REF] Macfarlane | Accumulation and partitioning of heavy metals in mangroves: A synthesis of field-based studies[END_REF]. Since A. marina diffuses oxygen in its rhizosphere, oxidation of reduced TM can occur, which mobilizes the TM that become bioavailable for direct absorption by the plant [START_REF] Thakur | Plant-driven removal of heavy metals from soil: uptake, translocation, tolerance mechanism, challenges, and future perspectives[END_REF]. [START_REF] Macfarlane | Accumulation and partitioning of heavy metals in mangroves: A synthesis of field-based studies[END_REF] showed a trend in higher root BCF for species that are less frequently inundated, which results in less anoxic soil (i.e. Kandelia spp.) and species with pneumatophores (i.e.

Avicennia spp.). The difference in BCF between the two species can also result from different metabolisms and therefore different physiological needs [START_REF] Yan | Accumulation and tolerance of mangroves to heavy metals: a review[END_REF]. Also, A. marina is able to excrete some TM in excess through the salt glands on its leaves as it was observed for Cu and Zn [START_REF] Naidoo | Ecophysiological responses of the mangrove Avicennia marina to trace metal contamination, Flora -Morphology, Distribution[END_REF]. In the review of [START_REF] Macfarlane | Accumulation and partitioning of heavy metals in mangroves: A synthesis of field-based studies[END_REF] on TM accumulation in mangroves, high variability of Cu, Pb, and Zn for each species were displayed but the means BCF of reviewed literature confirm the higher capacity of A.

marina to absorb TM through its roots. The Rhizophora genre has already been characterized in the literature as a low TM-accumulating species [START_REF] Peters | Ecotoxicology of tropical marine ecosystems[END_REF]. Consequently, there are differences in TM bioconcentration between species due to factors expressed above and different mechanisms are used by species in order to limit metallic stress such as the formation of Fe plaque, which is the case for A. marina.

Role of the iron plaque against metallic stress

The mean concentration of Fe plaque is two times higher on Avicennia's pneumatophores at the ultrabasic site. However, the high variability results in the absence of a significant difference (Fig. 5a). The variability can be explained by the fact that the Fe plaque was extracted from multiple pneumatophores in the same area. The age, length, and tree belonging can influence Fe plaque formation capacity. Higher mean Fe plaque formation is consistent with the higher concentrations of Fe obtained in the solid and dissolved phases of the soil at the ultrabasic site. More Fe in the soil, if in reduced form, enable more reactions between Fe 2+ and O2 to form the plaque. Multiple studies showed positive relationship between Fe plaque concentration on roots surface and reduced Fe in the soil [START_REF] Christensen | Formation of root plaques and their influence on tissue phosphorus content in Lobelia dortmanna[END_REF][START_REF] Pi | Effects of wastewater discharge on formation of Fe plaque on root surface and radial oxygen loss of mangrove roots[END_REF], 2011). Pi et al.

(2011) even showed that Fe plaque formation does not reach a threshold of Fe concentration in the soil since plaque formation occurs until the mangrove's death. Sequential extractions are though required in order to get the proportion of Fe in the reduced form to confirm what was presented in literature.

Iron plaque formation is extensive on the pneumatophores at both sites since more than 40% of Fe extracted from the pneumatophores come from the Fe plaque. Available Fe 2+ ions are phytotoxic for mangroves as they can cause DNA damages and a reduction of photosynthesis [START_REF] Nagajyoti | Heavy metals, occurrence and toxicity for plants: a review[END_REF].

Through radial oxygen loss (ROL) via pneumatophores, the formation of Fe plaque is an efficient mechanism to reduce Fe 2+ concentrations and uptake through the root system [START_REF] Youssef | Anatomical adaptive strategies to flooding and rhizosphere oxidation in mangrove seedlings[END_REF].

Our results suggest that ROL is significant enough to ensure important Fe 2+ oxidation in a mangrove with large concentrations of dissolved Fe.

Cobalt, Cr, Mn, Ni and Zn are retained in significant quantities in the Fe plaque at both sites (> 47%) (Fig. 5b). Iron oxides and hydroxides have a strong TM adsorption capacity due to their large specific surface area (> 200 m² g -1 ) [START_REF] Lin | Seedlings influenced by sulfur and iron amendments[END_REF]. Several authors reported the significant role of Fe plaque against metallic stress, mainly by trapping TM [START_REF] Taylor | Use of the DCB technique for extraction of hydrous iron oxides from roots of wetland plants[END_REF][START_REF] Machado | Trace metals in mangrove seedlings: role of iron plaque formation[END_REF][START_REF] Pi | Formation of iron plaque on mangrove roots receiving wastewater and its role in immobilization of wastewater-borne pollutants[END_REF][START_REF] Tripathi | Roles for root iron plaque in sequestration and uptake of heavy metals and metalloids in aquatic and wetland plants[END_REF]. The calculated percentages of TM immobilized in the plaque of A. marina are consistent with previous studies and show the significance of the Fe plaque against metallic stress for this species [START_REF] Machado | Trace metals in mangrove seedlings: role of iron plaque formation[END_REF][START_REF] Pi | Formation of iron plaque on mangrove roots receiving wastewater and its role in immobilization of wastewater-borne pollutants[END_REF][START_REF] Lin | Seedlings influenced by sulfur and iron amendments[END_REF]. For example in Brazil, more than 60% of immobilization on the surface of an Avicennia species, and between 25 and 60%, was observed for Mn and Zn, respectively [START_REF] Fonseca | Biogeoquímica de metais pesados na rizosfera de plantas de um manguezal do Rio de Janeiro[END_REF]. In a study concerning the role of Fe plaque on three mangroves species (Bruguiera gymnorrhiza, Excoecaria agallocha, and Acanthus ilicifolius), authors described a mean immobilization of TM in the following order: Mn > Ni > Zn > Cr [START_REF] Pi | Formation of iron plaque on mangrove roots receiving wastewater and its role in immobilization of wastewater-borne pollutants[END_REF]. These results are slightly different from what we observed here since Ni is the least trapped TM but not significantly compared to Zn and Cr (Fig. 5b). However, in this previous study by [START_REF] Pi | Formation of iron plaque on mangrove roots receiving wastewater and its role in immobilization of wastewater-borne pollutants[END_REF], TM immobilization order was speciesdependent as multiple studies exposed previously [START_REF] Tripathi | Roles for root iron plaque in sequestration and uptake of heavy metals and metalloids in aquatic and wetland plants[END_REF]. In addition, the experiment was set up in a laboratory in controlled conditions that are not always representative of in situ conditions, which has an influence on TM speciation.

More Fe plaque was measured on pneumatophores at the ultrabasic site; however, no significant difference between sites in percentages of TM retained in the plaque was observed (Fig. 5b). Two groups of TM can be identified from this observation; one composed of Co, Mn, and Ni, and the other one of Cr and Zn. Larger concentrations of Co, Mn, and Ni are retained in the plaque at the ultrabasic site, where there is more of these TM in the soil compared to the non-ultrabasic site. There are also larger concentrations of these TM in the DCB-treated pneumatophores (pneumatophores without Fe plaque). In this case, a greater volume of Fe plaque equals a greater volume of TM immobilized; however, in terms of percentage of immobilization (capacity of the plaque to immobilize TM), it is equivalent at both sites. This scenario is consistent with previous findings that show a positive correlation between Fe plaque concentrations and TM immobilization [START_REF] Pi | Formation of iron plaque on mangrove roots receiving wastewater and its role in immobilization of wastewater-borne pollutants[END_REF][START_REF] Tripathi | Roles for root iron plaque in sequestration and uptake of heavy metals and metalloids in aquatic and wetland plants[END_REF]. For Cr and Zn, the concentrations of TM in the treated pneumatophores and in the DCB extract (Fe plaque), are similar between both sites. Therefore, there is no positive relationship between Fe plaque concentration and TM immobilization. This observation may indicate a threshold of concentration of TM that can be retained in the plaque. Some studies highlighted the ability of the Fe plaque of the rice species Oryza sativa to trap TM such as Cr and Cd from the environment without affecting uptake and translocation of the TM [START_REF] Liu | Influence of iron plaque on uptake and accumulation of Cd by rice (Oryza sativa L.) seedlings grown in soil[END_REF][START_REF] Hu | Influence of iron plaque on chromium accumulation and translocation in three rice (Oryza sativa L.) cultivars grown in solution culture[END_REF]. In the present study, this means that Fe plaque concentration does not impact the amount of Cr and Zn that will be bioaccumulated in the pneumatophores. Fe plaque has never been previously studied in New Caledonian mangroves and further research must be conducted to confirm or disprove the results obtained.

Iron and pyrite accumulation within roots epidermis

Iron plaque formation is an efficient mechanism for A. marina to limit metallic stress by trapping TM and limiting their uptake by the roots as discussed earlier. Nevertheless, a fraction of TM passes through this physical barrier and can accumulate in the roots or be transferred to other plant organs. The amount of TM in the roots depend on the TM considered, its concentration in the soil, its mobility, the redox conditions and the mangrove species. Using SEM-EDX analysis, we looked for possible zones of accumulation of TM in the roots of both species. Fe was the only TM observed in roots of mangroves (Fig. 6). Iron is the most concentrated TM in roots with mean values between 2,486 and 17,125 mg kg -1 , which is between 15 and more than 1,500 times greater than the other TM (except Hg) (Table 1). The detection limit of the apparatus must restrain the observation of TM other than Fe.

Iron has only been detected in the epidermis of the roots of both A. marina and R. stylosa (Fig. 6). The epidermis is a barrier against physical damages and pathogens. The epidermis limits water loss and is the first line for water and nutrients absorption path by the roots [START_REF] De Granville | Aperçu sur la structure des pneumatophores de deux espèces des sols hydromorphes en Guyane[END_REF][START_REF] Reece | Plant structure, growth, and development[END_REF]. Iron is preferentially accumulated in the root system limiting transport to higher organs, which is why it will be more concentrated on, or in, the epidermis rather than in the cortex or vascular cylinder [START_REF] Machado | Trace metals in mangrove seedlings: role of iron plaque formation[END_REF]. Free Fe is highly toxic for plants and is therefore rarely observed on its own in tissues. Iron, mostly in its reduced form Fe 2+ , is bound to chelators and chaperones or present as Fe sequestering proteins such as ferritin and frataxin. Iron is essential for the synthesis of Fe-S clusters, fundamental in the structure of Fe-S proteins such as ferredoxins that intervenes in electron transfer in many metabolic reactions [START_REF] Morrissey | Iron uptake and transport in plants: the good, the bad, and the ionome[END_REF]. With the EDX elemental maps of mangroves roots, we observed clusters of Fe linked to S in the epidermis of A. marina's pneumatophores (Fig. 7b). However, the clusters were identified as framboidal pyrite. The sphere of FeS2 visible in Figure 7b seems attached to the cell walls. A hypothesis is that pyrite formed within the root from organic sulfur. Plants naturally fix sulfides in multiple forms (carbonates, amino acids, esters, polysaccharides, …) that can then produce H2S and will precipitate within the vascular tissues if Fe is present [START_REF] Vallentyne | Isolation of pyrite spherules from recent sediments[END_REF][START_REF] Altschuler | Sulfur diagenesis in everglades peat and origin of pyrite in coal[END_REF]. A suggested scenario is that pyrite formation within the pneumatophores of A. marina is a mechanism to limit Fe, and other chalcophile TM, transfer to higher organs. Pyrite crystals, framboidal and in lines, were previously observed in vascular channels of roots of Spartina alterniflora, a salt-marsh cordgrass of the East coast of the United States [START_REF] Giblin | Pyrite formation in marshes during early diagenesis[END_REF]. Framboids were also identified in the root vascular tissues of mangroves in the Everglades in Florida [START_REF] Altschuler | Sulfur diagenesis in everglades peat and origin of pyrite in coal[END_REF]). The explanation mentioned by [START_REF] Altschuler | Sulfur diagenesis in everglades peat and origin of pyrite in coal[END_REF] and [START_REF] Giblin | Pyrite formation in marshes during early diagenesis[END_REF] is that in-roots pyrite formation happens when the main source of sulfide within the soil is organic sulfide, which is not the case in intertidal ecosystems. To our knowledge, observation of pyrite crystals within vascular tissues has never been reported in mangroves. Further research must be conducted on the occurrence of those FeS2 crystals and on its drivers, soil properties, seasons, and species.

Conclusion

Despite their geographical proximity, two mangroves located on the semi-arid West coast of New Caledonia differ greatly in the properties of their soil and in the distribution of TM due to distinct watersheds. Mangrove soil located downstream an old Ni mine, the ultrabasic site, was characterized by TM concentrations higher than world average due to the laterized soil leached and eroded upstream. In addition, inputs of Fe oxy/hydroxide allows a good renewal of electron acceptors, and consequently, soil was characterized by suboxic conditions, which influences TM dynamics. The nature of the watershed should therefore be taken into account when studying geochemical processes and TM dynamics in mangroves. If significant differences were observed in TM concentrations in the soil of the two mangroves, TM concentrations in the roots did not seem influenced by the watershed inputs, but rather by the species. We suggest that TM uptake by those two mangroves were mainly driven by plant metabolisms and speciesrelated biochemical mechanisms. Iron and Ni were of the most concentrated TM in the roots of both species but showed low BCF possibly due to the lack of ions in the dissolved phase and the excessively large concentrations in the soil leading to potential uptake regulation by the plants. Uptake of TM by A. marina was greater than that of R. stylosa potentially due to a more permeable root system. Eventually, Fe plaque formation on pneumatophores of A. marina may be correlated to Fe concentration in the soil. Iron plaque plays an important role in limiting TM uptake by the plant up to 94% for Mn. Pyrite crystals were observed for the first time via SEM-EDX in the epidermis of pneumatophores of A. marina. We hypothesize that those framboids limit TM uptake by the plant via TM immobilization the same way as in mangroves soil. The acute investigation of pyrite crystals in root epidermis is considered for perspectives. Eventually, studies on TM species and locations within mangroves' organs should be further explored.

Figure captions

All figures are 2-column fitting images if possible. *** p < 0.001 for soil between sites † p < 0.05, † †p < 0.01 for roots between species

  TM concentrations(Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, and Zn) in soil extracts, biotic extracts (roots of R. stylosa, treated pneumatophores, and untreated pneumatophores of A. marina), DCB solutions, and porewaters were obtained via ICP-OES (Varian 730-ES) at the chemistry laboratory LAMA of the French Research Institute for Sustainable Development in New Caledonia (IRD) except for Hg, which was measured using a Hg analyzer (Brooks rand, Merx) at IRD for soil and biotic samples. Concentrations were obtained using a calibration curve previously prepared with the right matrices from a stock solution of TM at 1,000 mg L -1 . Certified reference materials were used to calculate a z-score for each TM. In the soil, for Co, Cr, Fe, Mn, Ni, and Zn, |z-score| < 2. Results are therefore validated. For Cu and Pb, 2 < |z-score| < 3.

Figure 1 .

 1 Figure 1. Map of the two study sites with the Dumbéa River and the old open-cast nickel mine. Stands of

Figure 2 .

 2 Figure 2. Depth profile of mean pH, Eh (mV), TOC (%) (n=3) and dissolved Fe (mg L -1 except for (d) µg L -1 ) (n=2) on both sites under both mangrove species.

Figure 3 .

 3 Figure 3. XRD spectra of soil at both sites under both mangrove species (a-d) and XRD spectra of rocks

Figure 4 .

 4 Figure 4. (a) Bioconcentration factors of TM in the pneumatophores of A. marina and roots of R. stylosa at both sites and (b) ratio of the dissolved/solid fractions (µg L -1 /mg kg -1 ) of TM except Hg in the soil.

Figure 5 .

 5 Figure 5. (a) Mean concentrations and standard deviations of iron plaque extracted from pneumatophores of

Figure 6 .

 6 Figure 6. SEM images of a cross-section of a pneumatophore from A. marina and elemental spectra obtained

Figure 7 .

 7 Figure 7. SEM images of framboidal pyrite (a) in a soil sample and (b) in a cross-section of the pneumatophore

Table 1 .

 1 Influence of sites on TM concentrations (mean ± SD) in soil between 6 and 12 cm deep (n=3 except Hg n=1), which corresponds to the depth at which root sections were taken, and influence of species on TM concentrations (mean ± SD) in roots (n=3 except Hg n=2) in mg kg -1 except for Hg in µg kg -1 .

		8,000
	(mg kg	6,000
	concentration	4,000	4,132
	plaque	2,000		1,863
	Iron
		0
			Ultrabasic	Non-ultrabasic

Acknowledgments

The authors acknowledge Kapeliele Gututauava and Franck Bouilleret for their technical help on site. Sarah Gigante and Valérie Medevielle are acknowledged for their technical support at the lab. The authors are grateful to Valérie Sarramegna for the knowledgeful discussions. Aurélie Monnin and Olivia Barthélémy are acknowledged for the technical assistance during SEM observations. The authors thank Leocadie Jamet and Monika Lemestre for Hg analysis and ICP-OES measurements, respectively. Eventually, the authors are grateful to the two anonymous reviewers who helped improve the manuscript with their constructive comments and suggestions.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-forprofit sectors.

Statistical analysis

Statistical analyses were performed on R studio software (version 1.2.5001). First, normality was checked with a Shapiro Wilk test. For comparison between species or study sites (two samples), parameters following a normal law were statistically verified with a Student's t-test with equal or unequal variances depending on F-test scores. For parameters not following a normality curve, the Mann-Whitney test was performed. All other statistical analyses with more than two samples were verified with one-way ANOVA analysis followed by a Tukey's HSD test for parameters following a normal curve. Other parameters were tested with a Kruskal-Wallis test followed by a Wilcoxon test. All tests were performed with a 95% confidence interval and n ≥ 3.

Results

Soil description

Percentages of TOC, pH, and Eh of the soil as well as concentrations of Fe in the dissolved phase (porewater) are presented for both species at both sites as a function of depth in Figure 2. Eh values along soil cores are significantly more negative at the non-ultrabasic site (p-value < 0.001) with an average value of -277 mV, whereas at the ultrabasic site the average value is -100 mV. The pH of the soil at both sites is slightly acidic (6 -6.6). The soil is significantly more acidic under R. stylosa (p-value < 0.001) with an average of 6.2 against 6.4 under A. marina. Even though there is no significant difference in pH overall between sites, pH is significantly lower at the non-ultrabasic site under R. stylosa compared to the ultrabasic site (p-value < 0.001). Negative correlations are obtained between Eh and pH for cores under R. stylosa only (-0.59 and -0.97) (SM 2).

The amount of TOC in the soil is larger at the non-ultrabasic site compared to the ultrabasic site with values ranging from 9.95 to 23.70%, with a mean value of 15.17%, and ranging from 6.91 to 18.20%, with a mean value of 11.71%, respectively. TOC is correlated negatively with pH for soil cores under A. marina (-0.73 and -0.71) (SM 2).

Concentrations of dissolved Fe are much higher at the ultrabasic site with an average value of 64 mg L - 1 , and with a large increase of dissolved Fe at a depth of 15 cm followed by a significant decrease (Fig. 2a