Alfred Obermayer

Jean-François Aubry

Nesrine Barnat
email: nesrine.barnat@theraclion.com

Extracorporeal Treatment With High Intensity Focused Ultrasound of an Incompetent Perforating Vein in a Patient With Active Venous Ulcers

Keywords: High intensity focused ultrasound, Perforating vein, Thermal ablation, Vein wall coagulation, Venous ulcer

Introduction: Endovenous techniques such as ultrasound guided foam sclerotherapy, thermal methods, or glues are generally recommended to occlude incompetent veins. However, these methods can be technically challenging and risky for patients with severe atrophic skin disorders like lipodermatosclerosis or atrophie blanche. A non-invasive alternative method can be high intensity focused ultrasound (HIFU), which has been shown to coagulate and occlude veins successfully. This case report details ultrasound guided HIFU to occlude noninvasively a refluxing perforator vein causing active ulcers.

INTRODUCTION

Current options for the treatment of incompetent perforating veins (IPVs) include minimally invasive techniques like endovenous thermal ablation (EVTA) or surgical options.

Regardless of the technique used, occluding IPVs is technically challenging compared

with the ablation of the great saphenous vein (GSV). 1 While EVTA for GSV incompetence has a success rate of > 96%, 1 lower success rates are reported for the ablation of IPVs (59% -90%). These higher closer failures of perforating veins are explained by the difficulties cannulating them. 1 Perforating veins are frequently deep, tortuous, and located close to damaged and ulcerated skin. 1 An extracorporeal approach using HIFU to treat an IPV causing active ulcers in an elderly patient is proposed.

CASE REPORT

A 95-year-old man presented with painful recurrent left leg ulcers after a six year ulcer free period. The patient was in relatively good general health but had a medical history of prior ischaemic stroke and coronary artery disease. In 2012, he had undergone a stripping of his left GSV and simultaneous shave therapy with mesh graft transplantation to treat the initial ulcer.

The clinical examination revealed in the left medial calf two active ulcers located in an area of severe lipodermatosclerosis, atrophie blanche, hyperpigmentation, and mild oedema (Fig. 1A). Both ulcers were exuding, with diameters of 10 mm. The Clinical, Etiologic, Anatomic and Pathophysiologic (CEAP) classification of his left limb was C2,3,4a,b,6,Ep,Ap,Pr,18.

Before treatment, a detailed ultrasound examination of the left limb was performed using a Terason scanner (Burlington, MA, USA).

Examination revealed a status postflush ligation and partial stripping of the GSV to knee level without recurrence. No deep venous incompetence was observed. To identify the origin of the venous leg ulcers, a "sourcing" technique described by Obermayer and Garzon was performed. [START_REF] Obermayer | Identifying the source of superficial reflux in venous leg ulcers using duplex ultrasound[END_REF] This technique, carried out with the patient standing, consists of softly compressing and releasing the ulcerated area and investigating, under duplex ultrasound, the routes of venous reflux penetrating into the ulcer area.

A refluxing posterior tibial perforating vein at a distance of 18 cm from the floor and with a systolic and diastolic outward flow > 1.5 seconds was identified as the source of the local venous hypertension (Fig. 1B,C) feeding superficial varicose veins. A diameter of 2.7 mm was measured at the level of the fascia.

The patient was placed in the Trendelenburg position and was treated in an outpatient setting.

A Sonovein device was used for the treatment. This ultrasound guided device encompasses a motorised treatment head mounted on an articulated arm. The treatment head (Fig. 2, right) is a removable part incorporating both an imaging transducer (7.5 MHz, 128 elements) and a therapy transducer (3 MHz, single element, diameter 56 mm, -6 dB focal spot width 0.5 mm) that can treat target tissues located at least 10 mm under the skin.

The system operates in conjunction with a single use disposable kit called EPack (Fig. 3). A fluid filled balloon was fixed to the transducer to ensure acoustic coupling between the transducer and the target. The coupling liquid was continuously circulated and cooled at 10°C to prevent skin burns.

The treatment head was first manually positioned in the region of interest and then the precise position was adjusted robotically (Fig. 4). To ensure the targeted vein was located at least 10 mm below the skin, 8 mL 0.1% lidocaine was injected subcutaneously under ultrasound guidance. This local anaesthesia was also performed to prevent pain and to compress the vein mechanically, which increases energy deposition on the vein wall. [START_REF] Barnat | Noninvasive vascular occlusion with HIFU for venous insufficiency treatment: preclinical feasibility experience in rabbits[END_REF][START_REF] Barnat | Efficacy and safety assessment of an ultrasound-based thermal treatment of varicose veins in a sheep model[END_REF][START_REF] Barnat | Design and validation of a non-invasive method for the treatment of varicose veins using high-intensity focused ultrasound[END_REF] Owing to the fragility of the skin and to avoid an infection, the injection was performed with a 4 21 G needle (0.80 × 120 mm; B Braun, Melsungen, Germany) inserted from dorsal, outside the lesion.

Thirty-one eight second pulses were delivered to the IPV at a mean ± standard deviation acoustic power 80 ± 21 W. Acoustic power was adjusted during the treatment according to the appearance of hyperechoic regions on the ultrasound image. [START_REF] Barnat | Design and validation of a non-invasive method for the treatment of varicose veins using high-intensity focused ultrasound[END_REF] A minimal pause time of 30 seconds was observed between each pulse to allow the skin to cool down.

After the procedure, a compression bandage was applied for seven days and the patient returned to his normal activities.

Clinical follow up appointments were scheduled at seven days and three months posttreatment.

A visual analogue pain score (from 0 to 10) was given to the patient before treatment, at day seven post-treatment, and at three months. A patient satisfaction questionnaire was additionally completed at the last clinical visit.

The patient initially reported a pain score of 3 before treatment and 0 on day seven and three months post-treatment.

Seven days postoperatively, the vein was not occluded and the ulcers were still active, but at three months the vein was occluded and the ulcers healed (Fig. 5B). The patient scored the treatment as very satisfactory. No complications occurred during the treatment or during the follow-up.

Approval was obtained to publish this case.

DISCUSSION

This article describes the first results of the treatment of IPVs with HIFU.

This technique, currently used for many clinical applications like the treatment of benign and malignant solid tumours, [START_REF] Kennedy | High-intensity focused ultrasound in the treatment of solid tumours[END_REF] uses high intensity focused beams to concentrate acoustic energy into a small soft tissue volume. The focusing of a high-power acoustic beam allows induction of high temperatures locally at the focus (approaching 100°C) producing a localised tissue ablation.

Owing to the heat and for patient comfort, a small amount (8 mL) of anaesthetic was injected before the sonications. The volume used was considerably less than the amount of tumescent anaesthesia infiltrated around truncal veins during EVTA (> 250 mL).

The use of HIFU to occlude veins has been investigated in preclinical studies. [START_REF] Barnat | Noninvasive vascular occlusion with HIFU for venous insufficiency treatment: preclinical feasibility experience in rabbits[END_REF][START_REF] Zhou | The effect of the scanning pathway in high-intensity focused ultrasound therapy on lesion production[END_REF] In particular, the ability of occluding veins of similar size (mean diameters of 2.0 -4.8 mm) has been demonstrated with the same device. [START_REF] Barnat | Noninvasive vascular occlusion with HIFU for venous insufficiency treatment: preclinical feasibility experience in rabbits[END_REF][START_REF] Barnat | Efficacy and safety assessment of an ultrasound-based thermal treatment of varicose veins in a sheep model[END_REF][START_REF] Barnat | Design and validation of a non-invasive method for the treatment of varicose veins using high-intensity focused ultrasound[END_REF] Dedicated chronic in vivo experiments were performed in a sheep model to assess the long term efficacy and safety of the procedure. [START_REF] Barnat | Efficacy and safety assessment of an ultrasound-based thermal treatment of varicose veins in a sheep model[END_REF] Eighteen saphenous veins (mean diameter 3.2 mm) were exposed to pulses similar to those delivered here (mean 84 W, eight seconds). Animals were followed up to 90 days posttreatment and after completion of the follow up vein segments were harvested and assessed microscopically. Histological findings revealed vein wall changes similar to the ones reported after EVTA (vein wall coagulation and fibrosis). In a human study with a six week follow up, Whiteley [START_REF] Whiteley | High intensity focused ultrasound (HIFU) for the treatment of varicose veins and venous leg ulcers -a new non-invasive procedure and a potentially disruptive technology[END_REF] reported that the closure rates observed in five patients treated with the Sonovein appeared to be as good as those after EVTA in the same short time with the major advantage of being non-invasive. In the same study, HIFU was delivered to treat varicose veins, venous leg ulcers, and other manifestations of venous reflux disease. [START_REF] Whiteley | High intensity focused ultrasound (HIFU) for the treatment of varicose veins and venous leg ulcers -a new non-invasive procedure and a potentially disruptive technology[END_REF] The mechanisms through which HIFU produces vascular occlusion are discussed in Barnat. [START_REF] Barnat | Design and validation of a non-invasive method for the treatment of varicose veins using high-intensity focused ultrasound[END_REF] Vascular occlusion is thought to occur as a result of numerous contributing factors, including vein wall collagen coagulation and endothelial damage. Transmural death of the cells in the vein wall may lead to permanent closure of the targeted vein by fibrosis. [START_REF] Whiteley | High intensity focused ultrasound (HIFU) for the treatment of varicose veins and venous leg ulcers -a new non-invasive procedure and a potentially disruptive technology[END_REF] Although EVTA has proven to be effective, these methods can be arduous and risky as vein wall perforation may occur. [START_REF] Quehe | Endovenous Celon radiofrequency-induced thermal therapy of great saphenous vein: a retrospective study with a 3-year follow-up[END_REF] With HIFU, as nothing is inserted into the vein, infection and bleeding risks are minimal. HIFU can be particularly interesting for patients with a risk of bleeding . [START_REF] Whiteley | High intensity focused ultrasound (HIFU) for the treatment of varicose veins and venous leg ulcers -a new non-invasive procedure and a potentially disruptive technology[END_REF] Contrary to EVTA methods, the occlusion does not seem to occur immediately but rather after a short delay of time (here, after seven days). This initial experience suggests that the Sonovein may be useful for ablation of refluxing veins.

Although this technique is new, early results are encouraging, as illustrated by the present case.

CONFLICTS OF INTEREST

Nesrine Barnat is an employee of Theraclion.

FUNDINGFigure 1 .

 1 FIGURE LEGENDS

Figure 2 .

 2 Figure 2. Sonovein high intensity focused ultrasound (HIFU) device.

Figure 3 .

 3 Figure 3. EPack. The kit comprises a prefilled pouch of degassed liquid, a balloon, and tubes to connect the balloon to the pouch. The pouch is incorporated in a dedicated bracket and placed in the cooling unit to cool the liquid; the balloon is mounted on the transducer; and the tubes are placed in the pumps, which allow the circulation of the liquid from the pouch to the balloon and back.

Figure 4 .

 4 Figure 4. (A) Positioning of the high intensity focused ultrasound transducer on the calf. (B) Robotic adjustment on the treatment screen of the therapy transducer position. To be properly centred on the targeted vein (white arrow), the focal point position is adjusted with the green directional command arrows. Note here the presence of the subcutaneous infiltration (white star) creating depth between the skin (pink line) and the anterior vein wall.

Figure 5 .

 5 Figure 5. Appearance of the left calf (A) before treatment and (B) three months after treatment.


Matthieu Ménager
email: matthieu.menager@univ-avignon.fr

Patricia Fernandez Esquivel

Paula Sibaja Conejo

The use of FT-IR spectroscopy and SEM/EDS characterization of slips and pigments to determine the provenances of archaeological ceramics: the case of Guanacaste ceramics (Costa Rica)

Keywords: Slips, Infrared spectroscopy, Provenance, Ceramics, Pigments

In this paper, the red and black pigments and slips of 63 Guanacaste ceramics were analyzed by Scanning Electronic Microscopy and infrared spectroscopy. All the pigment layers were made by dilution of a pure pigment in the slip layer. Red pigments were made using hematite, sometime in association with maghemite, mixed in the slip geomaterial. Black pigments were made of manganese oxide, sometime in association with magnetite.

Methodologically speaking, we clearly showed that the FT-IR analysis of the slip layer could allow a fast and pertinent discrimination of different sites of ceramic production according to their chemical compositions. Such approach could allow easier, cheaper and faster analytical analysis to answer to provenance study of archeological ceramics. In fact, the different ceramic types shared similar slip compositions with no correlation with the excavation site, showing that slip ceramics are relevant to trace Guanacaste ceramics in their exchanges or trades up to the Central Valley. Moreover, no local reproduction of Guanacaste styles in the central valley was noticed in this study. Meta-kaolinite and metasmectite, different types of tectosilicates as quartz and cristobalite, hematite and feldspar were notably detected in slip layers. The Agglomerative Hierarchical Clustering and Principal Component Analysis of slip infrared spectra showed the presence of three distinct clusters in great accordance with actual knowledge on the ceramic production in the area. Finally, the heating temperatures were estimated between 670 and 870°C during the fabrication processes, with almost no significant difference due to school or ceramic types.

Introduction

Color always had an important role in human societies for symbolic [START_REF] Crone | A History of Color: The Evolution of Theories of Light and Color[END_REF] or aesthetic [START_REF] Ball | The Aesthetics of Color: A Review of Fifty Years of Experimentation[END_REF] aspects. In many civilizations, specific colored materials were created from natural materials in order to improve art and representations on a large palette of supports.

Pre-Columbian cultures settled in Guanacaste (north of Costa Rica), in the archeological area of Greater Nicoya [START_REF] Lange | Lower Central America as Seen from Mesoamerica[END_REF], developed an important production of fine bichrome and monochrome ceramics until the arrival of Mesoamerican groups around 500 CE. After 500 CE, the ceramic production was expanded with polychromy and different designs of Mesoamerican origin [START_REF] Bishop | s Legacy of Research Support: The Prehispanic Ceramic Schools of Greater Nicoya[END_REF][START_REF] Day | New approaches in stylistic análisis: The Late Polychrome Period ceramics from Hacienda Tempisque[END_REF][START_REF] Healy | Greater Nicoya and Mesoamerica: Analysis of Selected Ceramics[END_REF]. These ceramics stand out for their white and cream slip layers with the use of red, orange, and black pigments in vessels with shaped like bowls, cups, plates and anthropomorphic and zoomorphic figurines [START_REF] Abel-Vidor | Principales tipos cerámicos y variedades de la Gran Nicoya[END_REF].

The most important study about the production and distribution patterns of the pottery of Greater Nicoya was made by Ronald L. Bishop and Frederick W. Lange [START_REF] Bishop | s Legacy of Research Support: The Prehispanic Ceramic Schools of Greater Nicoya[END_REF][START_REF] Bishop | Análisis de composición de la cerámica en el sur de America Central[END_REF][START_REF] Lange | La geología y arqueología de la cerámica prehistórica de la Gran Nicoya[END_REF]. In their publications, the elemental (Instrumental Neutron Activation Analysis) and petrographic compositions of more than 3000 ceramic pastes were analyzed in order to find geochemical and mineralogical patterns associated to their types and varieties. Bishop and Lange [START_REF] Bishop | s Legacy of Research Support: The Prehispanic Ceramic Schools of Greater Nicoya[END_REF] proposed the existence of 30 compositional groups linked to the existence of possible ceramic schools in Greater Nicoya, with three of them located in Guanacaste. According to these authors, Guanacaste Polychrome School II, (GN-04) was most probably located at the east of Guaitil, and was related to the production of Altiplano Polychrome, Birmania Polychrome, Mora Polychrome Chircot, and late Mora Polychrome Mora varieties. Near the coastal zone at the height of Tamarindo Bay, Middle Tempisque School (GN-05) most probably produced all the varieties of the Jicote Polychrome type [START_REF] Bishop | s Legacy of Research Support: The Prehispanic Ceramic Schools of Greater Nicoya[END_REF]. The location of the different schools proposed by Bishop and Lange is given in figure 1.

Guanacaste ceramics were clearly important goods in exchange and trade networks all over Costa Rica and Nicaragua. Indeed, the presence of such Guanacaste ceramics was documented in domestic and funeral contexts in many archeological sites located in the Central Valley of Costa Rica [START_REF] Creamer | Regional Exchange along the Pacific Coast of Costa Rica during the Late Polychrome Period, A. C. 1200-1550[END_REF][START_REF] Stone | The Archaeology of Lower Central America[END_REF][START_REF] Snarskis | Central América: The Lower Caribbean[END_REF][START_REF] Snarkis | Comentarios sobre el intercambio entre la Gran Nicoya y la Vertiente Atlántica y el Valle Central de Costa Rica en períodos precolombinos e históricos[END_REF]. The ceramic trade even increased from approximately 700 CE to 1520 CE, with Mora, Birmania, Papagayo, Altiplano and Jicote as the most frequent polychrome ceramic types [START_REF] Abel-Vidor | Principales tipos cerámicos y variedades de la Gran Nicoya[END_REF][START_REF] Corrales | El Pacífico Central de Costa Rica y el intercambio regional[END_REF][START_REF] Solís | Lomas entierros: Un centro político prehispánico en la cuenca baja del río Grande de Tárcoles[END_REF][START_REF] Valerio | Rescate arqueológico del sitio Llorente (SJ-51-Ll)[END_REF].

Another central question raised in the scientific literature is whether there was an exchange of polychrome ceramics from Guanacaste to the Central Region or whether there could have been local copies of this ceramic production [START_REF] Bishop | Análisis de composición de la cerámica en el sur de America Central[END_REF][START_REF] Vazquez | Arquitectura, caminos empedrados y cronología del sector principal del sitio Las Mercedes-1[END_REF]. In order to answer such scientific issue, physicochemical analyses are a truly pertinent solution as various studies showed the differences between elemental compositions of Gran Nicoya and Central Valley ceramics [START_REF] Salazar | Análisis de la cerámica policromada por fluorescencia de rayos X para estudios arqueológicos[END_REF][START_REF] Chapdelaine | Neutron activation analysis of archaeological ceramics from the Central Valley and Turrialba, Costa Rica[END_REF].

In this way, the production techniques of Guanacaste ceramics, and especially the materials used in the slip and pigment layers, could allow finding new fabrication patterns in order: (i) to assess the presence of copies of the Guanacaste types in the Central Valley, (ii) to strengthen, question or precise the theory of the schools of ceramic productions.

Lastly, Guanacaste ceramics are famous for their colored patterns and decorations. However, with the exception of our preliminary work [START_REF] Ménager | Los pigmentos naturales usados en Costa Rica: análisis preliminar de geomateriales y fragmentos cerámicos policromos de Guanacaste[END_REF], no research ever characterized the colored geomaterials used in slip and pigment layers. These colored materials are often slightly dependent on the local geology. In Costa Rica, clay materials can be composed, for example, of various mixtures of kaolinites, smectictes, allophanites, iron oxohydroxides (Hematite, goethite), halloysite, gibbsite, feldspars [START_REF] Alvarado Hernández | Clay minerals identified in soils of Costa Rica from 1931 to 2014: II. Major clay types described in soils with vertic, and oxidickaolinic properties[END_REF][START_REF] Bertsch | Properties, Geographic Distribution, and Management of Major Soil Orders of Costa Rica[END_REF][START_REF] Rossini | Application of the multi-element analysis by X-fluorescence and neutron activation to the characterization of an archaeological site[END_REF].

In this project, we analyzed 63 fragments of Guanacaste polychrome ceramic types from: (i) two Guanacaste archeological sites were selected: Palo Blanco (MNCR-G-800 PB) and Las Pilas (MNCR-G-850-LP), (ii) four sites in the Central Valley: Cariari (MNCR-H-134-CR), Chaparral (MNCR-A-359-CH), Barranca (MNCR-A-372-BA) and Alto del Cardal (MNCR-C-304-AC), (iii) one in the Central Caribbean: Nuevo Corinto (MNCR-L-72-NC). The selected ceramic types were Mora Polychrome with the varieties Guapote, Mono and Chircot, Altiplano Polychrome, Birmania Polychrome, Jicote Polychrome and Jicote variedad Felino, all of them produced between 800 CE to 1520 CE.

The Palo Blanco site is located in the lower basin of the Tempisque River, in the Palo Verde National Park in Guanacaste. The samples were taken from funerary contexts [START_REF] Chávez | Relación de las poblaciones antiguas con los recursos naturales inmediatos entre[END_REF]. The Las Pilas site, located in the Abangares area, is a multi-component site with funerary contexts. The analyzed samples come from burial 6 and stratigraphic excavations [START_REF] Aguilar | Informe de Campo, Laboratorio de Arqueología[END_REF]. In the western sector of the Central Valley, the Chaparral site is located, the samples come from the excavation of garbage and the Barranca site, the samples were obtained from test pits as a result of intensive prospecting. In the central sector of the Central Valley, the Cariari site is located; the samples were obtained from the excavation of burials and surface collections [START_REF] Beck | Reporte de campo, sitio Las Pilas. UCR 94[END_REF]. The Alto del Cardal site is in the eastern portion of the Central Valley, the samples came from a funeral and domestic context [START_REF] Morales | El camino precolombino del sitio Arqueológico Alto del Cardal C-304 AC[END_REF]. Finally, in the Nuevo Corinto site, the samples were obtained from domestic and funeral contexts [START_REF] Salgado González | El Sitio Nuevo Corinto (L-72-NC): Una aldea cacical[END_REF]. The locations of all archeological sites are given in figure 1. Methodologically speaking, ceramic provenance studies are principally based on mineralogical and elemental analysis of ceramic pastes and raw materials. In this study, we clearly showed that FT-IR analysis of slip layer could allow a fast and pertinent discrimination of different sites of ceramic production according to their chemical compositions. Such approach could allow easier, cheaper and faster analytical analysis to answer to provenance study of archeological ceramics.

The different objectives of this work are (i) to produce the first analyses of pigments and slip layers in Costa Rica and assess the material and techniques used, (ii) to use multivariate statistics in order to Group samples by similar chemical composition in order to find patterns of fabrication of Guanacaste ceramic found in Guanacaste and Central Valley sites using slip chemical compositions, (iii) to trace Gran Nicoya ceramics in the central region in order to validate the presence of a ceramic trade between the different archeological sites, (iv) to compare the group of similar composition to the ceramic school theory in Guanacaste.

Materials and methods

Archeological samples

All 63 Samples were part of the collection of the Archeology Laboratory of the Costa Rica university. The table 1

Infrared spectroscopy

Micro-sampling was performed with 0.55 mg of pigment, after lightly and carefully scraping the pigmented surface. The pigment mass was homogenized with 100 mg KBr and compressed to 7.5 T.cm -2 to form pellets.

The FT-IR spectrometer used was Perkin Elmer Frontier model, coupled to Spectrum software, which belongs to the Materials Science and Engineering Research Center (CICIMA). In addition, it was used in transmission mode. The spectra were collected in the range of (4000-400 cm -1 ), with a resolution of 4 cm -1 and 30 scans. The samples were directly analyzed with FT-IR.

All spectra were treated using the R software with the Chemospec packages. These spectra were baseline corrected using the Fillpeak algorithm, smoothed using Savitzky-Golay algorithm and normalized. Furthermore, the second derivative of infrared spectra were performed to improve the identification of minerals.

Scanning Electron Microscopy

Micro-fragments of the samples were used in order to prepare cross-sections of pigment and slip layers. An encapsulation in Epoxy Mount resin was performed using PLA sample holders made by 3D printing and silicone molds. The cross-sections were polished with aluminum oxide polishes (15 m and 3 m) and aqueous suspension of 0.25 m diamond powder using the EcoMet 30 equipment belonging to the Central American School of Geology. The cross-sections were cleaned during 20 min in distilled water in an ultrasonic bath. Gold coating of 10 nm was applied to cross sections in order to improve the image resolutions in scanning electron microscopy.

The cross-sectional analyses were performed with Hitachi S-3700 N scanning electron microscopy equipment which belongs to Microscopic Structures Research Center (CieMIC). The backscattered electron technique was used in COMPO Std. mode to choose the

Results and discussion

All ceramic sherds included 2 distinct layers: white slips and red or black pigments. All the pigment layers presented a similar infrared signal as their associated slip layer with new infrared absorption specific to the red or black geomaterials used as coloring agent, as shown for sample 5 (Mora Mora type, found at Cariari site) in figure 3. Such result revealed the use of a similar technique for the confection of the pigment layer: the dilution of a pure pigment in the slip layer.

Slip layers

In the slip layers, several geomaterial were identified by FT-IR spectroscopy (cf table 2 for main geomaterials): (i) Two distinct types of meta-clays: meta-kaolinite and meta-smectite, (ii) different types of tectosilicates as quartz and cristobalite, (iii) iron oxides, (iv) other types of minor minerals as anorthite, feldspar and calcite. All identifications were done according to the existent literature [START_REF] Gates | Infrared and raman spectroscopies of clay minerals[END_REF][START_REF]Iron oxides: From nature to applications[END_REF][START_REF] Hunt | The Oxford Handbook of Archaeological Ceramic Analysis[END_REF][START_REF] Balandier | Chemical analyses of Roman wall paintings recently found in Paphos, Cyprus: The complementarity of archaeological and chemical studies[END_REF] and comparison with standards from the laboratory as described in table 2.

Meta-clays were detected in all samples according to their main absorption band around 1040 ± 10 cm -1 for meta-smectite (cf figure 4) and 1080 ± 10 cm -1 for meta-kaolinite due to asymmetric stretching of Si-O bonds. Other characteristic bands were found around 1168 cm -1 ( ( --)), and between 465 and 475 cm -1 ( ( --)).

Three types of tectosilicates were identified: (i) quartz thanks to its characteristic doublets at 778 and 798 cm -1 ( ( -)), main absorption band at 1084 cm -1 ( ( -)), and confirmatory band at 694 cm -1 , (ii) cristobalite thanks to its main absorption band at 1098 cm -1 ( ( --)), its single band centered at 796 cm -1 , and other confirmatory bands around 1200 cm -1 , (iii) feldspar thanks to infrared bands at 1140, 1008, 642 and 586 cm -1 . Water adsorbed in slip and pigment layers induced a broad band around 3426 cm -1 and another around 1636 cm -1 respectively linked to H2O stretching and bending modes. It is important to point out that cristobalite is naturally present in the volcanic area of Guanacaste [START_REF] Ménager | Los pigmentos naturales usados en Costa Rica: análisis preliminar de geomateriales y fragmentos cerámicos policromos de Guanacaste[END_REF] and, thus do not come from the thermic degradation of quartz, occurring at high temperatures.

The presence of hematite was revealed in almost all ceramic slips with its characteristic band around 520-570 cm -1 which maximum vary in function of the percentage of substituted Al [START_REF] Kosmas | Relationship among Derivative Spectroscopy, Color, Crystallite Dimensions, and Al Substitution of Synthetic Goethites and Hematites[END_REF]. Confirmatory bands were taken at 634 cm -1 ( ( -)) and 470 cm -1 ( ( -)). The last one was often overlapped by meta-clays or tectosilicates signals.

Mineral

Multivariate statistic analysis of slip FT-IR spectra

The Agglomerative Hierarchical Clustering (AHC) dendrogram presented in figure 5 (b) showed the presence of three distinct clusters, associated with three distinct slip compositional groups (cf appendix A). This observation nicely fitted the 3 groups of samples observed on the graph of individuals of Principal Component Analysis (PCA) using the first two Principal Components (PC1 and PC2) representing 85.7% of total variance (cf figure 5 a)). The different clusters included the following ceramic types and varieties and main identified geomaterials (the mean FT-IR are presented in figure 4 and in appendix A) :

• Cluster 1: containing all MGp and MGb, 5 MMora, 1 MMono, 11 MP mostly composed by meta-kaolinite, quartz and the highest concentrations of hematite.

• Cluster 2: containing all JP and JF, composed of meta-smectite, cristobalite, feldspar and the lowest concentrations of hematite.

• Cluster 3: containing all the AP, BP and MCh, 4 MMono, 1 MMora, 3 MP mostly composed by a mix of meta-kaolinite and meta-smectite, quartz and variable concentrations of hematite. First of all, the ceramics were grouped by types or varieties either if they were excavated in Guanacaste or Central Valley sites, showing that they shared similar slip compositions with no correlation with the excavation site. These results indicated that slip ceramics are relevant to trace Guanacaste ceramics in their exchanges or trades up to the Central Valley. Moreover, no local reproduction of Guanacaste styles in the central valley was noticed in this study.

As noticed in figure 5 (a), the ceramics included in the clusters 2 and 3 nicely fitted the associations between ceramic types and varieties of the Middle Tempisque (GN-04) and Guanacaste Polychrome II (GN-04) ceramic schools described in introduction and proposed by Bishop and Lange [START_REF] Bishop | s Legacy of Research Support: The Prehispanic Ceramic Schools of Greater Nicoya[END_REF]. Moreover, in the cluster 2, all samples had an important level of similarity. In the cluster 1, all slip compositions only showed the presence of meta-kaolinite as meta-clay.

The different compositional groups shown in figure 5 (a) were discriminated according to the type of meta-clay, the concentrations of hematite and feldspar, and the presence of quartz or cristobalite, as noticed on the loading spectra of PC1 and PC2 presented in figure PC1 (74.3% of total variance) PC2 (11.4% of total variance) 6. Indeed, PC1 is highly correlated with the infrared bands of meta-smectite (1034 and 918 cm -1 ), hematite (430 and 544 cm -1 ) and quartz (1180, 1088, 800, 780 and 694 cm -1 ). The PC2 is linked to the FT-IR signal of cristobalite (1114 and 484 cm -1 ), feldspar (1002, 586 and 642 cm -1 ) and meta-kaolinite (1062, 1228, 452 cm -1 ).

Mora Polychrome of unknown varieties were named MP in this study. Using AHC clustering, 11 samples were associated to the clusters 1, and 3 samples were associated to the cluster 3. Linear Discriminant Analysis (LDA) was also used in order to validate these attributions using a supervised statistical model. The LDA was realized on the first four principal component (PC, cf figure 5) of the 49 ceramics from AP, BP, JP, JF, MCh, MGb, MGp, MMono, MMora types (cf table 1). We used the AHC clusters previously described as predictive class. This allowed us to model the linear combinations between PC and clusters in order to predict the group attribution (1, 2 or 3) of all MP samples. As previously discussed, these AHC groups contain specific ceramic types and varieties attributed to the presence of different workshops of fabrication in the area [START_REF] Bishop | s Legacy of Research Support: The Prehispanic Ceramic Schools of Greater Nicoya[END_REF]. Thus, in order to homogenize the types and varieties inside the different clusters, the MMono of cluster 1 was attributed to cluster 3 and the MMora of cluster 3 was attributed to the cluster 1 before the LDA analysis. The LDA model was then validated by Leave One Out Cross Validation (LOOCV) which matrix (appendix B) showed a 96% of correct predictions. The confusion matrix (data not shown) led to 100% of correct predictions. LDA predictions and AHC clustering led to the attribution of samples: (i) 104, 105, 106, 117,

, 108, 36 to cluster 3. This result is in perfect accordance with the good grouping of these samples with corresponding clusters in PCA analysis (5 a).

Red pigments

All red pigments were made using hematite mixed in the slip geomaterial, as noticed by the presence its specific identified infrared bands (540 and 430 cm -1 ) as shown in figure 8. As shown in figure 7, SEM/EDS analysis confirmed these identifications and detected up to 36% (relative molar concentration) of iron and significant amounts of silicon, oxygen and aluminum.

In addition to hematite, maghemite was detected in all MGb, MMora and MGp red pigments of the cluster 1. Maghemite could be detected thanks to its characteristic bands at 446 ( ( -)), 486 ( ( -)), 558 ( ( --)) and 624 ( ( --)) cm -1 (cf figure 8). It is worth noting that maghemite was also identified in the MMora present in the cluster 3.

Black pigments

All black pigments presented manganese oxides with its characteristic infrared bands at 456 ( ( -)), 606 and 634 cm -1 ( ( -)) as shown on figure 9. Indeed, black pigment layers from all ceramic types presented relative molar concentration up to 9.6% of Mn when analyzed using SEM/EDS. All black pigments, with the exception of those from Jicote ceramics, presented significant amount of hematite (band around 540 cm -1 ).

All black pigments of the cluster 2 (all JP and JF) presented a particular composition with mix of manganese oxide and magnetite. Magnetite could be identified by its characteristic band at 570 cm -1 (cf figure 9). Such result could be related to: (i) an anthropic origin, with a intentional mix of these two geomaterial, which could be specific of the black pigments of cluster 2, or (ii) a geological origin, with the use of geomaterial containing both pigments. Nevertheless, Mapping of Mn and Fe concentrations in Jicote black pigment layer showed the presence of different microstructures: (i) ones rich in Fe, (ii) other rich in Mn (cf figure10). Moreover, in a previous study [START_REF] Ménager | Los pigmentos naturales usados en Costa Rica: análisis preliminar de geomateriales y fragmentos cerámicos policromos de Guanacaste[END_REF], we characterized a black geomaterial from the nearby Guaitil area, which was traditionally used by indigenous local potters from ages to nowadays, and only detected manganese oxide as black pigment. All these data allowed to hypothesize an anthropic origin for the mix between magnetite and manganese oxide for Jicote ceramics. It is worth noting that, for the black pigments of Jicote ceramics, characteristic infrared bands of hematite could be hidden by magnetite and manganese oxides infrared signals.

Heating temperatures

Shoval & al [START_REF] Shoval | Analysis of thermal phases in calcareous Iron Age pottery using FT-IR and Raman spectroscopy[END_REF] showed the existing relation between wavelength position meta-clay principal absorption band and the heating temperature of associated clay. This relation is ̅ = 0.074 x + 1032 with ̅ in nm and in °C. For metasmectite, we used their data to build a new model using R software: = (( ̅ -1028)/(1.23 * 10 %&& )) '. (&) with ̅ in nm and in °C (p-value = 6 * 10 %+ , Adjusted , & = 0.983). These equations allowed us to establish direct correlations between meta-smectite and meta-kaolinite Si-O asymmetric stretching bands and heating temperatures.

According to our results (cf table 4), Guanacaste ceramics were most probably heated between 670 and 870°C during the fabrication processes, with almost no significant difference between school or ceramic types. Heating temperatures were calculated for all slip and pigment samples. It is important to point out the good level of similarity between the values calculated with meta-smectite and metakaolinite Si-O asymmetric stretching bands, showing the good reliability of the method.

On the archeological point of view, such range of temperature is coherent with the pre-Colombian ovens, open and buried, found in the Vidor Bahía Culebra site in Guanacaste [START_REF] Abel-Vidor | Dos hornos precolombinos en el sitio Vidor Bahía Culebra[END_REF]. According to the authors, such technology could allow reaching temperatures up to 900°C, in good agreement with the values given by other experimental archaeology experiments [START_REF] Daszkiewicz | Experimental Firing and Re-firing[END_REF][START_REF] Thér | Experimental Pottery Firing in Closed Firing Devices from the Neolithic Hallstatt Period in Central Europe[END_REF].

Clusters

Conclusion

In this paper, the red and black pigments and slips of 63 Guanacaste ceramics were analyzed by Scanning Electronic Microscopy and infrared spectroscopy. Red pigments were made using hematite, sometime in association with maghemite, mixed in the slip geomaterial. Black pigments were made of manganese oxide, sometime in association with magnetite.

All the pigment layers were made by dilution of a pure pigment in the slip layer. Metakaolinite and meta-smectite, different types of tectosilicates as quartz and cristobalite, hematite and feldspar were notably detected in slip layers. Impressive homogeneity of the compositions of the slips of Jicote type ceramics was observed with all 12 samples composed of meta-smectite, cristobalite, hematite and feldspar.

We clearly showed that slip layer could allow to discriminate different sites of ceramic production according to their chemical compositions. Indeed, the different ceramic types shared similar slip compositions with no correlation with the excavation site, showing that slip ceramics are relevant to trace Guanacaste ceramics in their exchanges or trades up to the Central Valley. Moreover, no local reproduction of Guanacaste styles in the central valley was noticed in this study. Meta-kaolinite and meta-smectite, different types of tectosilicates as quartz and cristobalite, hematite and feldspar were notably detected in slip layers. The Agglomerative Hierarchical Clustering and Principal Component Analysis of slip infrared spectra showed the presence of three distinct clusters in great accordance with actual knowledge on the ceramic production in the area.

The cluster 1 was composed of slips made of meta-kaolinite, quartz and the highest concentrations of hematite, and contained almost all Mora Polychrome Guapote and Mora Polychrome Guabal and Mora Polychrome Mora samples. Moreover, all red pigments of this cluster were composed of maghemite and hematite, which strengthen a common fabrication techniques, and potentially a common workshop, for all sherd of this cluster.

Meta-smectite, cristobalite, feldspar and hematite were found in cluster 2 slips in all ceramics from Jicote Polychrome and Jicote Polychrome Felino Their black pigments were the only one made of manganese oxide and magnetite, unlike all the black pigments of all other clusters. The ceramic varieties in this cluster were in good accordance with the Middle Tempisque proposed by Bishop and Lange [START_REF] Bishop | s Legacy of Research Support: The Prehispanic Ceramic Schools of Greater Nicoya[END_REF].

The slips from the cluster 3 were composed of a mix of meta-kaolinite and meta-smectite, quartz and variable concentrations of hematite, and included all Altiplano Polychrome, Birmania Polychrome and Mora Polychrome Chircot ceramics Interestingly, four FT-IR spectra of MMono slips were statistically attributed to this cluster. Such materials could be characteristic of the Guanacaste Polychrome II (GN-04) ceramic schools [START_REF] Bishop | s Legacy of Research Support: The Prehispanic Ceramic Schools of Greater Nicoya[END_REF].

For the slips of Mora Polychrome of unknown varieties (MP) eleven could be associated to the clusters 1, and three to the cluster 3 thanks to Agglomerative Hierarchical Clustering and Linear Discriminant Analysis. Two of the three MP samples associated with the cluster 1, also presented a mix of maghemite and hematite as red pigment.

Finally, the existing relation between the wavelength position of meta-clay principal absorption band and the heating temperature of associated clay allowed us to estimate heating temperature between 670 and 870°C during the fabrication processes, with almost no school or ceramic types significant difference.

Figure 1 :

 1 Figure 1: Map of the different archeological sites and possible locations of ceramic school as proposed by Bishop and Lange (2013).

Figure 2 :

 2 Figure 2: Pictures of different samples analyzed in this paper: a) 49, b) 51, c) 25, d) 47, e) 34, f) 109, g) 1, h) 40, i) 45.

Figure 3 :

 3 Figure 3: Infrared spectrum of slip and pigments layers of sample 5 (Type Mora Mora, Cariari).

Figure 4 :

 4 Figure 4: Mean infrared spectrum (bottom) and its second derivative (above) for all JP y JF analyzed. The gray area represents the lower and upper Gaussian confidence limits based on the t-distribution.

Figure 5 :

 5 Figure 5: a) Principal component analysis of the first derivative of slip infrared spectra with 95% confidence ellipse of the clusters identified by Agglomerative Hierarchical Clustering. b) Agglomerative Hierarchical Clustering of the first derivative of slip infrared spectra (Euclidean distance, Ward's method).

Figure 6 :

 6 Figure 6: Loading spectra of Principal Components 1 and 2 of the PCA presented in figure slip infrared spectra. The presented spectra are the non-derivative spectra obtained by the integration of the original loading spectra using R software using the cumsum function from base package.

Figure 7 :

 7 Figure 7: (left) Photography of the red pigment layer of sample 14 (MP, Palo Blanco) using Scanning Electron Microscopy with backscattering electron detector (400x). (right) Associated elemental compositions of slip and red pigment of sample 14 measured by the Energy Dispersive X-ray detector.

Figure 8 :

 8 Figure 8: (above) Results of the subtraction of the red pigment spectra by the above slip layer for samples 22 (MP, Chaparral), 25 (MGp, Chaparral), 47 (MMora, Nuevo Corinto) 51 (MGp, Nuevo Corinto) and 52 (MMono, Nuevo Corinto) with characteristic infrared bands of hematite and maghemite. (bottom) Results of the subtraction of the red pigment spectra by the above slip layer for samples 16 (JP, Palo Blanco), 31 (BP, Barranca) and 45 (JF, Las Pilas), 114 (AP, Alto del Cardal), 118 (MCh, Alto del Cardal) with characteristic infrared bands of hematite.

Figure 9 :

 9 Figure 9: (above) Results of the subtraction of the black pigment spectra by the above slip layer for samples 16 (JP, Palo Blanco), 18 (JP, Palo Blanco), 45 (JF, Las Pilas) and 46 (JP, Las Pilas) with characteristic infrared bands of manganese oxide and magnetite. (bottom) Results of the subtraction of the black pigment spectra by the above slip layer for samples 24 (MMora, Chaparral), 25 (MGp, Chaparral), 51 (MGp, Nuevo Corinto) 52 (MMono, Nuevo Corinto), 105 (MP, Alto del Cardal), 118 (MCh, Alto del Cardal) and 119 (BP, Alto del Cardal) with characteristic infrared signal of manganese oxide and hematite.

Figure 10 :

 10 Figure 10: (SEM) Photography of the black pigment layer of sample 39 (JP, Las Pilas) using Scanning Electron Microscopy with Secondary Electrons detector, (Mn -Fe) Associated mapping of Mn and Fe measured by the Energy Dispersive X-ray detector.

  describe the distribution of the samples among the different archeological sites. The pictures of different samples are given in figure2.

	Ceramic types	Acronyms	Archeological sites and associated samples
	Altiplano Polychrome	AP	(Palo Blanco) 17, (Barranca) 29, (Chaparral) 20,
			(Cariari) 3,6,8, (Alto del Cardal) 109, 110, 111, 112,
			114, 115, (Nuevo Corinto) 55, 56
	Birmania Polychrome	BP	(Las Pilas) 34, (Barranca) 27, 31, (Chaparral) 23, (Alto
			del Cardal) 113, 119
	Jicote Polychrome	JP	(Palo Blanco) 12,13,16,18, (Las Pilas) 39, 40, 41, 42,
			43, 44, 46
	Jicote Polychrome Felino	JF	(Las Pilas) 45
	Mora Polychrome	MP	(Palo Blanco) 14, 15, (Las Pilas) 35, 36, (Barranca) 26,
			(Chaparral) 19, 22, (Alto del Cardal) 104, 105, 106, 107,
			108, 117, (Nuevo Corinto) 54
	Mora Polychrome Chircot	MCh	(Cariari) 1,4, (Alto del Cardal) 116, 118
	Mora Polychrome Guabal	MGb	(Chaparral) 25
	Mora Polychrome Guapote	MGp	(Nuevo Corinto) 51
	Mora Polychrome Mono

MMono (Las Pilas) 37, 38, (Cariari) 9, (Nuevo Corinto) 49, 52 Mora Polychrome Mora MMora (Chaparral) 24, (Cariari) 5, (Nuevo Corinto) 47, 48, 50, 53 Table 1: Ceramic types and archeological sites of the samples analyzed in the present study.

Table 2 :

 2 Infrared characteristic frequencies (cm -1 ) of identified geomaterialsThe presence of meta-clays was strengthened by the SEM/EDS elemental analysis, characteristic of silicate minerals, showing that: (i) Si relative molar concentration varied between 23.2 and 31.3 %, and (ii) Al relative molar concentration varied between 5.5 and 8.7 %. Quartz and cristobalite crystals were also detected thanks to their characteristic elemental composition (as shown for a quartz microstructure of samples 48 in appendix C).An impressive homogeneity of the infrared spectra of the slips of Jicote type ceramics (Jicote Polychrome and Jicote Polychrome Felino) was observed as shown in figure4and table3. Indeed, most slips were made of a geomaterial composed of meta-smectite, cristobalite, hematite and feldspar. Some traces of quartz were observed in many Jicote samples. The figure4was made by calculating the mean of all infrared spectra, the low distribution of slip chemical compositions was underlined by the very thin interval of the mean confidence limits based on the t-distribution.

		Wavenumber (cm -1 )	Acronym	Color used in graphics	References
	Meta-clays
	Meta-clays	465-475, 1169, 1235	MC	Black	[31]
	Meta-kaolinite 1070-1090	MK	Blue	[31]
	Meta-smectite 1030-1050	MS	Green	[31]
	Oxide minerals
	Hematite	430, 470, 520-560, 634	Hem	Red	[30,34]
	Maghemite	444, 486, 556, 624	Mgh	Orange	[36]
	Magnetite	570	Mgn	Grey	[30]
	Manganese	454, 606, 640	Mn02	Black	[30,31]
	oxide
	Tecto silicates
	Cristobalite	480, 618, 695, 795, 1099, 1176, 1200	Cr	Yellow	[31,35]
	K-Feldspar	430, 586, 642, 1008, 1139	Feld	Purple	[31,35]
	Quartz	460, 513, 694, 778, 798, 1084, 1180	Qz	Brown	[31,35]
	Mineral	Wavenumber (cm -1 )	Acronym	Color used in graphics	References
	Meta-clays
	Meta-clays	465-475, 1169, 1235	MC	Black	[31]
	Meta-kaolinite 1070-1090	MK	Blue	[31]
	Meta-smectite 1030-1050	MS	Green	[31]
	Oxide minerals
	Hematite	430, 470, 520-560, 634	Hem	Red	[30,34]
	Maghemite	444, 486, 556, 624	Mgh	Orange	[36]
	Magnetite	570	Mgn	Grey	[30]
	Manganese	454, 606, 640	Mn02	Black	[30,31]
	oxide
	Tecto silicates
	Cristobalite	480, 618, 695, 795, 1099, 1176, 1200	Cr	Yellow	[31,35]

As detailed in table 3, slips from Mora Polychrome Mora, Guabal and Guapote, and different Mora Polychrome ceramics were made of meta-kaolinite with quartz in more than 80% of analyzed samples, sometimes in association with minor levels of meta-smectites. Moreover, more than 70% of Altiplano Polychrome, Birmania Polychrome and Mora Polychrome Chircot ceramic slips included meta-smectite, meta-kaolinite with quartz.

Table 3 :

 3 For each ceramic type, percentages of detection of every geomaterials identified by FT-IR spectroscopy and SEM/EDS (number of samples with detection of the mineral divided by the number total of sample for a specific ceramic type or variety).

Table 4 :

 4 Minimum and maximum heating temperatures estimated from the wavelength of Si-O asymmetric stretching band for AHC cluster. The values were calculated thanks to the data from[START_REF] Shoval | Analysis of thermal phases in calcareous Iron Age pottery using FT-IR and Raman spectroscopy[END_REF] for meta-kaolinites (MK) and meta-smectites (MS).

		Min (MK) Min (MS) Max (MK) Max (MS)
	1	676	650	730	730
	2		684		775
	3	676	684	757	807

measurement areas of the elemental composition. Energy-Dispersive-X-ray Spectroscopic (EDS) was performed with 120 s of measurement and the results were obtained using the Iridium Ultra software. The analysis conditions were 15 kV, 10-12 mm working distance, and high vacuum.

Acknowledgment

Our research was supported by a grant from the Universidad de Costa Rica, to the project "Los pigmentos naturales usados en Costa-Rica en el periodo precolombino" (B7241). We want to thank the Centro de Investigación en Ciencia e Ingeniería de Materiales (CICIMA) and the Centro de Investigación en Estructuras Microscópicas (CieMIC) for the support and the use of the different analytical tools. We also thank the Centro de Investigación en Antropología (CIAN) for his great support in the successful completion of these researches. The authors want to thank Silvia Salgado and Carolina Cavalini for the help during the ceramic type identification, Benjamin Acevedo for its advises on Costa Rican geology and Mavis Montero for her support during the analytical analysis. We also want to thank the reviewers for their comments and corrections in order to improve the quality of the manuscript.


Mercedes García-Sánchez
email: garcia.sanchez.mercedes@gmail.com

Gloria Andrea Silva-Castro

Alvaro Sanchez

Cesar Arriagada

Inmaculada García-Romera

Role of the extraradical mycelium of arbuscular mycorrhizal fungi combined with mycoremediated dry olive residue in Pb transport and plant protection

Keywords: Dry olive residue, Container system, Funelia floccose, Funneliformis mosseae, Hydrophobic polytetrafluoroethylene membrane, Lead (Pb)

This study aims to evaluate the role of the extraradical mycelium of arbuscular mycorrhizal fungi in Pb transport. We also investigate how these fungi, alone or combined with the mycoremediated dry olive residue (MDOR), provide protection against Pb. We established a container system consisting of a central compartment and two lateral compartments separated by a hydrophobic membrane. The central compartment was filled with sterilized soil in which wheat plants, inoculated and noninoculated with Funneliformis mosseae, were grown. The lateral compartments were filled with sterile, Pb-contaminated or MDOR-amended soil or combinations of both. In contrast to shoots and grains, wheat roots accumulated larger amounts of Pb with or without applications of MDOR. The extraradical mycelium (ERM) and the glomalin related protein content were significantly boosted by adding MDOR to Pb-contaminated soil samples. Wheat root biomass was decreased as the result of Pb contamination with no increases in plant phosphorous (P) uptake. However, MDOR, when added to Pbcontaminated soil samples, only boosted the accumulation of P in roots, with P content and biomass remaining unchanged in wheat shoots and grains. Our study highlights the role of the ERM in Pb transport its accumulation in wheat roots and how the protection effect exerted by AMF seemed to rely on MDOR application by increasing the P uptake rather than Pb.

Introduction

Lead (Pb), which is widely distributed in soils, is complexed with organic matter, adsorbed by clays and oxides and precipitated as carbonates, hydroxides and phosphates [START_REF] Epstein | EDTA and Pb-EDTA accumulation in Brassica juncea grown in Pb-amended soil[END_REF]. However, industrial activities, such as mining, smelting, the burning of fossil fuels and the manufacture of pesticides and fertilizers, are the main cause of lead soil pollution [START_REF] Sharma | Lead toxicity in plants[END_REF][START_REF] Tchounwou | Heavy metal toxicity and the environment[END_REF]. Arbuscular mycorrhizal fungi (AMF), which form a symbiotic relationship with over 80% of terrestrial plants [START_REF] Smith | The symbionts forming arbuscular mycorrhizas[END_REF], have been shown to take an active part in plant resistance to contamination by heavy metals such as Pb [START_REF] Arriagada | Contribution of arbuscular mycorrhizal and saprobe fungi to the tolerance of Eucalyptus globulus to Pb[END_REF][START_REF] Arriagada | Contribution of arbuscular mycorrhizal and saprobe fungi to the aluminum resistance of Eucalyptus globulus[END_REF]. The so-called growth dilution effect of AMF described by some authors is based on enhanced plant growth through higher phosphorous (P) uptake [START_REF] Arias | Effects of Glomus deserticola inoculation on Prosopis: Enhancing chromium and lead uptake and translocation as confirmed by X-ray mapping, ICP-OES and TEM techniques[END_REF][START_REF] Chen | A modified glass bead compartment cultivation system for studies on nutrient and trace metal uptake by arbuscular mycorrhiza[END_REF]. However, other authors have reported that the alleviation of plant metal toxicity by AMF might be associated with the immobilization of large quantities of metals in the extraradical mycelium (ERM) and their subsequent translocation from plant roots to shoots [START_REF] Nayuki | Cellular imaging of cadmium in resin sections of arbuscular mycorrhizas using synchrotron micro X-ray fluorescence[END_REF][START_REF] Rufyikiri | Uranium uptake and translocation by the arbuscular mycorrhizal fungus, Glomus intraradices, under root-organ culture conditions[END_REF][START_REF] Weiersbye | Micro-PIXE mapping of elemental distribution in arbuscular mycorrhizal roots of the grass, Cynodon dactylon, from gold and uranium mine tailings[END_REF]. This could be connected with the high cation exchange capacity (CEC) of the ERM which may favour metal adsorption on the surface of fungal hyphae [START_REF] Chen | A modified glass bead compartment cultivation system for studies on nutrient and trace metal uptake by arbuscular mycorrhiza[END_REF][START_REF] Joner | Metal-binding capacity of arbuscular mycorrhizal mycelium[END_REF]. Nevertheless, even if the ERM is involved in their transport to mycorrhizal roots, the metals could be stored in fungal structures i.e. spores or easily-extractable glomalin related soil protein (EE-GRSP), thus preventing their transfer across symbiotic surfaces between AMF and root cells [START_REF] Gao | Functions and application of glomalin-related soil proteins: A review[END_REF][START_REF] Göhre | Contribution of the arbuscular mycorrhizal symbiosis to heavy metal phytoremediation[END_REF][START_REF] Joner | Uptake of 109Cd by roots and hyphae of a Glomus mosseae/ Trifolium subterraneum mycorrhiza from soil amended with high and low concentrations of cadmium[END_REF][START_REF] Nayuki | Cellular imaging of cadmium in resin sections of arbuscular mycorrhizas using synchrotron micro X-ray fluorescence[END_REF][START_REF] Salazar | Pb accumulation in spores of arbuscular mycorrhizal fungi[END_REF].

On the other hand, despite the reported ability of AMF to explore metaldegraded environments their diversity and abundance, with the exception of some more resistant strains, can be depleted by increasing metal content [START_REF] Hildebrandt | Arbuscular mycorrhiza and heavy metal tolerance[END_REF][START_REF] Zarei | Molecular diversity of arbuscular mycorrhizal fungi in relation to soil chemical properties and heavy metal contamination[END_REF]. Organic amendments, such as mycoremediated dry olive residue (MDOR), have significantly increased the abundance of AMF populations and their capacity to adapt to different metals such as As, Cd, Pb and Zn, thus enhancing the nutrient status of wheat plants [START_REF] García-Sánchez | Risk element immobilization/stabilization potential of fungal-transformed dry olive residue and arbuscular mycorrhizal fungi application in contaminated soils[END_REF], 2019). MDOR is characterized by a high degree of organic matter humification and low levels of toxic phenol substances as a result of biological stabilization through the use of saprophytic fungi such as Funelia floccose, which produce a set of extracellular oxidoreductases [START_REF] Reina | Solid state fermentation of olive mill residues by wood-and dung-dwelling Agaricomycetes: Effects on peroxidase production, biomass development and phenol phytotoxicity[END_REF][START_REF] Sampedro | Solid-state cultures of Fusarium oxysporum transform aromatic components of olivemill dry residue and reduce its phytotoxicity[END_REF][START_REF] Sampedro | Short-term impact of dry olive mill residue addition to soil on the resident microbiota[END_REF][START_REF] Siles | Assessing the impact of biotransformed dry olive residue application to soil: Effects on enzyme activities and fungal community[END_REF]. Although it has been reported the suitability of combining MDOR application and AMF inoculation for recovering metal-degraded soils [START_REF] García-Sánchez | Implications of mycoremediated dry olive residue application and arbuscular mycorrhizal fungi inoculation on the microbial community composition and functionality in a metal-polluted soil[END_REF], the specific mechanisms involving the ERM of the AMF in presence of MDOR on Pb transport and in hosting plant response (in terms of plant protection, P uptake and yield) to Pb has been scarcely studied. Therefore, the hypothesis of the present study was aimed to investigate whether: i) the ERM of F. mosseae was directly involved in the transport of Pb to wheat roots, or by contrary, the Pb was immobilized in the external mycelia, ii) the MDOR application had a beneficial impact on the development of ERM, leading to increased Pb transport, and iii) the exogenous carbon (C) substances and nutrients supplied by MDOR might improve P uptake and plant yields.

Material and methods

Materials

Soil site description

The soil used in this experiment was obtained from the field "Cortijo Peinado", a farm in the province of Granada in southern Spain (37°13′N; 3° 45′W), at an altitude of 550 m. The soil was described as Haplic Regosol [START_REF] Ortega | Characterization of soil moisture and temperature regimes in southern Spain[END_REF], and its principal properties are represented in the Table 1. The climate in the region is Mediterranean with the mean annual precipitation about 357 mm with extended periods of drought.

Mean annual temperature is 15.1 °C; the coldest month is January (mean 6.7 °C), and the warmest month is July (mean 24.8 °C) (http://www.aemet.es).

Arbuscular mycorrhizal fungi (AMF) inocula

The mycorrhizal inoculum used, Funneliformis mosseae BEG 12 (Banque Européene des Glomales), was collected from Medicago sativa L. trap pot culture, consisting of soil, spores, mycelia and colonized root fragments (10 sporocarps g -1 , 1-5 spores per sporocarp).

Mycoremediated dry olive residue (DOR)

The mycoremediation of dry olive residue (DOR) was carried out using the fungus Funnelia floccose (Spanish Type Culture Collection, CECT 20449T). The fungal strain was pre-cultured on 2% malt extract agar (MEA) for 2 weeks at 24 °C to maintain the fresh inoculum. DOR, provided by Sierra Sur S.L. (Granada, Spain), was sterilized by autoclave three times at 121 °C for 20 min, frozen and stored at -20 °C for later use. DOR was mycoremediated under solid state fermentation (SSF) conditions as described elsewhere [START_REF] Reina | Solid state fermentation of olive mill residues by wood-and dung-dwelling Agaricomycetes: Effects on peroxidase production, biomass development and phenol phytotoxicity[END_REF]. To do this, four fungal agar plates, homogenized in 80 ml of sterile water, were used to pre-culture F. floccose for one week in a medium containing 18 g of barley seeds and 30 ml of sterile water.

Subsequently, the barley-seed media inoculated with F. floccose was mixed with sterilized DOR in a 1:1 ratio and moistened with sterile water. After four weeks of incubation at 28 ºC, the MDOR was heat-inactivated by autoclaving; thereafter, samples were sieved (5 mm mesh) and the remaining barley seeds were manually removed. The samples were stored at 4 ºC for later use. Prior to the experiment, MDOR, whose chemical composition is shown in Table 2, was characterized.

Experimental design and description of treatments

The experiment was carried out using the container system described by [START_REF] Mäder | Boundaries between soil compartments formed by microporous hydrophobic membranes (GORE-TEXR) can be crossed by vesicular-arbuscular mycorrizal fungi but not by ions in the soil solution[END_REF] with some modifications, which consisted of a central compartment (CC) and two lateral compartments (LCs), 24 cm long, 12 cm high and 2 cm wide, made of polychlrorure of vinyl (PVC) (Supplementary Fig. S1). The central container was separated from the two lateral containers by a screen system composed of 32 μm mesh nylon combined with polytetrafluoroethylene (PTFE) hydrophobic GORE-TEX ® membranes with nominal pore diameters of 5 μm (Fig. 1) which have been shown to efficiently inhibit the mass flow and diffusion of mobile ions in a soil solution in an abiotic system, they can be penetrated by the extraradical mycelium of AMF [START_REF] Mäder | Transport of 15 N from a soil compartment separated by a polytetrafluoroethylene membrane to plant roots via the hyphae of arbuscular mycorrhizal fungi[END_REF].

The experiment had a completely randomized factorial system with three factors of variation consisting of two levels. The first factor comprised soil inoculated with and without F. mosseae. The second one included contaminated soil with Pb or not, and the third was the soil with or without MDOR application (Table 3). The CC of the container system was filled with a mixture of soil and quartz sand (2:3, v/v) which was previously steam-sterilized. 15-d-old wheat plants were planted in the CC of the container systems and only one half received the F. mosseae inoculum (8 g) (Table 3). A similar amount of inoculum filtrate (Whatman no. 1 filter paper) containing soil microflora free of AM fungal propagules was added to the other half. The LCs of each container systems, AMF-inoculated (+ AMF) and non AMF-inoculated (-AMF) in the CC, was filled with sterile soil samples (Control), Pb-contaminated soil samples (+Pb), MDOR-amended soil samples (+ MDOR) or combined Pb-contaminated/MDOR-amended soil samples (+Pb + MDOR). Pb-polluted soil samples were artificially contaminated with an aqueous Pb (NO 3 ) 2 solution up to a final concentration of 3000 mg Pb kg -1 . MDOR was applied and manually mixed with the soil up to a final concentration of 50 g kg -1 , as previously suggested by [START_REF] Siles | Assessing the impact of biotransformed dry olive residue application to soil: Effects on enzyme activities and fungal community[END_REF]. Three replicates of each container system were stablished per each treatment. The experiment was conducted under greenhouse conditions (supplementary light at 25/19 °C and 50% relative humidity), and the wheat plants were watered weekly with 10 ml water without P [START_REF] Hewitt | Sand and water culture methods used in the study of plant nutrition., Sand and water culture methods used in the study of plant nutrition[END_REF] and grown for two months. At the end of the experiment, soil samples contained in each compartment (CC and LCs) were separately collected, and in the case of the LCs, the soil samples were manually mixed to obtain a composite soil. Subsequently, soil samples were homogenised, sieved (2 mm mesh) and kept at 4 ºC for further analysis. Wheat plants were also harvested and separated into roots, shoots and grains and then divided into two sub-samples. The first sub-samples were used to record plant biomass after dying the material at 105 ºC for 72 h. The second sub-sample was air-dried and analyzed for total Pb and P content.

Analyses

Analytical methods

For the content of Pb and P the aliquots (0.5 g) of soil samples or plant biomass were decomposed in a digestion vessel contained 8 ml of 65% HNO 3 and 2 ml of 30% H 2 O 2 . The mixture was heated in an Ethos 1 (MLS GmbH, Germany) microwaveassisted wet digestion system for 30 min at 220 ºC. After cooling, the digests were transferred to 20 ml glass tubes, which were filled to the mark with deionized water.

Each extraction was carried out in triplicate. Extracts were centrifuged in a Hettich Universal 30 RF (Germany) at 3000 rpm (i.e., 460 g) for 10 min at the end of each extraction procedure and the supernatants were stored at 4 ºC prior to analysis.

Inductively coupled plasma-atomic emission spectrometry (ICP-OES, Agilent 720, Agilent Technologies Inc., USA), equipped with a two-channel peristaltic pump, a Struman-Masters spray chamber, and a V-groove pneumatic nebulizer made of inert material, was used to determine the Pb and P contents of soil and plant digests.

Quantification of the root length colonization percentage and external mycelium

The percentage of root length colonization was estimated using the methodology described by [START_REF] Giovannetti | An evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots[END_REF] by randomly selecting fresh root systems which were cleared and stained [START_REF] Phillips | Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection[END_REF]. 3 g of fresh soil were sieved through 700 and 100 μm nylon mesh to retain the extraradical mycelium. The fungal hyphae in the 100 μm sieve were transferred to a nylon membrane (32 μm) and stained with fuchsine acid solution (0.05%); hyphae length was then quantified under a stereoscopic microscope at 100× [START_REF] Giovannetti | An evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots[END_REF].

Determination of easily extractable glomalin-related-soil protein (EE-GRSP)

The EE-GRSPs were determined after removing 1 g of soil with 8 ml of citrate buffer (20 mM, pH 7.0) followed by autoclaving at 121 °C for 30 min [START_REF] Wright | A survey of soils for aggregate stability and glomalin, a glycoprotein produced by hyphae of arbuscular mycorrhizal fungi[END_REF]. Samples were then centrifuged at 10,000g for 15 min and filtered through a Whatman No. 1 filter. Protein content was determined using the Bradford Bio-Rad Protein Assay (Bio-Rad Labs) with bovine serum albumin as standard [START_REF] Wright | Time-course study and partial characterization of a protein on hyphae of arbuscular mycorrhizal fungi during active colonization of roots[END_REF].

Statistical analysis

Data analyses were carried out using IBM SPSS Statistics version 26.0 software.

All data presented are the means of three replicates with standard deviation (mean ± DE). One-way analysis of variance (ANOVA), followed by the post hoc Tukey HSD test (p<0.05), were used to evaluate: i) the Pb transport and plant variables (P uptake and biomass) when AMF was present or absent and, ii) the ERC development (in terms of fungal hyphae density and EE-GRSPs) between the LCs and CC. Using one-way ANOVA, we statistically analysed the effect of Pb contamination, MDOR and the combination of MDOR and F. mosseae inoculation on: i) Pb accumulation in wheat plants, ii) fungal hyphae density, iii) EE-GRSPs, iv) the percentage of root colonization, v) P uptake and vi) plant biomass. Differences among treatments, which were analysed by post-hoc tests, are indicated by different letters.

Results and discussion

The role of the ERM in Pb transport

The transport of Pb from the lateral compartments to the central compartment through the ERM was evaluated by measuring Pb content in wheat plants. In the absence of F. mosseae (-AMF), wheat roots unexpectedly accumulated a relatively small amount of Pb after Pb-polluted soil samples were added to the LC (Table 4).

While this could indicate that Pb is diffused through the screen system, no statistical differences were found between the Control, + Pb, + MDOR and + Pb + MDOR treatments (Table 4). This could be related to the presence of trace Pb content in the soil used, with values observed to be below plant toxic limits (Table 1). Conversely, the presence of F. mosseae (+AMF) in the container system significantly increased Pb levels in wheat roots with respect to the container system without AMF (-AMF) when the soil samples were artificially contaminated (Table 4). After evaluating the translocation of Pb to the upper parts of wheat plants, interestingly, we detected no Pb in either wheat shoots or grains. This could point to the involvement of the F. mosseae ERM in the transport of Pb from the lateral compartment to wheat roots. This strategy might be considered as a plant protection mechanism which decreased the translocation of Pb to the upper parts of the plant in the presence of MDOR, as other authors suggested [START_REF] Arriagada | Contribution of arbuscular mycorrhizal and saprobe fungi to the tolerance of Eucalyptus globulus to Pb[END_REF].

The addition of MDOR (+ MDOR) to soil samples showed the same values for

Pb content regardless of the presence or absence of F. mosseae (-AMF/ + AMF) (Table 4). By contrast, we observed a sharp increase in Pb content when Pb-contaminated soil samples were treated with MDOR (Pb+-MDOR) in the presence of F. mosseae, which differed significantly from + Pb soil samples (Table 4). This could indicate that the nutrients supplied by MDOR application could be involved in the stimulation of the ERM which in turn favoured the transport of Pb through the ERM with its subsequent accumulation in roots decreasing the translocation of Pb to the upper parts of the plant in the presence of MDOR. However, this hypothesis requires further investigation.

Regardless of the soil treatment used, the development of the external mycelium and EE-GSRPs was less marked in the LCs than in the CC (Fig. 1a-b), given the presence of the F. mosseae inoculum in these soil samples. The exposure of AMF to Pb did not significantly reduce the external mycelium, whose values were similar to those for control treatments (Fig. 1).

F. mosseae clearly possesses a mechanism of protection against Pb toxicity

which presumably immobilizes Pb in fungal structures due to the great sorption and accumulation capacity of the ERM (García-Sánchez et al., 2016) (Fig. 1a). MDOR supplies soils with labile C compounds as a result of fungal transformation, which can be used as an energy source by microorganisms, as reported [START_REF] Siles | Assessing the impact of biotransformed dry olive residue application to soil: Effects on enzyme activities and fungal community[END_REF]. This is in line with our finding that MDOR (+MDOR), rather than the control treatment, greatly enhanced the external mycelium (Fig. 1a). The combined application of Pb and MDOR (+Pb + MDOR) also stimulated the development of ERM in Pb-contaminated soil samples (Fig. 1a). Thus, MDOR stimulates AMF development by increasing nutrient solubilisation, which boosts soil aggregates, thus favouring AMF hyphal growth, as described by [START_REF] García-Sánchez | Implications of mycoremediated dry olive residue application and arbuscular mycorrhizal fungi inoculation on the microbial community composition and functionality in a metal-polluted soil[END_REF].

According to [START_REF] Purin | The arbuscular mycorrhizal fungal protein glomalin: Limitations, progress, and a new hypothesis for its function[END_REF], glomalin plays an important environmental role in soil by boosting feedback between plant production, soil aggregation and external AMF hyphal growth. We observed no significant differences in glomalin content in the CC and LCs between the treatments tested except in the case of + Pb and +Pb +MDOR (Fig. 1b). The presence of Pb in soil samples greatly increased glomalin content in the CC as compared to control samples, while Pb levels in the CC were similar following the control and +Pb treatments (Fig. 1b). This could indicate that Pb is immobilized by surface complexation with cysteine-containing ligands of glomalin as reported elsewhere [START_REF] González-Chávez | The role of glomalin, a protein produced by arbuscular mycorrhizal fungi, in sequestering potentially toxic elements[END_REF]. Increased glomalin content is usually observed following the addition of organic sources, such as manure, crop stubble and compost [START_REF] Curaqueo | Inoculation with arbuscular mycorrhizal fungi and addition of composted olive-mill waste enhance plant establishment and soil properties in the regeneration of a heavy metalpolluted environment[END_REF]. This finding is in line with our results which found that MDOR increased glomalin content in both the CC and LCs, whose glomalin levels differed significantly from those observed in control samples (Fig. 1b). Contrary to expectations, the addition of MDOR to Pb-contaminated soil samples did not boost glomalin production significantly in LC with respect to +Pb treatments. This contradicts other studies which report that the quantity of glomalin extracted from soils is typically related to AMF hyphal density [START_REF] González-Chávez | Natural attenuation in a slag heap contaminated with cadmium: The role of plants and arbuscular mycorrhizal fungi[END_REF][START_REF] Lovelock | Using glomalin as an indicator for arbuscular mycorrhizal hyphal growth: an example from a tropical rain forest soil[END_REF]. However, we found that the increase in the external mycelium exceeded that in glomalin content when MDOR was added to Pb-polluted soils. Nevertheless, reduction in glomalin production could be due to the presence of certain chelating humic substances supplied by MDOR rather than to the presence of Pb.

Role of AMF in plant protection

We also evaluated the role of mycorrhiza in plant protection, which is well known to lead to an increase in biomass and P plant uptake. Some studies indicate that AMF have the ability to absorb P from adjacent soil which is rapidly translocated to the fungus-plant interface and subsequently absorbed by roots [START_REF] Smith | Mycorrhizal fungi can dominate phosphate supply to plants irrespective of growth responses[END_REF][START_REF] Smith | Functional diversity in arbuscular mycorrhizal (AM) symbioses: the contribution of the mycorrhizal P uptake pathway is not correlated with mycorrhizal responses in growth or total P uptake[END_REF]. This explains the well-known positive effects of AMF on plant P nutrition, especially under stressful conditions. Figure 2 shows the results of the analysis of P content and plant biomass in wheat roots, shoots and grains.

The F. mosseae inoculation resulted in an overall increase in P plant uptake in relation to the non-inoculated container system, which could be associated with the higher levels of wheat biomass (Fig. 2a-f). Interestingly, P uptake by wheat roots in the presence of AMF was not greatly affected by exposure to Pb, as similar P levels were found in both control and +Pb treatments (Fig. 2a). This could, in turn, be explained by the similar levels found for root biomass following these treatments (Fig. 2d). As suggested by others authors, our results could indicate that P uptake is superceded by Pb transport in soils that are highly contaminated by this metal [START_REF] Xiong | Bioaccumulation and physiological effects of excess lead in a roadside pioneer species Sonchus oleraceus L[END_REF]. ERM development was not greatly affected by exposure to Pb, which could be explained by its possible translocation to root cells rather than by its possible accumulation in vesicles and spores, as some authors have reported [START_REF] Salazar | Pb accumulation in spores of arbuscular mycorrhizal fungi[END_REF]. This hypothesis concurs with our finding that the presence of Pb did not have a significant effect on AMF-root colonization in soil samples (Table 4). [START_REF] Arriagada | Contribution of arbuscular mycorrhizal and saprobe fungi to the tolerance of Eucalyptus globulus to Pb[END_REF] have reported a similar finding with respect to Eucalyptus plants inoculated with Rhizophagus irregularis in soil highly contaminated by Pb. On the other hand, we observed no significant increase in wheat and grain P uptake in Pb-polluted soil samples (+Pb) as compared to control treatments (Fig. 2b), which is consistent with the unchanged levels of shoot and grain biomass (Fig. 2e).This could indicate that AMF play a major stabilizing role in plant protection by reducing Pb translocation to shoots. Thus, Pb, which is transported to the cytosol across the cell wall and cell membrane via active metabolism, is accumulated inside the cell and/or via passive metabolism by which Pb adheres to fungal surface molecules (sorption) [START_REF] Mishra | Recent advances in microbial metal bioaccumulation[END_REF], which could also explain the decrease in root biomass. However, the binding of Pb to fungal tissues associated with roots could be involved in creating a physical barrier against Pb translocation to the plant [START_REF] García-Sánchez | Physiological response of mycorrhizal symbiosis to soil pollutants[END_REF].

MDOR significantly improved P uptake by wheat roots and shoots (Fig. 2a, andb), which could be associated with increased biomass, but only in roots (Fig. 2d, e andf). The addition of MDOR to Pb-polluted soil samples (+Pb+MDOR) resulted in increased P uptake which proved to be higher than that observed after +Pb treatment (Fig. 2a). However, root biomass did not increase significantly, with +Pb and +Pb+MDOR treatments producing similar results. An increase in P content caused by MDOR might determine the transport of P and Pb through the ERM to wheat roots.

However, Pb could be transferred from fungal hyphae to root cells which, in turn, would lead to a reduction in root biomass. This reasoning concurs with our finding that MDOR did not increase AMF root colonization (Table 5). Conversely, no differences in wheat shoot and grain P content were observed between +Pb and +Pb+MDOR treatments, which, in turn, did not modify shoot biomass levels. This could point to possible competition between Pb and P for the same P transporter in plant plasma membranes, as some authors such, as [START_REF] Smith | Arsenic uptake and toxicity in plants: integrating mycorrhizal influences[END_REF], have reported with regard to other metals. However, AMF could induce a resistance mechanism based in the uptake of both, P and metals, through the same phosphate transporters or by contrary can discriminate between P and metals which in turn reduces the metal uptake via the ERM resulting in lower toxicity [START_REF] Christophersen | Unraveling the influence of arbuscular mycorrhizal colonization on arsenic tolerance in Medicago: Glomus mosseae is more effective than G. intraradices, associated with lower expression of root epidermal Pi transporter genes[END_REF]. In our case, MDOR could stimulate other AMF plant resistance mechanisms involving reduced Pb uptake due to the suppression of high-affinity P uptake systems.

Conclusions

This study highlights the feasibility of using a container system with a GORE-TEX ® hydrophobic membrane to evaluate the role of the F. mosseae ERM in Pb transport. Our results show that the ERM is responsible for Pb accumulation in wheat roots. F. mosseae also protected wheat plants by stabilizing Pb content in roots and by preventing its translocation to shoots and grains in the upper part of the plant. Pb may have been transferred by AMF to the cytosol through the cell wall and membrane via active metabolism and have been accumulated inside the cell and/or via passive metabolism, by which Pb adheres to fungal surface molecules (sorption) as a result of decreased root biomass. In addition, MDOR greatly improved P uptake which probably led to a reduction in Pb uptake. The development of the ERM was unaffected by exposure to Pb, while the nutrients supplied by MDOR significantly increased fungal hyphae density and glomalin production. Our study highlights the transport of Pb through the ERM, its accumulation in wheat roots and how the increase in the uptake of P rather than Pb by MDOR is a plant protection mechanism triggered by AMF. 4 The Pb content (mg kg -1 ) accumulated in wheat roots, shoots and grains grown in the central compartment (CC) of the container system inoculated or not with F. mosseae in presence of: i) sterile soil (control) or ii) Pb-polluted soil (+Pb), or iii) MDOR-amended soil (+MDOR), or iv) Pb-polluted/MDOR amended soil (+Pb +MDOR) in the lateral compartment (LC) at the end of the experiment.

Treatments

Fig. 1 .

 1 Fig. 1. External mycelium (a) and easily-extractable glomalin-related soluble protein

Fig. 2 .

 2 Fig. 2. P content (mg kg-1) and dry biomass (mg) in roots, shoots and grains of wheat

  Fig 1

  detection limit Data expressed as mean and standard deviation (n = 3). Same letters indicated not statistical significance at p<0.05 between treatments. *Asterisk shown statistical significance at p<0.05 in presence or absence of F. mosseae.

Table 1

 1 Chemical properties of soil (n = 3).

	*Total organic carbon (TOC).
	*Water soluble organic carbon (WSOC).
	*Total nitrogen (TN).
	(Siles et al., 2014)

Table 2

 2 Main characteristics of MDOR.

	MDOR

Table 3

 3 Experimental design.

	Treatments	Soil samples in each compartment
		CC	LC (both sides)
	Control	̶ AMF-inoculated soil	Sterile soil
		+ AMF-inoculated soil
	+ Pb	̶ AMF-inoculated soil	Pb-polluted soil
		+ AMF-inoculated soil
	+ MDOR	̶ AMF-inoculated soil	MDOR-amended soil
		+ AMF-inoculated soil
	+ Pb + MDOR	̶ AMF-inoculated soil	Pb-polluted and MDOR amended
		+ AMF-inoculated soil	soil samples

Table

Table 5

 5 Percentage of AMF-root colonization.

	Treatments	% Mycorrhizal
		colonization
	Control	43.0 ± 8.3
	+MDOR	44.8 ± 1.7
	+Pb	39.0 ± 5.5
	+Pb+MDOR	42.0 ± 5.2

Acknowledgements

The study was funded by the Spanish Ministry of Science and Innovation (project RTI2018-094327-B-I00). We also wish to thank Michael O'Shea for proofreading the article.


Zhenyu Yao

Xing Lin

Remi Chauvin
email: chauvin@lcc-toulouse.fr

Lianhui Wang

Emmanuel Gras

Xiuling Cui

Communication Phosphine-phosphonium ylides as ligands in palladium-catalysed C2-H arylation of benzoxazoles

Keywords:

As balanced electron-rich P,C-chelating ligands, phosphine-phosphonium-ylides are considered for their ability to in situ promote palladium-catalysed direct sp 2 -C-H arylation. Using methyl phosphonium salts of 2,2'-bis(diphenylphosphino)-1,1'-binaphtyl ("methyl-BINAPIUM") as ylide precursors under optimized reaction conditions, arylation of benzoxazole was found to proceed in moderate to high yield to give functional 2-aryl benzoxazoles. A strong anion effect of the non-salt free ylide was evidenced (TfO -> I -> PF6 -≈ salt-free). This first example of phosphonium ylides as ligands in catalytic C-H activation extends the prospect of their general implementation in homogeneous transition metal catalysis.

Transition-metal-catalyzed direct C-H bond functionalization have experienced tremendous developments over the past decade, allowing novel key step approaches in the synthesis of natural products and therapeutic agents [1]. These reactions have required catalysts with high kinetic reactivity to ensure broad substrate scope, lower reaction temperature and catalyst loading, short time and favourable product distributions. Ligand development has also played a central role. While the efficiency of phosphine and N-heterocyclic carbenes (NHCs) as σ-donating ligands of catalytic transition metal centers has been widely exemplified [2,3], general guidelines for the design of suitable ligand scaffolds to promote C-H arylation are still lacking. In this context, however, beyond electron-balanced monodentate phosphoramidites [4] or imidazolium ylides [4b], bidentate amino/imino-amides, such as mono-protected amino acids (MPAAs), were shown to be behave as LX N,N-ligands in enantioselective sp 3 -C-H arylation, or through reverse polarity from aryl boronates [5]. Noteworthy is the recently reported use of a P,C-ligand for Pd-catalysed 2-arylation of benzoxazoles (actually a phosphine-NHC ligand in a labile PNC pincer framework) [6]. Such ligands enable access to a structurally diverse range of valuable products via C-H bond activation with superior chemo-, regio-and enantio-selectivity. But the potential of ligands in improving catalyst efficiency of C-H bond activation remains to be further explored. As a new prospect of our continuing studies on direct sp 2 -C-H bond activation [7], we envisaged the implementation of particular P,C-ligands, i.e. phosphino-phosphonium ylides [8], hereafter referred to as "PhosphYls" (Scheme 1). These ligands were shown to be stronger σ-donating ligands than NHCs [8b].

By design, suitable PhosphYls are of limited structural variety, as they must be derived from monoalkyl-triarylphosphoniums, with a single type of acidic P + CH unit with low steric hindrance and moderate pKa value, such as those of the P + CH3 moiety, to allow selective deprotonation and efficient coordination (Scheme 1). Under these requirements, the simplest PhosphYls are ylides of phosphonium salts of o-bis(diphenylphosphino)benzene (dppbz) such as L1•HX, which were shown to provide catalysts for malonate C-allylation (Pd) [9], asymmetric alkene hydrogenation or ketone hydrosilylation (Rh) [10], and theoretical olefin metathesis (Ru) [11]. Alternative PhosphYls satisfying the above requirement are methylenephosphorane derivatives of 2,2'-bis(diphenylphosphino)-1,1'-binaphtyl (BINAP), such as the methyl-BINAPIUM ylide L2 [12], used in Rh-catalysed hydogenation and hydrosilylation [13], or its methoxycarbonyl-stabilized versions, Yliphos (R = CO2Et, Scheme 1) used in Pd-catalysed C-allylation [14] (not to be confused with YPhos, ylido-phosphines acting as strongly donating P ligands in Pd or Au catalysts) [15]. As the low pKa values of Yliphos favours partial de-coordination of the stabilized free ylide [17], exploratory investigations prompted the choice of the rac-methyl-BINAPIUM ylide L2 as a benchmark PhosphYl. On the tracks of pioneering works [16] and recent advances in Pd-catalysed C-H arylation of heterocycles [17], the transformation was chosen as a model to further evaluate the ability of PhosphYls to form in situ catalysts. Scheme 1. Phosphine-phosphonium salts and ylides there of (PhosphYls) as LX-chelating ligands of late transition metals M (e.g. Ru, Rh, Pd).

A R T I C L E I N F O A B S T R A C T X PPh 2 X = I,
Indeed, while their electron-donating ability is expected to facilitate the step of the aryl halide oxidative addition, their bulky chelating characters appear appropriate for steric protection of the Pd center.

With the view to accessing functional hetero-aromatic systems without resorting to an ortho-directing group, the reaction of the benzoxazole substrates 1 with various aryl halides 2 was considered [7]. The target 2-aryl-benzoxazole products 3 indeed exhibit versatile properties [18], going from antibacterial [19], cholesterol ester transfer protein inhibition [20], adenosine receptor antagonism [21], amyloidogenesis inhibition [22], and thioflavine-like binding affinity to β-amyloid plaques [23], to fluorescence in the solid state [24].

As a soluble Pd(II) salt widely used in C-H activation catalysis also exhibiting propensity to form Pd-C(sp 3 ) bonds [25], Pd(OAc)2 was envisaged as catalyst precursor, to which non-saltfree PhosphYls could be added, after in situ deprotonation of parent phosphoniums. Considering the possible effect of the anion X -in non-salt-free ylides, well known in the Wittig reaction [26], several phosphonium salts L2•HX were compared (X= I, TfO, PF6).

Benzoxazole 1 and bromobenzene 2a, or 4-bromoanisole 2b, were selected as the model reactants. From a preliminary screening under various conditions, the following conditions were adopted: DMSO as a solvent, Cs2CO3 as a base and Pd(OAc)2 as catalyst precursor at 50 °C for 14 h (Table 1). At the outset, commercially available monodentate phosphines, PPh3, PCy3, PtBu3, were first selected as standard reference ligands, giving 3a in 48%, 45% and 37% yield, respectively (entries 1-3). Bidentate phosphines BINAP, dppm, o-dppbz, dppe and dppf also gave medium yields of 53%, 44%, 51%, 47% and 36%, respectively (entries 4-8). Under the same conditions, using the methylphosphonium salt L1•HX (X = I, TfO, PF6) of o-dppbz as pro-ligand (non-salt-free PhosphYls L1/HX or L2/HX were separately prepared by deprotonation of L1•HX or L2•HX with nBuLi in DMSO solution during 15 min at room temperature), the coupling product 3a was isolated in 53%, 59% and 43% yield, respectively (entries 9-11). These results are similar to those obtained by bidentate phosphines, and then it was decided to change the o-dppbz backbone to the BINAP backbone. The use of the BINAPIUM ylide generated from L2•HI gave 3a in an improved yield of 65% (entry 12). The same ylide L2 generated from L2•HOTf gave 3a in a much higher yield of 93%, revealing a dramatic counterion effect (entry 13). This was confirmed by the result of L2⋅HPF6, giving 3a in a twice lower yield of 50% (entry 14), thus showing a correlation of the yield with the association effect between the anion and the Pd and/or P + centers [27].

A systematic screening of the reaction conditions was then undertaken (Table 1). In the absence of any potential P-ligand, the reaction was found to proceed with 16% yield only (entry 15). It was also found that both the solvent and base are of critical importance. By reducing the amounts of Cs2CO3 from 1.0 equiv. to 0, the yields in 3a are reduced to 41%, 33% and 0% respectively (entries 16 -18). Upon replacement of Cs2CO3 by weaker bases, such as Et3N or PPh3, product was not observed (entries 19 and 20). The use of a stronger base, such as NaHMDS, t BuOK or MeONa, gave lower yields in the range of 27%-39% (entries [21][22][23]. When the same bases were used in THF instead of DMSO, the yields dropped to 12%-20% (entries [24][25][26]. Other solvents, such as THF, DMF, MeCN, EtOH or NMP, also resulted in a dramatic decrease of the yield (< 15%, details see the Supporting information). a Reactions performed using 1.0 equiv. of 1, 1.2 equiv. of 2a, 5 mol% of Pd(OAc)2, 5 mol% of PhosphYL L1 or L2, on a 0.4 mmol scale in 2 mL of dry solvent, N2, 50 ℃, 14 h. b Isolated yields. c 5 mol% of salt-free ylide was used (preparation of salt-free ylide described in Supporting information). d 5 mol% of LiOTf was added.

The yield in 3a was decreased from 93% (entry 13) with the salted ylide (L2/LiOTf) to 46% (entry 27) with the salt-free ylide, similar to the 50% yield (entry 14) obtained with the pseudo-saltfree ylide L2/LiPF6 (contrary to TfO -, PF6 -is totally inert with respect to the Pd and P + centers). Under the same conditions, 1 equiv. of LiOTf was added to the salt-free ylide L2, the yield in 3a was restored to 80%, thus demonstrating the key role of LiOTf (entry 28). The effect of LiOTf could be attributed to the interaction ability of the TfO -anion with the P + and/or the Pd(II) centers, by either electrostatic contact or coordination bonding [27].

With the optimized reaction conditions in hand, the scope of the substrates was examined (Scheme 2). Benzoxazole 1 reacted smoothly with bromobenzene 2a and its derivatives 2b-2t to give the anticipated products 3a-3t in moderate to high yields (35%-93%). Aryl bromides with a methyl group at the para-or metaposition (2c-2d) gave the corresponding products 3c and 3d in 82% and 79% yields, respectively. When the methyl group was attached at the ortho-position, a lower 54% yield was obtained, revealing the steric hindrance of the reacting position. The electron-withdrawing substituent CF3 at the para-position of 2e allowed isolation of the aryl-benzoxazole 3e in a slightly higher yield (84%), while electron-donating OMe counterpart has the opposite effect on the corresponding product 3f (70%). Nevertheless, both weakly electron-withdrawing (F, Cl) and strongly electron-donating (NMe2, t Bu) substituents gave higher yields (81%-88%), showing that the limiting step is not, at least not always, an SNAr-like oxidative addition process of the C-Br bond to the electron-rich Pd center (a favored by electronwithdrawing substituents on the aryl ring) [28]. For substituent F, however, the classical trend is restored through the relative reactivity of the para and ortho aryl bromide derivatives 2i and 2m (giving 3i and 3m in 81% and 80% yields) vs. the meta derivative 2k (giving 3k in 35% yield). This trend is also observed for the CF3 substituent, giving 41% yield in 3q for the meta position, vs. 84% yield in 3e for the para position. For a non-fluorinated electron-withdrawing substituent, the position effect of the formyl group remains consistent but much less dramatic, giving 80% yield in 3r for the meta position, vs. 86% yield in 3s for the para position. For the donating OMe substituent, a remarkable counter-effect of the position is observed: while para-and meta-bromoanisoles led to the expected products 3f and 3p in 70% and 62% yields, respectively, the more hindered ortho-bromoanisole 2o gave 3o with a significantly higher yield of 80%, revealing an assistance of the OMe group. The isosteric, but non-coordinating orthobromotoluene 2b gives 3b in 54% yield only. 2-Bromonaphthalene and brominated heterocycles such as 2bromopyridine, 2-bromothiophene, and 3-bromothiophene were also found to be suitable substrates, affording the corresponding products 3t and 3v-3x in 60%-73% yields. Scheme 2. Substrate scope. All reactions were carried out using 1 (0.4 mmol), 2 (0.48 mmol), Pd(OAc)2 (5 mol%), L2/LiOTf (5 mol), Cs2CO3 (2 equiv.) in DMSO (2.0 mL) at 50 o C for 14 h. Isolated yields.

On the basis of the previous literature reports and above experimental results, a tentative mechanism is proposed in Scheme 3. The PhosphYl ligand L2 could first coordinate in situ to the Pd(II) center, prior to or after in situ reduction of Pd(II) (e.g. by the phosphine end of L2 or DMSO) to Pd(0) in a zwitterionic palladate complex A similar to previously invoked. [7]. Nevertheless, the conversion σ/π-C → 3 + A could also proceed via a Pd-C-oxazolyl intermediate F from which the products would be directly released by reductive elimination, as invoked in the quinoline series [9,30]. The propositions remain, however, quite speculative, as preliminary attempts did not allow isolation of any pre-catalytic L2/Pd complex (mixture of several unidentified species were observed by 31 P/ 1 H NMR spectra).

In conclusion, the disclosed C-H arylation allows 2-arylbenzoxazoles to be produced in moderate to high yields over a night at 50 °C. The use of the particular PhosphYl BINAPIUM ylide L2 proves to be superior to the use of classical phosphine ligands. The in situ catalytic system happens to be general for arylbromide substrates and specific hetero-unsaturated C-H substrates, i.e. benzoxazole. It is indeed found to be ineffective for related benzothiazole and imidazole substrates under standard conditions, but is thus selective. Considering the adjustable steric and electronic features of the ylide structure and the variety of ways to generate it from phosphonium precursors, prospects of optimization are open, in particular for extending the substrate scope. In this context, the axially chiral BINAPIUM backbone allows envisaging applications in enantioselective catalysis from prochiral substrates, e.g. in Cammidge-like coupling of 1-halonaphthalenes with dissymmetric bulky (hetero)cycles [31]. Beyond the scope of the present report of in situ catalysis investigations, further efforts in coordination chemistry will be undertaken with the view to elucidating the actual structure of the catalyst. Finally, further extension of the potentialities of generic PhosphYl ligands in transition metal catalysis of other organic transformations deserves continuing investigations.

  Scheme 3. Putative mechanism for PhosphYl-Pd-catalysed 2-arylation of benzoxazole: alternative to the depicted Pd(0)/Pd(II) cycle, a Pd(II)/Pd(IV) cycle might also be envisaged.

Table 1

 1 Ligand effect in Pd-catalysed 2-phenylation of benzoxazole 1 with bromobenzene 2a.

				a
		N				[Cat.] 5 mol%		N
	1	O	H	Br	2a	Cs 2 CO 3 , DMSO, N 2 , 50 o C	O	3a
	[Cat.]: From Pd(OAc) 2 + {Ligand precusor + nBuLi (15 min)}(3 h)
	Entry Ligand	X	Base (equiv.)	Solvent		Yield
									(%) b
	1	PPh3	-		Cs2CO3 (2.0)	DMSO		48
	2	PCy3	-		Cs2CO3 (2.0)	DMSO		45
	3	P t Bu3	-		Cs2CO3 (2.0)	DMSO		37
	4	BINAP	-		Cs2CO3 (2.0)	DMSO		53
	5	dppm	-		Cs2CO3 (2.0)	DMSO		44
	6	o-dppbz	-		Cs2CO3 (2.0)	DMSO		51
	7	dppe	-		Cs2CO3 (2.0)	DMSO		47
	8	dppf	-		Cs2CO3 (2.0)	DMSO		36
	9	L1	I		Cs2CO3 (2.0)	DMSO		53
	10	L1	TfO	Cs2CO3 (2.0)	DMSO		59
	11	L1	PF6	Cs2CO3 (2.0)	DMSO		43
	12	L2	I		Cs2CO3 (2.0)	DMSO		65
	13	L2	TfO	Cs2CO3 (2.0)	DMSO		93
	14	L2	PF6	Cs2CO3 (2.0)	DMSO		50
	15	-		-		Cs2CO3 (2.0)	DMSO		16
	16	L2	TfO	Cs2CO3 (1.0)	DMSO		41
	17	L2	TfO	Cs2CO3 (0.5)	DMSO		33
	18	L2	TfO	-	DMSO		N.R.
	19	L2	TfO	NEt3 (2.0)	DMSO		N.R.

Acknowledgments

We gratefully acknowledge the financial support from the National Natural Science Foundation of China (Nos. 21572072 and 21602064), 111 Project (No. BC2018061), and Postgraduate's (Y.Z. Yao) Innovative Fund in Scientific Research of Huaqiao University.