H Hu

M Pauly

O Felix

G Decher
email: decher@unistra.fr

Spray-assisted alignment of Layer-by-Layer assembled silver nanowires: a general approach for the preparation of highly anisotropic nano-composite films

The present article focuses on the build-up and the properties of oriented silver nanowire monolayer films and Layer-by-Layer assembled multilayer films. We describe the templatefree oriented spray-assisted assembly of silver nanowires at solid/air-interfaces using Grazing Incidence Spraying, a simple and versatile approach that allows the formation of highly oriented thin films with a tunable density and in-plane orientation. Depending on the spraying conditions the nematic order parameter, which describes the angular spread of misaligned nanowires, can be as high as 0.98 (a value of 1.00 corresponding to a perfectly parallel alignment). The combination with the Layer-by-Layer assembly allows building multilayer thin films possessing in-plane anisotropy. In order to demonstrate that the local alignment does not cancel out on the macroscopic scale but leads to direction-dependent properties, we use linearly polarized UV-Vis-NIR spectroscopy to probe the selective excitation of the transverse and longitudinal localized plasmon resonances of the nanowires. The polarization efficiency of the thin films increases strongly with the in-plane density, the degree of orientation, and the number of silver nanowire layers. Multilayer films containing 4 layers of nanowires oriented in the same direction reach a polarization efficiency of up to 97% in the near-infrared region.

Introduction

The development of nanocomposite materials of high compositional and structural complexity, for example bio-inspired materials, is generally thought to be very important but not much explored due to the lack of appropriate methods for their preparation. 1 Interesting materials properties arise either from the composition of a material (i.e. the number and the chemical nature of its constituents) and/or from the complexity of its structure. 2 However, the number of different constituents in man-made materials is presently limited and their hierarchical organization is often very simple. With respect to the preparation of multimaterial thin films with a high level of control over the spatial positioning of their constituents, Layerby-Layer (LbL) assembly has gained its merits as a simple and highly versatile nanofabrication method. 3,[START_REF] Seyrek | Polymer Science: A Comprehensive Reference[END_REF] Multifunctional coatings with unique biomedical, electrical, optical, mechanical and magnetic properties can routinely be prepared by LbLassembly [START_REF] Seyrek | Polymer Science: A Comprehensive Reference[END_REF][START_REF] Srivastava | [END_REF] as it allows the combination of a large variety of components such as polyelectrolytes, spherical nanoparticles, 6 nanorods 7 and nanowires 8 in a single LbL-film. While the sequence of components in layered multi-material films can be very well controlled by LbL-assembly, tuning of the in-plane anisotropy has not yet been achieved. This has been a severe drawback since one-dimensional (1D-) nanomaterials, such as nanotubes, nanorods and nanowires, have been receiving increasing attention in the last few years because of their anisotropic physical properties and their potential applications in opto-electronic nanodevices. [9][10][11] Among the broad range of inorganic 1D-nanomaterials, silver nanowires (AgNWs) are one of the most investigated ones 10,12 as they could find applications as transparent conductive electrode materials, 13 plasmonic waveguides, 9,14 antennas 15 or sensors. 16,17 A variety of methods, 18 including Langmuir-Blodgett deposition, 19,20 blown bubble film transfer, 21 dip-coating, 22 H-bar dip-coating, 23 doctor-blade casting, 24 capillary printing 25 and the use of electrical or magnetic fields, 26 have been suggested to control the assembly and inplane alignment of rod-like nano-objects. Other techniques use flow-induced shear in fluidic cells, 27 in a capillary, 17,28 or by evaporating spray droplets arriving on hot surfaces. 29 However, these methods are limited in their ability to yield uniformly aligned films over large areas, they are restricted to small groups of components or they fail to produce compositional control along the z-direction. Recently, we have introduced "Grazing Incidence Spraying" (GIS) as a new method for the preparation of oriented monoand multilayer films of cellulose microfibrils 30 or of monolayers of metallic nanowires and nanorods. 31 While it is somewhat related to "blow-alignment", 32 or to "combing", 33 GIS is based on spray-assisted shear-induced assembly and can be combined with the LbL-approach in order to extend it toward the preparation of in-plane anisotropic multilayer films. In a previous study, 31 we have shown that oriented monolayer thin films of 1D-nanoparticles such as gold nanorods and AgNWs can be efficiently oriented using GIS. We have also shown that the quality of alignment increases with the length of the deposited nano-objects. Here we use GIS for the controlled alignment of AgNWs in mono-and multilayer architectures for preparing well-aligned thin films of controlled thickness homogeneous over large areas. In order to demonstrate that the local topological orientation of 1D-nano-objects induces macroscopic anisotropic properties, we use linearly polarized UV-Vis-NIR spectroscopy to probe the selective excitation of the transverse and longitudinal localized plasmon resonances of the nanowires. The anisotropic character of the physical properties (i.e. the polarization efficiency) of these mono-and multilayer architectures strongly increases with the in-plane density, the degree of orientation, and the number of AgNW layers.

Results and discussion

Monolayers of oriented silver nanowires

AgNWs were synthesized by a polyol reduction procedure as previously reported in the literature (for a detailed description, see the ESI †), 17,34 which results in nanowires that have a cross section of 47 ± 6 nm and a length of 4.2 ± 1.5 μm, as determined by Transmission Electron Microscopy (TEM) (Fig. S1 †). The oriented deposition of AgNWs on different surfaces was carried out by spraying the nanowire suspension at an angle of 15° onto a receiving surface using the GIS approach (Fig. 1a), which results in a dense monolayer of aligned AgNWs (Fig. 1b). AgNW monolayers were prepared on glass slides or on silicon wafers, both of which were previously coated with a layer of poly(ethyleneimine) (PEI).

The distribution of the angle θ between each nanowire principal axis and the spraying direction is extracted from the Scanning Electron Microscopy (SEM) pictures using the plugin OrientationJ 35 developed for ImageJ, 36 which is based on the analysis of the structure tensor in a local neighbourhood (Fig. 1c-e and S2 †). A 2D nematic order parameter S can be calculated from this angular distribution

S2D = ⟨2 cos 2 θ -1⟩ (1)
where the chevrons denote the weighted average over the distribution of θ, S = 0 corresponds to a random distribution (isotropic films, i.e. θ is equally distributed between 90° and -90°) and S = 1 corresponds to a perfectly parallel orientation (fully anisotropic films, i.e. θ = 0° for all AgNWs). The highest ordering of AgNWs (Fig. 1) was obtained at 1 cm downstream from the impact point of the spray jet on the substrate (S = 0.98, Fig. 1b andc). This corresponds to ∼97% of the nanowires aligned within ±15° of the spraying direction, and to ∼66% of the nanowires aligned within ±5°. The degree of orientation strongly depends on the distance away from the impact point along the flow direction. For instance, S is reduced to 0.87 at 1.5 cm away from the spray jet impact point. Generally speaking, S was above 0.80 for all the samples made with AgNWs following the method used here. We believe that the spraying at small angles produces a thin liquid film on the receiving surface whose flow across the surface in the direction away from the nozzle exercises sufficient shear for orienting the 1D-nanomaterials during their adsorption fromthe suspension. The nanowire density in the monolayer, and thus the in-plane plasmonic coupling 37 between different AgNWs in the same layer is conveniently tuned by varying the spraying time (Fig. 2). The surface coverage was varied between 15% for 10 seconds spraying, and up to 60% when the spraying is maintained for a few minutes (Fig. 2d). AgNWs only adsorb in areas where a PEI-coated interface is available (Fig. 2a-c), and do not form an adlayer on top of already adsorbed AgNWs. In competitive random sequential adsorption, a jamming coverage will be reached that depends on the size and shape of the adsorbing species and at which the surface coverage reaches a plateau. This adsorption mechanism also explains why the density is lower around misaligned nanowires or deformed nanowires (see for instance the circled areas in Fig. 2c). The extinction spectrum of AgNWs displays two peaks in the UV-Vis region and a broad band extending in the near-infrared (NIR) region due to the localized surface plasmon resonances (LSPR). For AgNWs in suspension (Fig. 2e), two peaks at 349 and 377 nm are observed. Kottman et al. have observed that silver nanowires with a non-circular cross-section display multiple transverse resonance peaks. 38 These peaks are commonly assigned to the outof-plane quadrupole resonance and the out-of-plane dipole resonance of nanowires respectively, 20,39 although this assignment has, to the best of our knowledge, not yet been confirmed by simulations. A broad band starting at ∼650 nm and extending towards the near infrared region can be assigned to the longitudinal mode of the LSPR. UV-Vis extinction with unpolarized light has also been measured for a disordered sample obtained by dropcasting and an oriented monolayer (Fig. 2e). The transverse LSPR peaks are significantly broadened and slightly redshifted due to plasmonic coupling between neighbouring AgNWs. The total extinction increases with the spraying time as the AgNW density increases in the monolayer (Fig. 2f), in particular the extinction measured at 378 nm (i.e. at a transverse LSPR peak, Fig. 2g). Furthermore, a new peak at ∼450 nm appears for spraying times above 50 seconds. The intensity of this peak increases for longer spraying times (i.e. for a higher AgNW density on the surface), and it can be assigned to a new mode arising from the coupling of dipolar transverse LSPR modes of individual nanowires. Polarized UV-Vis-NIR spectra of oriented AgNW arrays are used to evidence the macroscopic anisotropic character of the optical properties due to the local anisotropy. Indeed, both the longitudinal and the transverse modes of the localized surface plasmon resonance are independent of the polarization of the incident light in the non-oriented film (Fig. S3 †). When the nanowires are aligned, strong polarization effects are observed with respect to the polarization directions of the longitudinal and transverse modes (Fig. 3). The polarized extinction was measured as a function of the angle ɸ between the incident light beam polarization plane and the orientation direction (Fig. 3a andb). For ɸ = 0° (i.e. polarization parallel to the long axis of the nanowires), the longitudinal LSPR mode is excited as seen from the high extinction in the NIR. In contrast, for ɸ = 90° (i.e. polarization perpendicular to the long axis of the nanowires), the transverse LSPR modes are excited resulting in a high extinction in the UV-visible region and in the appearance of a small peak at 450 nm related to the coupling of transverse LSPR modes of individual nanowires. 37 Consequently, for intermediate polarization angles both modes are excited. The extinctions at 378 nm and 1500 nm are depicted in Fig. 3c in a polar plot as a function of the polarization angle ɸ. Fig. 3b and c illustrate nicely that oriented nanowire films are wavelength-dependent polarizers, as light is polarized in perpendicular directions below and above 425 nm. Another consequence is the high contrast of the reflected intensity of oriented nanowire thin films under crosspolarizers, demonstrating the macroscopic homogeneity of the optical properties (see Fig. S4 †).

Multilayers of oriented silver nanowires

As the orientation process has been demonstrated to be efficient for the build-up of an aligned AgNW monolayer, we used the Layer-by-Layer approach in order to fabricate multilayer films. PEI, poly(sodium 4-styrenesulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) were used as spacer layers between the nanowire layers, and the architecture of multilayer samples can be denoted as PEI/AgNW/[PEI(PSS/PAH)5PSS/PEI/AgNW]n-1, n indicating the number of individual nanowire layers.

Fig. 4 (a) Cross-section SEM pictures of oriented thin films composed of n = 2, 4, 6 and 8 AgNW layers. (b) Unpolarized extinction spectra of thin films of different thicknesses. (c) Extinction at 380 nm and thickness measured from the cross-section SEM pictures as a function of silver nanowire layer number n. (d) Extinction spectra for light polarized parallel (full lines) and perpendicular (dashed lines) to the orientation direction for various layer numbers n. (e) Polarization efficiency for various layer numbers n (see eqn (2)).

The multilayer build-up was monitored by cross-section SEM (Fig. 4a) and by determining the extinction (Fig. 4b) as a function of the number of deposited layers. Note that the term "AgNW layer" refers to the sequential adsorption of LbL films, but that such films resemble more a "woodpile"-like structure than a regularly layered stack. However, it appears clearly from the SEM pictures (Fig. 4a) that the film thickness increases proportionally to the number of AgNW deposition cycles, with a growth increment of ∼50 nm per repeating unit (Fig. 4c), which fits well with the combined thickness of the AgNWs and of the polyelectrolyte spacer layers. This linear growth is corroborated by the increase of the extinction at 380 nm as the number of deposited AgNW layers increases (Fig. 4c). The two transverse LSPR bands are broadened and strongly overlap, and a new broad extinction band appears at around 450 nm whose intensity grows as the number of layers increases, which arises from plasmon coupling between neighbouring layers. 37 Similarly to the oriented monolayer discussed above, the optical response of oriented multilayer thin films is polarization-dependent. Fig. 4d displays the extinction of the monolayer and multilayers with n = 2-4 for ɸ = 0° and ɸ = 90°. Similarly to monolayer films, the absorbance in the NIR range is high when the longitudinal LSPR band is excited and the absorbance in the near-UV range is high when the transverse LSPR bands are excited using the appropriate polarization directions in each case. The polarization efficiency (P.E.) is conveniently defined as the normalized difference between the transmittance of light polarized parallel (T∥) and perpendicular (T⊥) to the direction of nanowire orientation.

𝑃. 𝐸. = ! ∥ "! " ! ∥ #! " (2)
The polarization efficiency shows a positive maximum at the position of the transverse LSPR bands in the near-UV range, and becomes more and more negative when extending towards the NIR (Fig. 4e). Furthermore, the polarization efficiency increases strongly with the number of deposited AgNW layers. P.E. is equal to 31% at 347 nm and -57% at 1500 nm for a single layer of aligned nanowires, and reaches 92% and -97% for the 4-layer sample in the UV and NIR respectively. Note that P.E. does not increase much when adding a fourth AgNW layer to a multilayer composed of three AgNW layers because P.E. is already getting close to its maximum value of 100%.

Conclusions

In summary, we have shown that GIS is a very easy and versatile approach for orienting AgNWs. The resulting anisotropic thin films display optical properties that are in turn highly anisotropic. Moreover, multilayer thin films can be fabricated by combining this method with the Layer-by-Layer technique. The degree of orientation is high, with at least 95% of the nanowires being oriented within ±15° with respect to the main direction.

It should be highlighted that GIS is a process that is very easy to implement and scale up, as it does not require costly equipment or complicated sample preparation. Once the parameters are optimized, deposition of a layer takes a few tens of seconds, which can probably be reduced to shorter times by increasing the nanowire/substrate interaction. A single static spraying nozzle as used in this study allows covering homogeneously a few mm 2 , and this can already be scaled-up to several cm 2 using a mobile nozzle while the scale-up to m 2 is expected to be possible either by using an array of nozzles, or by scanning the nozzle(s) over the substrate. Such homogeneous anisotropic large area coatings are difficult to prepare by other alignment techniques such as Langmuir-Blodgett or doctor-blading. Finally, the tunability is very high, as GIS can in theory be applied to any 1D-nanomaterial on any type of substrate (oxide, metallic, polymeric, flexible or not, etc.) provided that there is an attractive interaction between the substrate and the deposited nanowires/nanorods (which can be adjusted by playing with substrate/nano-object surface functionalization). The combination with the LbL approach 3-5 furthermore opens perspectives towards multi-material architectures, in which different (nano)materials can be deposited in each layer, having independent control on the composition, density and orientation of each layer. As an example, Fig. S5 † shows a 2-layer thin film in which the second AgNW layer is aligned perpendicularly to the first one. One could imagine even more complex architectures, for instance by varying the angle between the layers or the number of layers. The LbL approach is also well known as a technique ideally suited for the fabrication of multi-material composite thin films that can comprise a large variety of materials. 40 The combination of the tunable inplane anisotropy demonstrated in this paper and the well-established control of the layer sequence (i.e. anisotropy in the direction of the layer normal) offered by LbL assembly allows envisioning multi-material nanoscale functional thin films that would enable new applications for example in optoelectronics, (bio)chemical sensing or for transparent conductive electrodes and solar cells.

Materials and methods

Chemicals

Poly(ethyleneimine) (PEI, 𝑀 $ )))) ≈ 60 000 g•mol -1 ), poly(sodium 4-styrenesulfonate) (PSS, 𝑀 % ))))) ≈ 70 000 g•mol -1 ), poly(allylamine hydrochloride) (PAH, 𝑀 %

))))) ≈ 15 000 g•mol-1), poly(vinylpyrrolidone) (PVP, 𝑀 %

))))) ≈ 40 000 g•mol-1), silver nitrate and glycerol were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). Sodium chloride was purchased from Carl Roth GmbH (Karlsruhe, Germany). All the chemicals were used without further purification. The aqueous solutions were prepared with ultrapure water (resistivity = 18.2 MΩ•cm, Milli-Q Gradient system, Millipore, Molsheim, France). PEI solutions were freshly prepared by direct dissolution of 2.5 mg•mL-1 of the polymer in ultrapure water. PSS solutions were prepared in NaCl solution (0.5 M) at a concentration of 0.62 mg•mL-1. PAH solutions were prepared in NaCl solution (0.5 M) at a concentration of 0.29 mg•mL-1.

Synthesis of Ag nanowires

Silver nanowires were synthesized by a polyol reduction procedure previously reported in the literature (for detailed description, see the ESI †). 17,34 Briefly, a glycerol solution of AgNO3, NaCl and PVP is heated from room temperature to 210 °C, which leads to Ag+ reduction and to the formation of silver nanowires that typically possess a pentagonal cross-section. The wires, coated in situ with PVP, form a stable suspension in water. The synthesis as performed in our laboratory results in nanowires which have a cross section of 47 ± 6 nm and a length of 4.2 ± 1.5 μm, as determined by Transmission Electron Microscopy (TEM) (see Fig. S-1 †).

Oriented deposition of silver nanowire layers

An aqueous suspension of silver nanowires (0.12 mg•mL -1 ) was sprayed on PEI-coated substrates (either glass slides or silicon wafers) using a home-made spraying system (see the ESI †) equipped with 2-fluid nozzles (internal diameter: 300 μm, Spraying Systems, Wheaton, IL) for 200 seconds unless otherwise specified. The angle between the central axis of the cone of the spray jet and the receiving substrate was fixed at α = 15° (Fig. 1a), and the distance between the nozzle and the substrate was held at a constant distance of 1 cm. The liquid flow rate was fixed to 1 mL min-1 and the air flow rate to 30 L min-1, which produces a cloud of droplets with a diameter in the range of 5-20 μm as determined by Phase Doppler Interferometry (PDI, data not shown here). Following deposition, the thin film was rinsed with Milli-Q water and dried using air flow.

For silver nanowire multilayer build-up, a polyelectrolyte multilayer spacer was deposited between each silver nanowire layer using air-pump spray cans (Roth, Lauterbourg, France). Each polyelectrolyte solution was sprayed for 10 seconds, followed by a 10 seconds rinsing step. Layer pairs of PSS and PAH were selected as spacer layers between the nanowire layers. The polyelectrolyte deposition sequence thus consisted of 1 layer of PEI, followed by 5 alternate layers of PSS and PAH, followed by another layer of PSS and finally a layer of PEI. The subsequent silver nanowire layer was sprayed under the same conditions as those used for the first layer, and the whole process can be repeated several times. The architecture of all multilayer samples discussed here can therefore be denoted as PEI/AgNW/[PEI(PSS/PAH)5PSS/PEI/AgNW]n-1, n indicating the number of individual nanowire layers.

Characterization

The nanowire size distribution was determined from the images obtained with a Tecnai G2 FEI Transmission Electron Microscope (TEM). Thin films were imaged by Scanning Electron Microscopy (SEM) using a JEOL 6700F microscope equipped with a field emission gun (FEG) and operating at 3 kV. The image analysis was performed using ImageJ. 36 The orientation analysis was done with OrientationJ, 35 a plugin developed for ImageJ, which is based on the analysis of the structure tensor in a local neighbourhood (see Fig. S-2 † for a detailed description of the orientation analysis procedure). UV-Visible-Near Infrared spectroscopy was carried out with a Cary 5000 spectrometer (Agilent) in dual beam configuration. The extinction for the thin film samples was measured through a circular aperture with 5 mm diameter and a blank substrate was used as a reference. A Glan-Taylor polarizer has been used for polarized UV-Vis-NIR spectroscopy.

Fig. 1

 1 Fig. 1 (a) Schematic representation of the GIS deposition technique. (b) SEM picture of an AgNW monolayer deposited on PEI-coated silicon wafer. (c) Orientation analysis of the dotted square portion of panel (b), in which the nanowires are colored according to their orientation. (d) Color-code used for the orientation analysis. (e) Distribution of the nanowire angle θ relative to the spraying direction.

Fig. 2

 2 Fig. 2 (a-d) SEM pictures of AgNW monolayers deposited for 10 s (a), 50 s (b) and 800 s (c), and surface coverage measured on SEM pictures as a function of the spraying time (d). The circled areas in panel (c) highlight regions in which the nanowire density is lower around misaligned nanowires. (e) UV-Vis-NIR extinction spectra of the nanowire suspension, a disordered AgNW thin film and an oriented AgNW monolayer thin film. (f) UV-Vis-NIR extinction spectra of oriented monolayer thin films deposited for 10 s, 50 s, 100 s, 200 s and 400 s, and (g) extinction at 377 nm as a function of spraying time.

37

 37

Fig. 3

 3 Fig. 3 (a) Scheme describing the polarized UV-Vis-NIR extinction measurement, in particular the angle ɸ between the light polarization plane and the nanowire orientation direction. (b) UV-Vis-NIR extinction spectra of an oriented nanowire monolayer thin film measured for various polarization angles ɸ. (c) Polar plot of the extinction at 378 nm (transverse LSPR) and 1500 nm (longitudinal LSPR) of an oriented AgNW monolayer thin film as a function of the polarizer angle ɸ.

Acknowledgements

The authors thank Marc Schmutz and the Electron Microscopy Platform (ICS, France) for TEM measurements, Cedric Leuvrey (IPCMS, France) for SEM measurements, and Martin Brinkmann (ICS, France) for giving access to his optical microscope. M.P. thanks the "Chaire d'Excellence" program of CNRS and the Université de Strasbourg for partial funding. G.D. thanks the Institut Universitaire de France for financial support. †Electronic supplementary information (ESI) available:

Detailed description of the experimental procedure, TEM pictures of the silver nanowires and their size distribution, description of the electron microscopy image analysis procedure, polarized optical properties of a non-oriented thin film, optical micrographs between crossedpolarizers of the oriented silver nanowire layers, and the SEM picture of a thin film composed of two AgNW layers with different orientation directions. See DOI: 10.1039/c6nr08045f


Lory-Anne Baker
email: l-anne.baker@etu.uca.fr

David G Biron

Fanny Millan

Olivier Voldoire

Vincent Breton

Elisabeth Allain

Carlos E Wetzel

Luc Ector

Aude Beauger

D G B In

L E Orcid

The substrate, a key factor or not, to explain the species diversity of diatom communities in mineral springs

Keywords: diversity of diatom community, substrate influence, community drivers, mineral springs

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Diatoms are a widely distributed group of unicellular algae that can colonised most of the habitats of aquatic ecosystems (ex.: lakes, rivers, estuaries, seas, oceans, springs) on earth (Mann and Drop 1996). For instance, benthic diatoms can grow in habitats of an aquatic ecosystem by attaching to substrates such as rock (epilithon), silt surface (epipelon), sand (epipsammon), plant (epiphyton) and wood (epidendron) (Round 1991). The assumption on a relationship between the substrate type and the species diversity or assemblage of benthic diatom communities in an aquatic ecosystem was assessed by many surveys in freshwater aquatic ecosystems like rivers and lakes. Some of these studies concluded that the substrate type should influence both species diversity and assemblage of benthic diatom communities in aquatic ecosystems [START_REF] Cox | Has the role of the substratum been underestimated for algal distribution patterns in freshwater ecosystems?[END_REF][START_REF] Sabater | Community dynamics and metabolism of benthic algae colonizing wood and rock substrata in a forest stream[END_REF][START_REF] Soininen | Benthic diatom communities in boreal streams: community structure in relation to environmental and spatial gradients[END_REF][START_REF] Potapova | Choice of substrate in algae-based water-quality assessment[END_REF][START_REF] Medlin | Multivariate analyses document host specificity, differences in the diatom metaphyton vs. epiphyton, and seasonality that structure the epiphytic diatom community[END_REF].

Based on previous studies and on their results, Round (1991) suggested that substrate topology could drive the benthic diatom assemblage (community) colonising a given substrate. Therefore, attached diatoms are mostly found on hard surfaces (ex.: rock, wood) while motile ones are predominant on thin particles such as silt. [START_REF] Townsend | The role of substrate type on benthic diatom assemblages in the Daly and Roper Rivers of the Australian wet/dry tropics[END_REF] suggested that benthic diatom assemblages were similar on hard substrates such as sand, wood and rock but not for the species abundance. On the other hand, they concluded that benthic diatom assemblages on vegetation substrates were in general dissimilar. [START_REF] Stevenson | Variation in diatom community structure among habitats in sandy streams[END_REF] analysed during two summers the diatom assemblages at two streams of Michigan State in the USA. They observed that the species abundances varied among microhabitats, and also that the differences of diatom species in studied communities were higher between rivers rather than habitats at a same sampling site. Moreover, many studies observed no significant difference between diatom communities from various substrates for the species composition [START_REF] Sullivan | Similarity of an epiphytic and edaphic diatom community associated with Spartina alterniflora[END_REF][START_REF] Millie | Studies on Lake Erie's littoral algae; Host specificity and temporal periodicity of epiphytic diatoms[END_REF][START_REF] O'quinn | Community structure dynamics of epilithic and epiphytic diatoms in a Mississippi stream[END_REF]. For instance, [START_REF] Jüttner | Diatoms as indicators of river quality in the Nepalese Middle Hills with consideration of the effects of habitat-specific sampling[END_REF] showed that there were similarities of species diversity between habitats during their surveys on Nepalese streams.

If there are several studies on the influence of substrate types on the species diversity or assemblage of benthic diatom communities in rivers and lakes, few were done on springs.

However, these aquatic-terrestrial ecosystems are widespread. The total number of springs on earth is estimated at more than 57 x 10 6 including approximately 10 5 thermal springs (Cantonati et al. 2012a). Besides, springs are present in different contexts [START_REF] Kresic | Types and classifications of springs[END_REF] and have diverse mineral compositions due to their origin and lithology. Some springs were used for medical purposes and also for drinking because of their special mineral composition. Thus, spring water catchment systems were made to collect underground water in many countries. Moreover, springs are also important because of their abundant and peculiar biodiversity (Cantonati et al. 2012a). Usually, springs are physically small biotopes where environmental variables are considered as stable. In fact, their stability depends on the spring type or configuration which can modified their endogenous chemical parameters. Some authors focus on the influence of substrate on benthic diatom assemblages in springs [START_REF] Cantonati | Diatom communities of springs in the southern Alps[END_REF]Cantonati et al. 2009Cantonati et al. , 2012b;;Wojtal and Sobczyk 2012;Lai et al. 2019b).

For their survey at Sardinia (Italy), Lai et al. (2019b) concluded that diatom assemblages in these thermo-mineral springs formed separated clusters and are not similar for three substrates: rock, cobbles and sediments. In contrast, the results of [START_REF] Cantonati | Diatom communities of springs in the southern Alps[END_REF] suggested that there were no differences between diatom assemblages growing on rock and plants. Wojtal and Sobczyk (2012) that also highlights that diatom distribution follows environmental variables like pH, salinity, trophic level and physico-chemical parameters. According to these last observations, the study of 16 springs and 5 streams of Dolomiti Bellunesi National Park in Italia determined that environmental variables explained 40% of the variability of diatom species diversity and substrate 3%.

Similar conclusion was obtained by

Therefore, these results suggest that macrohabitats have more impact on diatom assemblages than microhabitats [START_REF] Cantonati | The role of environmental variables in structuring epiphytic and epilithic diatom assemblages in springs and streams of the Dolomiti Bellunesi National Park (south-eastern Alps)[END_REF]. In order to bring new insights on the influence of substrate on benthic diatom assemblages in mineral springs, the epilithic and epipelic diatom communities of two springs located in Auvergne region were sampled monthly between July 2018 and June 2020 in order to test the spatio-temporal relationships between the substrate type and the species diversity.

Material and methods

Sampling sites

Two mineral springs, so called La Montagne and Mariol, were used for this survey (Figure 1).

Both are rich in bicarbonate and sodium, and belong to the springs of Châteldon, Vichy and the Saint-Yorre basins which take origin into the eastern border faults. The first one, La Montagne, is located in Châteldon (E 741086.699, N 6542796.817 in Lambert 93). At this spring, water is taken from the ground and discharged into a former building constructed during the bottling period (1859 -early 1900s). The second spring studied, Mariol, is located in the city of Mariol (E 739134.341, N 6546926.368 in Lambert 93) at 5 km from Châteldon and takes part of the South of Vichy basin. At this site, the water springing in a basin 50 cm above the ground. Both springs are located outside of the protected catchment area of the Vichy hydrotherapy industry and therefore accessible for sampling.

Physical and chemical analyses of water

Eighteen samples were taken between July 2018 and June 2020. Dissolved oxygen (%), conductivity (µS.cm -1 ) and water temperature (°C) were measured in situ using an Ysi ProODO oxygen probe and a multi-parameter WTW probe FC 340i. The carbonate concentration (HCO3 -) was also measured using a Hach carbonate kit. Additionally, during each sampling, a water sample was collected for further laboratory chemical analysis using a high pressure ion chromatography technic. First, these samples were filtered using Whatmann GF/C filters. For cation and anion analysis, a Thermo Scientific Dionex ICS1100 and a Thermo Scientific Dionex Aquion system were respectively used to measure concentrations (mg.L -1 ) in lithium (Li + ), sodium (Na + ), ammonium (NH4 + ), potassium (K + ), magnesium (Mg 2+ ), calcium (Ca 2+ ), fluoride (F -), chloride (Cl -), nitrite (NO2 -), nitrate (NO3 -), phosphate (PO4 3-).

Sampling of diatom communities

Both epipelic (on mud) and epilithic (on pebble) samples were collected from the two springs during 18 months between July 2018 and June 2020. "Mud" is referred to fine sediment with low quantity of silt, and "pebble" is referred to basalt stone (classified particles size: 64-128 mm, [START_REF] Malavoi | Description standardisée des principaux faciès d'écoulement observables en rivière : clé de détermination qualitative et mesures physiques[END_REF]. The epipelic sample depends of the kind of substrate presents on each site. Thus diatoms were sampled from a mix of mud and litter at La Montagne spring and from a mix of organic matter and non-hardened calcium carbonate from the travertine at Mariol spring. For the first site, the sediments were recovered by scraping off the first millimetres of mud directly with the vial, and for the second one, by brushing the non-hardened deposits. Epilithic samples were collected on basalt pebbles previously recovered from Allier river that were sterilised and placed in spring water. Epilithic algal communities were sampled by brushing the attached algae from the upper surface of submerged pebble using [START_REF] Douglas | The ecology of the attached diatoms and other algae in a small stony stream[END_REF] method.

Slide preparation and microscopy

Small sub-samples of epipelic and epilithic raw material were prepared for light microscopy (LM) observation according to the method of [START_REF] Prygiel | Guide méthodologique pour la mise en oeuvre de l'Indice Biologique Diatomées NF T 90-354[END_REF]. Samples were cleaned using hydrogen peroxide (H2O2, 35%) and hydrochloric acid (HCl, 37%), and rinsed several times with distilled water. Cleaned material was diluted with distilled water to avoid excessive concentrations of diatom valves on the slides. Then, a drop of dried, clean material was mounted in Naphrax®. LM observations and morphometric measurements were done using a Leica® DM2700M microscope with a 100x oil immersion objective using a differential interference contrast. For each slide, 400 valves were enumerated on random transects. Diatom identification was based on different taxonomical works (Krammer and Lange-Bertalot 1997a, 1997b, 2000a, 2000b, 2004;[START_REF] Krammer | Diatoms of Europe[END_REF][START_REF] Krammer | Diatoms of Europe[END_REF][START_REF] Krammer | Diatoms of Europe[END_REF][START_REF] Lange-Bertalot | Navicula sensu stricto, 10 Genera separated from Navicula sensu lato Frustulia[END_REF][START_REF] Kulikovskiy | Diatom assemblages from Sphagnum bogs of the world. I. Nur bog in northern Mongolia[END_REF][START_REF] Hofmann | Diatomeen im Süßwasser-Benthos von Mitteleuropa: 2 Corrected Edition[END_REF]Wetzel et al. 2015;[START_REF] Lange-Bertalot | Freshwater Benthic Diatoms of Central Europe: Over 800 Common Species Used in Ecological Assessment[END_REF]. Once the counting was completed, the relative abundance of diatom species was calculated.

Data analysis

Physical and chemical parameters

Abiotic data do not follow a normal distribution; thus there were standardised using z-score normalisation ((x-mean)/standard deviation) for Principal component analysis (PCA) [START_REF] Abdi | Principal Component Analysis[END_REF]. This transformation allows also to compare variables that initially had different units. From the 18 months of sampling, a stepwise regression was done in order to retain the most significant environmental variables from the dataset using XLSTAT 2020 3.1 software. As an outcome, conductivity, Cl -, NO3 -, F -and HCO3 -were retained as the most relevant parameters. Because of their ecological importance in spring ecosystems and on diatoms communities [START_REF] Patrick | Ecology of freshwater diatoms and diatom communities[END_REF]: temperature, dissolved oxygen, pH, anthropogenic indicators (NO2 -, NO3 -, PO4 3-) and a geological indicator (Na + ) were added to the environment variables selected through the stepwise regression. Then, a PCA was performed on these parameters to detect temporal and local differences among samplings. Statistical difference between Mariol and La Montagne samples was statistically tested with the non-parametric Krukal-Wallis test based on the Cartesian coordinates of the PCA samples points. Related to our observations during the survey, it seems that La Montagne spring could be affected by high precipitation events due to its configuration. To take into consideration the evolution of water conductivity and daily precipitations (data from US1116 AGROCLIM, INRA, Centre de Recherches PACA) during the sampling period, boxplots and graphs were also done with XLSTAT 2020 3.1 version. The correlation between these variables was tested by calculating Pearson correlation coefficient.

Statistical analysis of diatom data

Three diversity indices: Species richness (R), Shannon-Wiener index (H) and Evenness (J), were calculated for each spring. For each index, the comparison between substrate types was done by using the Wilcoxon test to verify the null hypothesis (no effect) of substrate. Further, to compare the composition of the diatom communities between substrates and considering the large sample size, the Jaccard dissimilarity index [START_REF] Jaccard | Distribution de la flore alpine dans le Bassin des Dranses et dans quelques régions voisines[END_REF]) was the most appropriate index. This one only takes account of the species shared between the two assemblages and the number of unique species of each assemblage. So, when the matrix of dissimilarities was generated with the function vegedist of the R package "vegan" (version 2.5-7), the arguments of the function were configured to convert the species relative abundances matrix into a binary matrix (0: absence, 1: presence) before running the distances matrix. A second matrix with the abundances-based jaccard dissimilarity index "ab.jaccard" (or chao jaccard) described in [START_REF] Chao | Abundance-based similarity indices and their estimation when there are unseen species in samples[END_REF] was also generated. The ab.jaccard distances is not present in vegan package so the dist.ldc function of the package "adespatial" version 0.3-14 [START_REF] Dray | Community ecology in the age of multivariate multiscale spatial analysis[END_REF] was used to generate a dissimilarity matrix with this index. These matrices were used for a non-metric Multidimensional Scaling ordination analysis (nMDS) performed to explore the similarity of diatom assemblages between the two substrates (pebble or mud) located in a same spring (XLSTAT 2020 3.1 version). Kruskal-Wallis stress was calculated to assess the nMDS adjustment quality (stress>0.20: bad adjustment, 0.10<stress <0.20: adequate, 0.05<stress<0.025: good, stress<0.025: excellent and stress=0: perfect). To complete the nMDS analysis and validate the significance of the differences between samples, a one-way analysis of similarities (ANOSIM) was used [START_REF] Clarke | Non-parametric multivariate analyses of changes in community structure[END_REF]. The ANOSIM were also done with Jaccard distances matrix and ab.jaccard distances. The percentage of contribution of three factors (location, season, substrate) to the variability of diatom species diversity in springs was determined by using a Non-parametric MANOVA analysis (i.e. ADONIS (Permutational MANOVA)) based on Jaccard or ab.jaccard dissimilarity index using the function Adonis of the vegan R package.

Results

Physical and chemical analyses of La Montagne and Mariol springs

The physical and chemical measures done at Mariol and La Montagne springs during the 18 months of sampling are reported in Tables 1, 2 and3 Montagne spring are related to high concentrations of HCO3 -and high conductivity. Indeed, La Montagne waters have a conductivity that varies significantly contrary to Mariol (Table 1).

While Mariol spring water emerges in a basin 50 cm above the ground, La Montagne spring water flows from a cavity on an abandoned building floor and can merge with streaming waters. As a consequence, we hypothesised that the variations in conductivity of this spring were linked to precipitations leading to spring waters more affected by dilutions.

Figure 3 shows respectively the evolution of water conductivity and daily precipitations during the 18 months of sampling at La Montagne spring. When the daily precipitations reach 5.4 mm in April 2019, the water conductivity decreases until 1138 µS.cm -1 while the maximum of water conductivity during the months before was 1849 µS.cm -1 . Since mid-October 2019 to February 2020, a rainy period occurred with daily precipitations high resulting in low conductivity (<800 µS.cm -1 ) except in mid-November. Considering the PCA (Figure 2), the samples of April 2019, October to December 2019, January and February 2020 correspond to samples associated with this period of low conductivity, increased precipitations (Figure 3) and also high concentrations of nitrates, phosphates, and sulphates (Figure 2). However, the correlation between conductivity and precipitations on sampling day indicates that the relationship between these two variables is low (Coefficient of determination: R²=0.231, p-value=0.043).

Influence of substrate on diatom communities' diversity

A total of 201 diatom taxa were identified on the two sites (Supplementary data 1). Species richness was respectively 168 and 134 for La Montagne and Mariol springs. Average species richness on epipelic samples were respectively 15 and 11 species for La Montagne and Mariol while epilithic richness was respectively, 19 and 12 (Figure 4). The results of Wilcoxon test on species richness and on Shannon index indicate significant difference between mud (epipelon) and pebble (epilithon) (p-value<0.05) at La Montagne when no difference was detected at Mariol (p-value>0.05) (Figure 4). Diversity index allows to estimate the "uncaptured richness". Shannon-Wiener diversity index takes into account the species richness and the evenness of diatoms. It varies between 0 and 1: 0 corresponds to a community where one species is totally dominant and 1 to a community where each species has similar repartition (Figure 4). The results of Wilcoxon test on Evenness index show that there is significant difference between species due to differences of species abundances only at Mariol (Figure 4). At the two sites, 5 taxa had a relative abundance > 1%. At Mariol, diatom assemblages were dominated by Planothidium frequentissimum Lange-Bertalot, Crenotia angustior (Grunow) Wojtal and Achnanthidium rivulare Potapova & Ponader (Table 4). At La Montagne, the dominant species were Planothidium frequentissimum, P. lanceolatum (Brébisson ex Kützing) Lange-Bertalot, Humidophila perpusilla (Grunow) R.L.Lowe, Kociolek, J.R.Johansen, Van de Vijver, Lange-Bertalot & Kopalová and Crenotia angustior (Table 4).

The nMDS (Figure 5) was performed based on ab.Jaccard matrix with species. Samples were projected in two dimensions and those with similar community structures are spatially close.

Graphically, there is no clear distinction between the assemblages from pebble and mud. 5). The contribution of season factor was 12.15 %* (jaccard) and 14.30 % (ab.jaccard) for La Montagne, and 8.5 % (jaccard) and 8.39 % (ab.jaccard) for Mariol. When the samples of Mariol and La Montagne were analysed together, location explained 10.14 % *** (jaccard) and 19.74%*** (ab.jaccard) when substrate explained 6.14 %*** (jaccard) and 18.47 %*** (with ab.jaccard) of the variability (Table 5). Percentages of contribution of seasonality are similar when analysis were performed with jaccard distances (5.8% *) or ab.jaccard distance (5.32 %) (Table 5).

Discussion

Mariol and La Montagne are two different springs

The environmental variables measured at La Montagne and Mariol springs clearly reveals the specificity of each spring which is in accordance with their origin (Figure 2). Mariol is characterised by high concentration of fluoride [START_REF] Risler | Description et classification géologique des sources minérales et thermales du Massif Central. Description and geological classification of the mineral and thermal springs of the Massif Central[END_REF]). This observation is in line with the composition of the Vichy basin, where the fluoride concentration can reach 9 mg.L -1 . The Vichy basin springs are originated from the same deep reservoir where the temperature is estimated at 135-150°C [START_REF] Risler | Description et classification géologique des sources minérales et thermales du Massif Central. Description and geological classification of the mineral and thermal springs of the Massif Central[END_REF]. In contrast, higher carbonate concentrations are detected in La Montagne spring. This is a particularity of the Massif Central where there is a preponderance of bicarbonate rich waters [START_REF] Risler | Description et classification géologique des sources minérales et thermales du Massif Central. Description and geological classification of the mineral and thermal springs of the Massif Central[END_REF]. Related to the high conductivity of La Montagne, this spring is more mineralised than Mariol. Considering the different variables, this last spring appears more stable. Indeed, the waters catchment in an elevated bowl leads to a more stable environment across the time at the spring emergence. At La Montagne, conductivity varies significantly over time linked to the precipitations. Indeed, when large episodes of precipitations as for example between October 2019 and February 2020 lead to decrease of conductivity due to spring water dilution. Furthermore, the increase of anthropogenic indicators (NO2 -, NO3 -, PO4 3-) in the samples from October 2019 to February 2020 shows that La Montagne is affected by human activities. In fact, Châteldon is an area where there are some agricultural lands. The configuration of La Montagne site is favourable to the mixing of spring water and the watershed that hosts livestock farming. The watershed upstream of La Montagne emergence appears to have a great influence on its water.

According to the observations and measures at the two springs, La Montagne and Mariol are two springs with different characteristics and represent two distinct biotopes.

Substrate influence on richness

In this study, two types of substrates were considered: epilithic communities were collected from pebble as hardened substrate, and epipelic communities from a mix of mud and litter (La Montagne) or a mix of travertine and litter (Mariol) as non-hardened. Previous works done on the influence of substrate on diatom communities suggest that specific richness was lower on pebble [START_REF] Cantonati | Diatom communities of springs in the southern Alps[END_REF][START_REF] Cantonati | The role of environmental variables in structuring epiphytic and epilithic diatom assemblages in springs and streams of the Dolomiti Bellunesi National Park (south-eastern Alps)[END_REF]Lai et al. 2019b). However, at La Montagne the specific richness (S) is higher on epilithic communities (S=19) than epipelic ones (S=15) while it is similar on both substrates at Mariol. La Montagne and Mariol springs are two different biotopes with distinct configurations. As quoted before, La Montagne is a spring characterised by time-dependent physico-chemical properties due to disturbance by precipitations. The speculation that can be made is that in absence of great changes of physical and chemical parameters, species richness seems to be more similar between substrates. Otherwise, when the biotope is less stable, the richness is higher on hard substrate than on non-hardened ones. This can be related to the topography of the substrate. For example, in La Montagne mud can be more sensitive to the water flows than pebbles. Close relationship between richness and the degree of biotope disturbance is documented in the literature [START_REF] Odum | Fundamentals of Ecology[END_REF][START_REF] Dudgeon | Freshwater biodiversity: importance, threats, status and conservation challenges[END_REF]Cantonati et al.2012a). Additionally, there are more species at La Montagne than at Mariol which seems to be inconsistent with this last observation. In fact, heterogeneity of the environment is another parameter that needs to be take into account. Stable and homogeneous environment displays less diversity [START_REF] Ramade | Ecologie fondamentale-Eléments d'écologie. 4 e édition[END_REF]. At Mariol, the absence of fluctuations of physical and chemical variables and its configuration suggest that this spring represents a stable and homogeneous biotope. This can explain the lower diversity observed at this spring.

Substrate influence on diatom communities

At La Montagne spring, the diatom communities were mostly dominated by Planothidium frequentissimum, Humidophila perpusilla, Crenotia angustior and P. lanceolatum.

Planothidium frequentissimum and C. angustior were also dominant at Mariol with Achnanthidium rivulare. The two springs show similarities through their dominant species.

When studying the diatom communities of some springs in Auvergne (France) and Sardinia (Italy), Lai et al. (2019a) reported that P. frequentissimum was found in others springs of Auvergne and was also counted among the most abundant species in some of them. Crenotia angustior was present in springs of Europe like the Mofette spring which is in the south of Poland [START_REF] Noga | Mofette springunusual environment for microbial life on the example of diatoms[END_REF]. Humidophila perpusilla colonised environment with low light intensity [START_REF] Lange-Bertalot | Freshwater Benthic Diatoms of Central Europe: Over 800 Common Species Used in Ecological Assessment[END_REF]. Achnanthidium rivulare has affinity for water with relatively low calcium and high chloride concentrations [START_REF] Potapova | Two common north American diatoms, Achnanthidium rivulare sp. nov. and A. deflexum (reReimer) Kingston: Morphology, ecology and comparison with related species[END_REF].

Achnanthidium rivulare have different ecological preferences but are abundant in rivers with low-alkalinity and low phosphorus concentration [START_REF] Potapova | Two common north American diatoms, Achnanthidium rivulare sp. nov. and A. deflexum (reReimer) Kingston: Morphology, ecology and comparison with related species[END_REF][START_REF] Lange-Bertalot | Freshwater Benthic Diatoms of Central Europe: Over 800 Common Species Used in Ecological Assessment[END_REF].

The nMDS was done on a dissimilarity matrix based on ab.jaccard index. No clear distinction of substrate samples was observed. In agreement with this result, the ANOSIM analyses completed with samples from the springs, show that there is a low difference between samples from different substrates. These results are similar to [START_REF] Potapova | Choice of substrate in algae-based water-quality assessment[END_REF] that demonstrates that algal assemblages are similar on hard substrate (rocks and wood) and soft substrates (sediments) in 551 Russian rivers samples. [START_REF] Stevenson | Variation in diatom community structure among habitats in sandy streams[END_REF] also demonstrate that there is no significant difference between epipelic, epipsammic and epiphytic diatom communities of two rivers in northern Michigan.

The Adonis test that consists in a permutation multivariate ANOVA (PMANOVA) completes the first results and determines which factors explain the best the communities variability.

Location and season which are spatial and temporal factors known to influence the diversity were taken into account in a same ADONIS test. Jaccard dissimilarity index is used to compare taxonomic composition of two assemblages of community. Its operates with the number of species shared between the two assemblages and the number of unique species of each assemblage. The ab.jaccard distances (or Chao.jaccard) considers not only the taxonomic composition but also the species abundances and thus, evaluates similarities between samples diversity. Considering only the taxonomic composition of samples, substrate explained 6.14 % of the variance when location explained 10.1%. Location is the factor that better explained the difference of diatom assemblages composition between springs. As mentioned above, physical and chemical variables are different at Mariol and La Montagne springs making two different biotopes (Figure 2). The link between the PCA and the Adonis test suggests that environmental variables of the studied sites explained more the variability of diatom assemblages than the type of substrate. [START_REF] Cantonati | The role of environmental variables in structuring epiphytic and epilithic diatom assemblages in springs and streams of the Dolomiti Bellunesi National Park (south-eastern Alps)[END_REF] observed that environmental parameters explain 42% of the diatom assemblage variabilities when substrate explains only 3%. The sampling of 21 springs and streams in this study increases the statistical power and allows to conclude that macrohabitat which is the ecosystem's location and its abiotic factors have more impact on the variability of diatom assemblages than microhabitat such as the substrate type. Several studies have shown that environmental and spatial variables have more influence on diatom community variations than substrate [START_REF] Soininen | Benthic diatom communities in boreal streams: community structure in relation to environmental and spatial gradients[END_REF][START_REF] Soininen | Environmental and spatial control of freshwater diatoms -A review[END_REF]. Moreover, [START_REF] Sabater | Some factors affecting distribution of diatom assemblages in Pyrenean springs[END_REF] concluded that ionic composition of springs is the major factors that determine diatoms diversity as well as temperature, conductivity, alkalinity that reinforcing the results. Season is the parameter that explains the variability the least well. Same conclusion was reached when [START_REF] Moresco | Periphytic diatom as bioindicators in urban and rural streams[END_REF] analysed periphytic diatoms of two Brazilian streams. The addition of species abundances allows the consideration of abundant and rare species in the samples dissimilarity computation. Interestingly, in this case, substrate explained 18.74 % and location 19.74 % of the variability. The percentage of contribution of each of these two factors increases with the addition of species abundances but location remains the factor than explains the most the variability between diatoms communities. Also, the contribution percentages of these two factors became closer. This suggests that substrate can explain the difference between diatom communities' diversity as much as location. [START_REF] Townsend | The role of substrate type on benthic diatom assemblages in the Daly and Roper Rivers of the Australian wet/dry tropics[END_REF] observed that communities were similar between rock and sand but abundances were different according to the substrate. These results suggest that substrate have less impact on diatom communities composition than location but have influence on species abundances. The comparison of species distributions at each site allowed to estimate the contribution of season and substrate in diatom communities' distribution.

However, several studies reported that species richness is higher during spring and summer.

But others assume less seasonal effect [START_REF] Cantonati | Longitudinal and seasonal differentiation of epilithic diatom communities in the uppermost sections of two mountain spring-fed streams[END_REF]. Seasonal effects on benthic diatoms were studied in river and lead to the conclusion that the change of river flow related to season affects diatoms community [START_REF] De Fabricius | Distribution patterns of benthic diatoms in a Pampean river exposed to seasonal floods: the Cuarto River (Argentina)[END_REF]. At La Montagne, seasonal effect was higher than at Mariol particularly with the jaccard index. Seasonality seems contribute more than substrate to diatom assemblages composition. This could be related to the disturbance of La Montagne water by precipitations. The relation between precipitation frequency and season can explain the result that was obtained with the Adonis test. When comparing communities by taking account of the species abundances, season didn't contribute more than substrate to diatoms community structuration. This observation is similar for the two springs studied.

Conclusion

La Montagne and Mariol springs are two different biotopes and this is reflected in their diatom communities composition. Their physico-chemical particularities are linked to the geological origin of the underground water. Mariol is a more stable spring than La Montagne due to the absence of great fluctuations of physical parameters. In contrast, La Montagne spring shows conductivities that highly vary due to precipitations. Thus, this spring shows less stability of its waters. At La Montagne and Mariol springs, low difference between epipelic and epilithic diatom communities is underlined. The variability of composition in diatom communities was better explained by the factor (location). When comparing the diversity of diatom communities, location is the factor that explained most of the variabilities between communities. Thus, environmental variables (summarised by Location) have significantly higher importance as drivers of diatom taxonomic composition than season and substrate, but the substrate seems to have influence on the species abundances as much as the location.

Wetzel CE, Ector L, Van de Vijver B, Compère P, Mann DG. 2015. Morphology, typification and critical analysis of some ecologically important small naviculoid species (Bacillariophyta). Tables: Table 1. Physical variables (mean ± SD) for La Montagne and Mariol springs considering the 18 months sampled.

  . The PCA (Figure 2) performed on environmental variables explained 53.96 % of the variance (axis 1: 32.26 % and axis 2: 21.69 %). The axis 2 clearly separates La Montagne and Mariol samples in two groups (Kruskal-Wallis test: p<0.0001). Mariol samples were associated with high concentrations of fluoride and high temperatures (upper part of the first factorial plan). Compared to Mariol, La Montagne samples formed a disperse cluster in the lower part of the factorial plan. Samples collected in April 2019, October to December 2019, January and February 2020 are associated with high concentrations of Cl -, SO4 -, NO3 -and PO4 3-. Most of the samples of La

  ANOSIM tests show a weak difference (*: p ≤ 0.05, **: p ≤ 0.01, *** p ≤ 0.001) between pebble and mud at La Montagne (R=0.28*** (jaccard) and R=0.32*** (ab.jaccard)), and also a similar result was obtained at Mariol ( R=0.31*** (jaccard) and 0.25*** (ab.jaccard)). The Adonis tests (Table 5) considering La Montagne or Mariol springs and both were performed to determine the percentage of variability explained by each factor. Significance of the ADONIS test results was indicated by *: p ≤ 0.05, **: p ≤ 0.01, *** p ≤ 0.001. The analysis shows that the substrate factor contributed to 10.56 % *** (jaccard) or 30.72 %*** (ab.jaccard) of the variability at La Montagne, and 9.4 %*** (Jaccard) and 25.74%** (ab.jaccard) at Mariol (Table

Fottea. 15

 15 (2):203-234. doi:10.5507/fot.2015.020 Wojtal AZ, Sobczyk L. 2012. The influence of substrates and physicochemical factors on the composition of diatom assemblages in karst springs and their applicability in water-quality assessment. Hydrobiologia. 695: 97-108. doi:10.1007/s10750-012-1203-0

Figure 1 .

 1 Figure 1. Location (a) in France of the two mineral springs studied in Auvergne Region (b): Mariol (c) and La Montagne (d, e).

Figure 2 .

 2 Figure 2. Principal component analysis (PCA) shows the samples distribution based on physical and chemical variables of the Mariol and La Montagne site, monitored from July 2018 until June 2020. The Kruskal-Wallis test based on Cartesian coordinates of the samples points of the PCA validate statistical difference between samples of Mariol and La Montagne by the axis 2 (p<0.0001).

Figure 3 .

 3 Figure 3. Evolution of the daily precipitations and of water conductivity for the La Montagne mineral spring.

Figure 4 .

 4 Figure 4. Boxplot showing results of (A) Species richness, (B) Shannon, (C) Evenness diversity index calculation for diatom for two substrates (Epilithon or Epipelon) communities at Mariol and La Montagne. Wilcoxon test: **: p<0.05, ***:p<0.005, NS: non-significant.

Figure 5 .Figure 1 .

 51 Figure 5. Non-metric multidimensional scaling (nMDS) ordination plot based on species composition of diatom communities for two substrates (Epilithon or Epipelon) at Mariol and La Montagne springs. The matrix distances is based on ab.jaccard distances.

Figure 2 .

 2 Figure 2. Principal component analysis (PCA) shows the samples distribution based on physical and chemical variables of the Mariol and La Montagne site, monitored from July 2018 until June 2020. The Kruskal-Wallis test based on Cartesian coordinates of the samples points of the PCA validate statistical difference between samples of Mariol and La Montagne by the axis 2 (p<0.0001).

Figure 3 .

 3 Figure 3. Evolution of the daily precipitations and of water conductivity for the La Montagne mineral spring.

Figure 4 .

 4 Figure 4. Boxplot showing (A) Species richness, (B) Shannon, and (C) Evenness diversity index for diatom communities living on two substrates (Epipelon (red) or Epilithon (blue)) at Mariol and La Montagne. Wilcoxon test: **: p<0.05, ***: p<0.005, NS: non-significant.

Figure 5 .

 5 Figure 5. Non-metric multidimensional scaling (nMDS) ordination plot based on species composition of diatom communities for two substrates (Epilithon (blue) or Epipelon (red)) at Mariol and La Montagne springs. The matrix distances is based on ab.jaccard distances.

Table 1 .

 1 Physical variables (mean ± SD) for La Montagne and Mariol springs considering the 18 months sampled.

Table 2 .

 2 Concentrations (mg.L -1 ) of ions (indicators of deep water and geological nature) (mean ± SD) for each site.

Table 3 .

 3 Concentration (mg.L -1 ) of pollutant ions (mean ± SD) for each site.

Table 4 .

 4 Relative abundances of the dominant species with an abundance >1% in at least one sample for Mariol and La Montagne springs.

Table 5 .

 5 NPMANOVA (Adonis) results for unifactorial analysis of substrate, location and season variables made with Jaccard and ab.jaccard distances for Montagne and Mariol springs or the springs together performed on taxa with an abundance >1% in at least one sample. The

	significance of the results is indicated by (*) p < 0.05, (**) p < 0.01, (***) p < 0.001.
	Supplementary

Table 1 .

 1 Species list of diatoms at Mariol and La Montagne springs.

Table 2 .

 2 Concentrations (mg.L -1 ) of ions (indicators of deep water and geological nature) (mean ± SD) for each site.

	Sites	Li 2+	Na +	K +	Mg 2+	Ca 2+	F -	HCO3 -
	La Montagne	0.59 ± 0.59	118.56 ± 55.81	16.23 ± 8.29	27.92 ± 17.48	153.70 ± 74.67	1.38 ± 0.58	822.83 ± 410.06
	Mariol	0.51 ± 0.02	144.87 ± 7.15	10.80 ± 2.69	4.01 ± 0.44	33.00 ± 3.33	4.75 ± 0.43	504.61 ± 54.15

Table 3 .

 3 Concentration (mg.L -1 ) of pollutant ions (mean ± SD) for each site.

	Sites	Cl -	NH4 +	NO2 -	NO3 -	PO4 3-	SO4 2-
	La Montagne	8.93 ± 9.00	0.34 ± 0.40	0.02 ± 0.00	3.41 ± 4.93	0.11 ± 0.07	12.31 ± 4.05
	Mariol	2.82 ± 0.71	0.40 ± 0.37	0.02 ± 0.01	0.08 ± 0.05	0.04 ± 0.01	6.73 ± 0.48

Table 4 .

 4 Relative abundances of the dominant species with an abundance >1% in at least one sample for Mariol and La Montagne springs.

	La Montagne	Mariol

Table 5 .

 5 NPMANOVA (Adonis) results for unifactorial analysis of substrate, location and season variables made with Jaccard (JD) and ab.jaccard (ab.J) distances for Montagne and Mariol springs or the springs together performed on taxa with an abundance >1% in at least one sample. The significance of the results is indicated by (*) p < 0.05, (**) p < 0.01, (***) p < 0.001.

	Sites	La Montagne and Mariol	La Montagne		Mariol
		R 2 (J)	R² (ab.J)	R 2 (J)	R² (ab.J)	R 2 (J)	R² (ab.J)
	Substrate (2 modalities)	6.14 % ***	18.47 % ***	10.56 % ***	30.72 % ***	9.4 % ***	25.74 % **
	Season (4 modalities)	5.85 % *	5.32 %	12.15 % *	14.30 %	8.5 %	8.39 %
	Location (2 modalities)	10.14 % ***	19.74 % ***	-		-

Acknowledgements

This study was funded by a grant from the Centre National de la Recherche Scientifique (CNRS, NEEDS, project Acronym: DISCOVER). L.A.B. was supported by a Ph. D. grant from the CNRS. Funding for this research was partly provided to C.E.W. and L.E. in the framework of the project DIATOMS (LIST -Luxembourg Institute of Science and Technology). We acknowledge financial support from CNRS-INEE within the context of the Zone Atelier Territoires Uranifères. We also acknowledge the anonymous reviewer and Patrick Gasqui (INRAE, UR EpiA -Unité de Recherche d'Épidémiologie Animale, France)

for their help and advises for the statistical analyses of the results.

Disclosure statement

No potential conflict of interest was reported by the authors.


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Mathilde Causse

Breeding tomato hybrids for flavour: comparison of GWAS on lines and F1 hybrids

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Online EUCARPIA congress 2021 -Breeding: the key to innovative solutions -23-26 aug 2021 Online

Breeding tomato hybrids for flavour: comparison of GWAS on lines and F1 hybrids

Estelle Bineau 1, 2 , José Luis Rambla 3 , Santiago Priego 3 , Alexandre Hereil 1 , Frédérique Bitton 1 , Clémence Plissonneau 2 , Antonio Granell 2) in a test cross panel involving the previous panel plus 44 elite cherry tomato lines crossed with a common big fruited line. High and consistent heritabilities were assessed for most VOCs in the two panels, and the line and test cross values for VOC content were strongly correlated. In the two panels, cluster analysis on 46 VOC content matched the metabolic pathways known from the literature. Results suggested a global additivity pattern for most VOC genetic control. We performed genome wide association studies (GWAS) on the line and test cross panels separately, along with a third GWAS on the test cross subset carrying the subset of 121 F1 hybrids corresponding to the line panel. We identified 205, 183 and 138 associations, respectively. Although less than 10% associations overlapped between line and F1 hybrid panels, we identified numerous overlapping associations for VOCs belonging to the same metabolic pathway within each panel. With this study, we highlighted the interest of testcross panel to create tasty F1 hybrid varieties. The clusters of associations in the F1 hybrid panel are new levers to improve tomato flavour by regulating several key VOCs simultaneously, while following only a few genomic regions in the breeding material. We selected seven chromosome regions fitting numerous criteria meant to facilitate breeding. For instance, a 4Mbp region on chromosome 3 carried F1 specific associations for 14 different VOCs derived from the lipid metabolism, eight of which having an impact on tomato overall flavour. This region is supported by several candidate genes also highlighted in previous studies. Key words: Tomato, breeding, flavour, volatiles, GWAS.


Antoinette Fayad

Wassim El Malti
email: wassim.elmalti@aum.edu.kw

Bruno Ameduri
email: bruno.ameduri@enscm.fr

Ali Alaaeddine
email: alikassem.alaaeddine@ul.edu.lb

Synthesis and characterization of novel vinyl ethers that bear various functional groups

Keywords: vinyl ether, transetherification, catalysis, palladium, NMR

The synthesis of new functionalized vinyl ethers (VE) by transetherification reaction between ethyl vinyl ether (EVE) and different alcohols is presented in this study. An air-stable palladium catalyst generated in situ efficiently catalyzed the reaction leading to various VE in a single step with good conversion % (50-82%) and good yields (up to 75%). The reaction conditions were optimized in terms of the initial EVE/alcohol molar ratio, alcohol concentration, solvent, presence and nature of the ligand, amount of the catalyst, and choice of the metal precursor. All VE were characterized using 1 H and 13 C NMR spectroscopy.

Introduction

Enol ethers or vinyl ethers (VEs) are a class of chemical compounds that display ether and vinyl functional groups. These compounds have found numerous applications as key reagents in organic reactions such as the Diels-Alder reaction [1] and Claisen rearrangement [2], thanks to the remarkable reactivity of their electron-rich carbon-carbon double bond and the range of substituents that can bear [3,4]. They have been extensively polymerized under cationic initiation to produce polymeric materials endowed with excellent properties used in various industrial applications such as adhesives, paints, coating, and the synthesis of stimulisensitive materials [5][6][7][8][9][10][START_REF] Mueller | In Coatings Technology Handbook (3rd Edition)[END_REF][START_REF] Couture | [END_REF]. Despite their simple structure, no universal method has been found to carry out their synthesis. Nevertheless, many methods have been developed to prepare vinyl ethers, particularly catalyzed transetherification using transition metals. Winternheimer et al. reported the main available pathways to synthesize vinyl ethers [2]. In the 1930's, Reppe and Wolff developed a process to form VEs from acetylene and primary alcohols under rigorous pressure and temperature conditions and using a large amount of basic catalyst [13,14]. Then, four additional synthetic pathways have been developed for the chemical conversion of alcohols to vinyl ether: i) addition onto acetylene [13,14], ii) phase transfer catalysis based on Williamson reaction between an alkoxide and 2-chloroethyl vinyl ether [15], iii) modification of vinyl acetate in the presence of an iridium complex [16,17], and iv) transetherification [18].

The latter strategy has been widely studied in the presence of either mercuric acetate [19,20] or palladium acetate [21][22][23][24] catalysts producing various vinyl ethers in fair to good yields.

Complexes of palladium acetate and trifluoroacetate with bidentate ligands, such as 1,10phenanthroline, have proven to efficiently catalyze most of the vinyl ethers preparations, and its catalysis mechanism was proposed by Handerson and Schlaf [23]. They successfully used (L-L) Pd(OAc) 2 catalyst, where L-L stands for 2,2'-bipyridyl or 1,10-phenanthroline chelate, for the transfer vinylation of protected monosaccharides. The efficiency of palladium (II) complex in transfer vinylation has been initially described by McKeon and Fitton [25]. To the best of our knowledge, only two other examples of catalyzed transfer vinylation were reported in literature: vinylation of steroids [22] and synthesis of glycidol vinyl ether [26]. In 2010, Pichavant et al. described the synthesis of a vinyl ether by transetherification catalyzed by a palladium(II) complex in the presence of ethyl vinyl ether in dichloromethane [27]. The iridium-catalyzed reaction of vinyl acetate with alcohols later appeared as a complementary method to palladium-catalyzed transetherification [16,17].

In 2013, our group published the transetherification between ethyl vinyl ether and various alcohols leading to the corresponding original vinyl ethers with 40-84% yield in a single step synthesis [18,[28][29][30][START_REF] Alaaeddine | ACS Symposium Series[END_REF][32][33]. A series of vinyl ether monomers that bear diverse functional groups, such as halogenomethyl, amino, carbonate, oligo(ethylene oxide) side-groups, and oligo(fluoroether) chains, were prepared and characterized by NMR spectroscopy. The an excess of either vinyl ether or alcohol.

In this work, a detailed study on the transetherification reaction of ethyl vinyl ethers was carried out. The aim was to prepare new vinyl ethers with various side chain functional groups (Scheme 1) and can be widely used in the synthesis of polymeric materials and other applications. These vinyl ethers were synthesized from the corresponding alcohols through transetherification of ethyl vinyl ether catalyzed by an organometallic complex generated in situ (Scheme 1). Several reaction parameters were studied: the initial EVE/alcohol molar ratio, alcohol concentration, nature of the solvent, presence and nature of the ligand, amount of the organometallic complex, and choice of the metal precursor.

Results and Discussion

In this study, liquid vinyl ethers bearing various side chains were prepared from four different alcohols (Scheme 1). Furfuryl alcohol was chosen to undergo a series of transetherifications reactions to determine the optimal experimental conditions (Scheme 2 The presence of the ligand played an essential role in the transetherification reaction.

This was proven in entry 1 that was carried out without ligand, for which no transformation was observed. Using 1-phenyl-1,2-ethanediol (entry 2) and triphenylphosphine (entry 3) ligands did not lead to the desired product. However, the 1,10-phenanthroline ligand showed a remarkable catalytic activity of Pd(OAc) 2 with 69% conversion, obtained after 24 hours of stirring at 20 °C (entry 4).

In order to assess the impact of the metal used in the catalyst, the employment of Zn(OAc) 2 and Co(OAc) 2 complex catalysts did not result in the formation of the desired product (entries 5-6). However, using Pd(OAc) 2 led to the formation of the desired vinyl ether with 69% conversion. These results align with B. Boutevin et al. work. They demonstrated that the 1,10-phenanthroline-palladium (II) acetate complex led to vinyl ethers by transetherification of ethyl vinyl ether in fair % yields [15].

While studying the effect of the solvent nature, moderate conversions were observed with 1,4-dioxane or toluene (entries 9-10). On the other hand, poor conversions were obtained using THF and DMF (entries 7-8). Dichloromethane was observed to be the best solvent for this reaction, with the highest conversion % (entry 4). This variation in the catalytic activity can be related to the solubility of the catalyst, which differs from one solvent to another depending on the solvent polarity. A slight decrease in the catalytic activity (66% conversion) was observed when the reaction was conducted without solvent (entry 11).

It was noticed that the increase in the initial EVE/alcohol molar ratio from 6 to 12 eq. shifted the equilibrium in favor of the desired vinyl ether product (entries 4 and 12).

Furthermore, an increase in the number of EVE equivalents for more than 12 did not benefit the % conversion (entry 13). This may be due to the dilution effect. Thus, 12 eq. of EVE was sufficient to promote this reaction.

The effect of the initial alcohol concentration was examined as well. 69% conversion was obtained with a concentration of 2.2 mol/L (entry 4). A similar % conversion was obtained when the alcohol concentration was raised to 4.4 mol/L (entry 14), showing that the maximum possible % conversion was already reached with a concentration of 2.2 mol/L.

Dropping the [alcohol] to 0.5 mol/L (entry 15), lessened the conversion to 37%. Thus, medium concentration (≈2.2 mol/L) was good enough to be used for the catalyst to be effective and give high % conversions.

Since the presence of the ligand was determined to be crucial, the variation of the number of equivalents of 1,10-phenanthroline was studied. When the number of the 1,10phenanthroline ligand equivalents was augmented from 1.0 to 1.5, it improved the conversion from 65 % to 69% (entries 4 and 17). In entry 18, a slightly lower % conversion was obtained with 2 ligand equivalents (63%). Furthermore, adding 3 equivalents of the ligand resulted in an apparent reduction of the % conversion (entry 19).

A significant increment in the conversion % was marked by increasing the amount of catalyst from 1 to 2% (entries 4 and 20). Adding a 5% catalyst (entry 21) did not show any notable advantage. However, the 10% catalyst (entry 22) marked a slight drop in the % conversion, indicating that a 2% catalyst is sufficient to achieve the highest conversion % in this transetherification reaction.

A series of vinyl ethers (1-4) was then synthesized by transetherification catalyzed by 2% palladium (II) acetate generated in situ. The reactions were carried out in dichloromethane in the presence of 1.5 equivalents of 1,10-phenanthroline and excess of EVE (12 eq.). The reaction was stirred at room temperature for 24 hours. The results are summarized in Table II. purity), DMF (anhydrous, 99.8% purity), and toluene (anhydrous, 99.8%) were purchased from Euriso-top, JTBaker, Riedel de Haen and Merck, respectively. THF was dried over sodium/benzophenone and distilled before using it.

Characterization by Nuclear Magnetic Resonance

The NMR spectra were recorded at room temperature on a Bruker AC 300 instrument using either d-chloroform or d-acetone as solvents and tetramethylsilane as reference.

Coupling constant and chemical shifts (δ) are given in hertz (Hz) and part per million (ppm), respectively. The experimental conditions for recording 1 H and 13 C NMR spectra were as follows: flip angle 90° (or 30°), acquisition time 4.5 s (or 0.7 s), pulse delay 2 s (or 2 s), and a number of scans 128 (or 512).

The catalyst solution was initially prepared by dissolving palladium (II) acetate (91.00 mg, 4.00*10 -4 mol) in 2 mL of dichloromethane. A solution of 1,10-phenanthroline (110.20 mg, 6.00*10 -4 mol) in dichloromethane (2 mL) was then charged drop-wise. The reaction medium was stirred at room temperature for 30 minutes to generate the palladium catalyst in situ. Then, a solution of 2-(hydroxymethyl) furan (2.00 g, 0.02 mol) and ethyl vinyl ether (17.30 g, 0.24 mol) in dichloromethane (5 mL) was added to the catalytic solution. The reaction mixture was stirred at room temperature for 24 hours. The % conversion was tracked by withdrawing a 0.2 ml sample and drying it under vacuum to eliminate the EVE residue.

The conversion was 69%. The purification of the synthesized EV was conducted by adding 50 mL of distilled water; the solution was extracted with dichloromethane (2 x 50 mL). The organic phase was then dried over Na 2 SO 4 , filtered, and the solvent was evaporated under vacuum to give the desired product (1) in the form of a brown liquid (1.46 g, 59% yield).

The same procedure for (1) was applied to 3,4,5-trimethoxybenzylalcohol (2.00 g, 0.01 mol) and ethyl vinyl ether (8.65 g, 0.12 mol) that were dissolved in 2.5 ml of dichloromethane. The solution was then added to the catalytic solution that was prepared by palladium (II) acetate (112.50 mg, 5.00*10 -4 mol) and 1,10-phenanthroline (135.15 mg, 7.50*10 -4 mol) in 2 mL of dichloromethane. The 1 H NMR analysis of an aliquot in CDCl 3 showed 82% conversion. The product was then purified in the same manner as described for

(1).

The same procedure for (1) was applied to tetraethylene glycol (2.00 g, 0.01 mol) and ethyl vinyl ether (8.65 g, 0.12 mol) that were dissolved in 2. The same procedure for (1) was applied to dianhydro-D-glucitol (0.50 g, 3.42*10 -3 mol) and ethyl vinyl ether (2.92 g, 0.041 mol) that were dissolved in 2.5 ml of THF. The solution was then added to the catalytic solution that was prepared by palladium (II) acetate (0.038 mg, 1.70*10 -4 mol) and 1,10-phenanthroline (0.045 mg, 2.55*10 -4 mol) in 2 mL of CH 2 Cl 2 . 1 H NMR analysis of an aliquot in acetone-d6 showed 50% conversion. The product was then purified in the same manner as described for (1). 6-(Vinyloxy)-hexahydrofuro [3,2-b]furan-3-ol (4) was obtained as an orange-yellow liquid (0.25 g, 42% yield).

CONCLUSION

Several functionalized vinyl ethers were successfully synthesized by the transetherification catalyzed by a palladium complex generated in situ with conversions ranging between 50 and 82 and in 40-75% yields. Different experimental reaction parameters were optimized with a series of reactions carried out at room temperature for 24 hours using a high EVE excess. All vinyl ethers were characterized by 1 H and 13 C NMR spectroscopy. The newly synthesized VEs could bear various functional groups that can potentially induce remarkable properties and different polymerization applications.

3 Scheme 1 .

 31 Scheme 1. Synthesis of vinyl ethers bearing various functional groups by transetherification of the corresponding alcohol and ethyl vinyl ether

Scheme 3 .

 3 Scheme 3. Transetherification of bis(2-2(hydroxyethoxy)ethyl)ether and EVE leading to either mono-functional VE (3) or bis-functional VE (3')

Figure 1 . 1 HFigure 2 . 13 C

 11213 Figure 1. 1 H NMR spectrum of 2-(vinyloxymethyl) furan (1) (CDCl 3 , 20 °C, 300 MHz), * represents the signal of the alcohol precursor remaining traces

  Ethyl vinyl ether, cobalt (II) acetate tetrahydrate, zinc (II) acetate, palladium (II) acetate, 1,10-phenanthroline, 1,2-phenylethanediol, triphenylphosphine, 2-(hydroxymethyl) .9%), THF (anhydrous, 99.9%), sodium sulfate, were purchased from Sigma Aldrich. Deuterated chloroform (99.96 atom % D), 1.4-dioxane (anhydrous, 99.8%

Table I .

 I Transetherification of EVE and furfuryl alcohol in different reaction conditions a

								).
						M(OAc) 2 (X%)
		O	OH	+	O	solvent Ligand (Y eq.)	O	O
						25 °C, 24 h		(1)
	Scheme 2. Synthesis of 2-(vinyloxymethyl) furan by transetherification of the furfuryl alcohol and ethyl vinyl
	ether
		Using our typical procedure, the following key parameters were investigated: the
	initial EVE/alcohol molar ratio, alcohol concentration, nature of the solvent, presence and
	nature of the ligand, amount of the catalyst, and choice of the metal precursor. The results are
	summarized in Table I.
	Entry M(OAc) 2 (X%)		Ligand (Y eq.)	Solvent	Initial EVE/ alcohol molar ratio	[alcohol] (mol/L)	Conv. b (%)
	1	Pd(OAc) 2 (2%)		-		CH 2 Cl 2	12	2.2	0
	2	Pd(OAc) 2 (2%)	1-Phenyl-1,2-ethanediol (1.5)	CH 2 Cl 2	12	2.2	0
	3	Pd(OAc) 2 (2%)	Triphenylphosphine (2)	CH 2 Cl 2	12	2.2	0
	4	Pd(OAc) 2 (2%)	1,10-phenanthroline (1.5)	CH 2 Cl 2	12	2.2	69
	5	Zn(OAc) 2 (2%)	1,10-phenanthroline (1.5)	CH 2 Cl 2	12	2.2	0
	6	Co(OAc) 2 (2%)	1,10-phenanthroline (1.5)	CH 2 Cl 2	12	2.2	0
	7	Pd(OAc) 2 (2%)	1,10-phenanthroline (1.5)	DMF	12	2.2	8
	8	Pd(OAc) 2 (2%)	1,10-phenanthroline (1.5)	THF	12	2.2	16
	9	Pd(OAc) 2 (2%)	1,10-phenanthroline (1.5)	Dioxane	12	2.2	50
	10	Pd(OAc) 2 (2%)	1,10-phenanthroline (1.5)	Toluene	12	2.2	57
	11	Pd(OAc) 2 (2%)	1,10-phenanthroline (1.5)	-	12	2.2	66
	12	Pd(OAc) 2 (2%)	1,10-phenanthroline (1.5)	CH 2 Cl 2	6	2.2	60
	13	Pd(OAc) 2 (2%)	1,10-phenanthroline (1.5)	CH 2 Cl 2	24	2.2	57

a Reaction conditions : Solvent or bulk (entry 11), 24 h, 20 °C, M(OAc)2 (X %), ligand (X eq /Pd(OAc)2).

b % conversion was assessed by

1

H NMR spectroscopy; Conv. (%) = x 100, where ∫i represent the integral of the signal centered at i ppm in 1 H NMR spectrum

Table II .

 II Transetherification of primary alcohols that bear various side groups with ethyl vinyl ether a , telechelic bis vinyl-ether (3') was formed as a major product, and the % yield dropped from 50 to 40 (entry 3). Finally, the transetherification of dianhydro-D-glucitol was exceptionally performed in THF/CH 2 Cl 2 due to its low solubility in dichloromethane. The

	desired vinyl ether (4) was obtained with 50 % conversion and 42 % yield (entry 4).
	Entry HO	(	O	ROH OH ) 3	O 12 eq.	VE Pd(OAc) 2 (2%, 5%) 1,10-Phenanthroline(1.5 eq./Pd) CH 2 Cl 2 , 24 h, 25 o C	Pd(OAc) 2 (x%) O O + O O	3 3	Conv. b (%) OH O	Yield c (%)
	1		O		OH		2-(vinyloxymethyl) furan (1)	2		69	59
					CH 2 OH
	2	H 3 CO			OCH 3	1,2,3 trimethoxy-5-(vinyloxymethyl) benzene (2)	2		82	75
					OCH 3
	3	HO		O	OH		2-(2-(vinyloxy)ethoxy) ethanol (mono-3)	2		75	50
					3		(2-(2-(vinyloxy)ethoxy)ethoxy) ethene (bis-3')	5		61	40
	4 d				O H	OH	6-(vinyloxy)-hexahydrofuro[3,2-b]furan-3-ol (4)	2		50	42
		HO H	O
									reaction carried out in THF
		2-(Vinyloxymethyl) furan (1) was prepared under the optimal conditions by
	transetherification catalyzed by a palladium (II) complex generated in situ. 69% conversion
	and 59% yield were obtained (entry 1). 1,2,3-trimethoxy-5-(vinyloxymethyl) benzene (2) was
	synthesized under the same conditions with 82% conversion and 75% yield (entry 2). The
	transetherification of tetraethylene glycol and ethyl vinyl ether (Scheme 3) led to mono-vinyl

a Reaction conditions: 20 °C, 24 h, Pd(OAc)2 (2%) generated in situ, 1,10-phenanthroline (1.5 eq /Pd(OAc)2).

b Conversion was assessed by 1 H NMR spectroscopy; Conv. (%) = x 100. c isolated yield. d ether (3) with 75% conversion calculated based on the 1 H NMR analysis of the reaction mixture. Surprisingly, in a random experimental trial, when the amount of the catalyst was raised to 5%

  1 H NMR (300 MHz, acetone-d6) δ (ppm): 6.49 (dd, -CH=CH 2 , 1H, H3, 3 J trans =14.37 Hz, 3 J cis =6.87 Hz), 4.10 (dd, CH a H b =CH, 1H, H1, 3 J cis =6.76 Hz, 2 J gem =2.03 Hz), 3.90 (dd, CH a H b =CH, 1H, H2), 4.50-3.50 (cyclic CH 2 and CH, 8H). 13 C NMR (300 MHz, CDCl 3 ) δ (ppm): 151.00 (-CH=CH 2 , 1C, C2), 88.90 (cyclic CH, 2C, C4 and C7), 78.00 (CH-O-CH=, 1C, C3), 72.00 (-CH-OH, 1C, C6), 82.50 (CH=CH 2 , 1C, C1), 76.50 et 73.10 (cyclic CH 2 , 2C, C8 and C5).

ACKNOWLEDGEMENTS

The authors thank Ecole Nat. Sup. de Chimie de Montpellier (ENSCM) and Ecole doctorale à l'université Libanaise (EDST) as well as the Université Libanaise-programme de recherche for the financial support. Pr. Bernard Boutevin (ENSCM), and Dr. Ali Hachem and Dr. Iyad Karameh (Université Libanaise) are also acknowledged for their inputs.