Keywords: Ag thin films, W under layer, surface morphology, wetting property

Silver thin films with thickness ranging from 3 nm to 33 nm were sputter deposited onto silicon wafers and tungsten layers. Those W layers were previously synthesized in the same DC magnetron sputter deposition system with various experimental conditions (argon pressure, target to substrate distance) in order to stabilize different surface morphologies. SEM observations and AFM images showed that the growth mode of Ag films is similar on Si substrates and on the smoothest W layers, whereas it is modified for rough W layers made of sharp grains. The effect of the W layer morphology on Ag film growth was clearly evidenced when the deposition took place at high temperature. It is seen that performing the deposition onto substrates of various morphologies allows to tailor the wetting property of the Ag deposit.

Introduction

It is well known that wetting properties of materials depend on their chemical nature and their roughness/morphology. The role of the surface roughness has widely been studied and it has been found to have opposite effects: increasing the roughness of a chemically hydrophobic surface will enhance its hydrophobicity, while increasing the roughness of a hydrophilic surface will further its hydrophilicity [START_REF] Raibeck | Investigating environmental burdens and benefits of biologically inspired self-cleaning surfaces[END_REF]. It has also been shown that surface state composed of superposition of two roughness patterns at different length scales, and fractal roughness may lead to superhydrophobicity, characterized by a contact angle higher than 150° [START_REF] Bhushan | Natural and biomimetic artificial surfaces for superhydrophobicity, self-cleaning, low adhesion, and drag reduction[END_REF]. Studies have been conducted on the roughness scales that would promote superhydrophobicity [START_REF] Oner | Ultrahydrophobic Surfaces. Effects of Topography Length Scales on Wettability[END_REF][START_REF] Boinovich | Hydrophobic materials and coatings: principles of design, properties and applications[END_REF]. It has been found that nano-roughness associated to micropatterns could promote this property, for instance by studying biological samples such as Lotus leaves [START_REF] Cheng | Effects of micro-and nano-structures on the self-cleaning behaviour of lotus leaves[END_REF]. This kind of multimodal roughness has been successfully obtained on various metal surfaces [START_REF] Fu | Fabrication of super-hydrophobic surfaces on aluminum alloy substrates[END_REF][START_REF] Hang | Super-hydrophobic nickel films with micro-nano hierarchical structure prepared by electrodeposition[END_REF]. It is interesting to note that by properly texturing the surface, superhydrophobicity has been obtained on hydrophilic substrates, showing the major role played by the morphology on the wetting property [START_REF] Xi | The preparation of lotus-like super-hydrophobic copper surfaces by electroplating[END_REF].

Depending on the targeted application, hydrophobic or hydrophilic surfaces are requested. Hydrophilicity is preferred when strong interaction with aqueous media is necessary, like for example, antibacterial property or anti-fogging [START_REF] Liu | Bio-inspired superoleophobic and smart materials: Design, fabrication and application[END_REF]. On hydrophilic surfaces the presence of a tightly adherent layer of water may help avoiding oil adsorption, like in anti-graffiti coatings [START_REF]Hydrophobicity, Hydrophilicity and Silane Surface Modification by Barry Arkles[END_REF]. Hydrophobicity and superhydrophobicity is associated to water repellency, anti-fouling, anti-adhesion and self-cleaning [START_REF] Nimittrakoolchai | Deposition of organic-based superhydrophobic films for anti-adhesion and self-cleaning applications[END_REF]. Usually for a surface to be self-cleaned a high static contact angle is not the only required parameter, a low sliding angle is also necessary, that means the contact angle hysteresis has to be low [START_REF] Liu | Bio-inspired superoleophobic and smart materials: Design, fabrication and application[END_REF]. On this kind of surfaces, the droplets may roll in addition to slide, which facilitates removal of contaminant particles and waste, which are weakly link to the material [START_REF] Boinovich | Hydrophobic materials and coatings: principles of design, properties and applications[END_REF].

However in some cases, a self-cleaning effect has been reported for superhydrophilic surfaces, like for TiO2-based compounds [START_REF] Guan | Relationship between photocatalytic activity, hydrophilicity and self-cleaning effect of TiO2/SiO2 films[END_REF]. Self-cleaning property of TiO2 has been investigated by many authors and has been found to be also related to its photo-catalytic property [START_REF] Fujishima | Titanium dioxide photocatalysis: present situation and future approaches[END_REF][START_REF] Lin | Design and fabrication of antireflective nanoparticulate thin films with superhydrophilic self-cleaning properties on glass substrate[END_REF]. Thanks to its superhydrophilicity, dusts and organic contaminants formed by UV decomposition of oils… etc. are able to be efficiently removed from the surface under rainwater, i.e. under a water flow.

In the aim to synthesize antibacterial and easy-to-clean coatings, the wetting property of magnetron sputtered Ag thin films deposited onto substrates exhibiting various morphologies was studied. Ag was chosen for its well-known broad-spectrum antibacterial ability, excellent biocompatibility and satisfactory stability [START_REF] Zheng | Introduction of antibacterial function into biomedical TiNi shape memory alloy by the addition of element Ag[END_REF][16][START_REF] Xinping | Evaluations of Antibacterial Activity and Cytotoxicity on Ag Nanoparticles[END_REF]. The idea is to build a twoscale roughness, in order to further the removal of water that is used during the cleaning step in the targeted application.

Magnetron sputter deposition was used among PVD techniques because most of the materials can be efficiently sputtered and many parameters can be varied (input power, gas pressure, target-substrate distance) to control the film properties. For instance, biasing the substrate would allow assisting the film growth by plasma ion bombardment, which is known to enhance the density and lower the roughness. High peak power impulses (HiPIMS) could also be used in order to induce ionization of the sputtered vapor, which could further broaden the accessible range of film characteristics.

In the preliminary study reported in this paper, conventional DC (direct current) magnetron sputtering was used to investigate, first, the growth of Ag on a flat silicon wafer, and second to synthesize W layers, that were used as textured substrates. W thin films deposited by magnetron sputtering have often been investigated [START_REF] Wang | Density measurement of W thin films coating by combination of ion beam analysis and scanning electron microscopy[END_REF][START_REF] Shen | Residual stress, microstructure, and structure of tungsten thin films deposited by magnetron sputtering[END_REF][START_REF] Wang | Deposition and structure of W-Cu multilayer coatings by magnetron sputtering[END_REF][START_REF] Sun | Microstructure and Mechanical Properties of Nanocrystalline Tungsten Thin Films[END_REF]. W was chosen in the present work because, depending on the deposition conditions, films with very different features could be obtained, ranging from dense and flat to porous, columnar and rough [START_REF] Karabacak | -phase tungsten nanorod formation by oblique-angle sputter deposition[END_REF][START_REF] Khedir | Morphology control of tungsten nanorods grown by glancing angle RF magnetron sputtering under variable argon pressure and flow rate[END_REF]. Moreover it exhibits good hardness, which is one of the required properties, and has already been deposited in our sputtering apparatus [START_REF] Wang | Deposition and structure of W-Cu multilayer coatings by magnetron sputtering[END_REF]. The influence of the substrate morphology onto the Ag film characteristics was studied by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Contact angle measurements were performed on Ag on Si (to study the intrinsic features of Ag films grown by magnetron sputtering), W on Si (to evidence wetting properties introduced by the sublayer) and Ag on W/Si (final two-scale roughness coatings) in order to evidence the parameters (surface roughness and/or chemistry effect) governing the wetting property.

To our knowledge, it is the first time that a stacking of layers is used to modify the wetting property of an antibacterial deposit in the aim to improve its cleaning efficiency.

Materials and methods

Ag films and W under-layers were deposited onto Si wafers by DC (direct current) magnetron sputtering of pure Ag (99.99%) and W (99.99%) 4" target, respectively. The substrates (Si wafers and W/Si) were placed on a heatable-rotating substrate holder 12 cm away from the target, in a stainless steel vacuum chamber evacuated down to 10 -4 Pa. Deposition conditions of silver remained unchanged throughout the study: Ar gas pressure was set to 2.5 Pa, and input power to 50 W. The deposition time was varied from 30 s to 5 min, corresponding to deposited thicknesses from about 3 to 33 nm. For thinnest deposited films, the thickness is calculated from the deposition rate determined on thickest films. In an attempt to investigate a wide range of Ag morphologies, deposition was also carried out at different temperatures: 230 and 400°C.

In order to synthesize layers with various morphologies, sputtering of W was carried out at different Ar pressures (from 0.25 to 5 Pa) and target-to-substrate distances (11-12 cm). Both parameters (or P*d) determine the number of collisions that sputtered W atoms undergo in the gas phase, which plays a role on their kinetic energy as they reach the substrate [START_REF] Wang | Deposition and structure of W-Cu multilayer coatings by magnetron sputtering[END_REF][START_REF] Karabacak | -phase tungsten nanorod formation by oblique-angle sputter deposition[END_REF][START_REF] Khedir | Morphology control of tungsten nanorods grown by glancing angle RF magnetron sputtering under variable argon pressure and flow rate[END_REF]. As often reported in literature for PVD techniques, the energy brought by depositing atoms influences the film structure [START_REF] Thornton | Influence of apparatus geometry and deposition conditions on the structure and topography of thick sputtered coatings[END_REF][START_REF] Anders | A structure zone diagram including plasma-based deposition and ion etching[END_REF][START_REF] Petrov | Microstructural evolution during film growth[END_REF]. This is especially the case for W, which has been found to form dense smooth films at low pressure and columnar, porous, rough films at high pressure. The input power during W sputter deposition, which is another parameter that could influence the film morphology [START_REF] Wang | Density measurement of W thin films coating by combination of ion beam analysis and scanning electron microscopy[END_REF][START_REF] Wang | Deposition and structure of W-Cu multilayer coatings by magnetron sputtering[END_REF], was set to 500 W in this study. Indeed, the aim of this work was not to perform a parametric study of W growth, but to synthesize W layers exhibiting a large range of morphologies. This was possible in our experimental configuration with a fixed power. Typical deposition time was 10 min, leading to layers of 300 to 800 nm thickness. These layers were used as substrates for the deposition of Ag thin films.

After deposition, le the morphology of Ag on Si, W on Si and Ag on W/Si samples were analyzed using a FEG-SEM (Carl Zeiss SMT, Supra-40) and an AFM from Digital Instrument, Nanoscope III using silicon cantilevers (300 kHz). AFM images have been recorded using tapping mode. Only height images are displayed. RMS roughness was estimated using image analysis of the AFM 1µm*1µm images. It is important to note that the aim here is to correlate a parameter standing for the surface morphology to the wetting properties of the samples rather than determining a true value of the roughness RMS. X ray diffraction (Brüker D8 Advance, λCu = 1.5606 Å) in -2 configuration was also carried out on W layers, in order to completely characterize the substrates on which Ag will be deposited. Static contact angle measurements were performed using a Digidrop apparatus from GBX (http://www.gbxonline.com/). A 2 µl distilled water drop was deposited onto the surface and the contact angle was determined by analysis of the recorded images using ImageTool software (http://imagetool.software.informer.com/). Three measurements were done on each sample.

Results and discussion

Ag growth onto Si wafer

In a first run of experiments, Ag was sputter deposited onto the flat surface of Si wafers. Films from 3, 6, 16 to 33 nm in thickness have been synthesized; corresponding SEM and AFM images are shown on Figure 1. 3 and 6 nm thick thin films (Fig. 1a andb) appear continuous on both SEM and AFM images, and a tiny fine structure is visible, close to SEM resolution, reason why the 3 nm thick film could not be imaged by this technique. It seems that the Ag deposit completely wet the Si wafer. On the 16 nm thick film (Fig. 1c), particles of various sizes and shapes are visible, and the 33 nm thick film is composed of 25-30 nm spherical particles (Fig. 1d). Very good agreement is found between SEM and AFM analyses.

From the above observations it appears that a change occurs in the growth mode of silver between 6 nm and 16 nm thick films. It turns from the covering of the substrate by a wetting Ag film (2D layer), to the formation of 3D islands. This kind of Stranski-Krastanov mode has already been observed for Ag growth on substrates of different kinds: Si (001) [START_REF] Glueckstein | Surface unwetting during growth of Ag on Si(001)[END_REF] and ZrNiCuAl metallic glass [START_REF] Jing | Stranski-Krastanov-like growth of an Ag film on a metallic glass[END_REF]. In this second study, 3D islands are detected above 6 ML, i.e. for thicknesses above 11 nm, which is in good agreement with our results, since we observed 3D islands for 16 nm thick film. Jing et al evidenced that 3D islands are composed of several fragments indicating the presence of grain boundaries, which suggests that they are polycrystalline [START_REF] Jing | Stranski-Krastanov-like growth of an Ag film on a metallic glass[END_REF]. The same kind of observation can be made on AFM images of both 16 and 33 nm thick films in the present work in Figure 1.

On 16 nm thick film (Fig. 1c), 100 nm clusters exist with smaller ones of various sizes and shapes, which is very different from what is seen on the 6 nm thick film. Moreover, voids seem to appear between smallest clusters, and the roughness that was very low before (0.6 nm on 3 and 6 nm thick films) increases up to 2.1 nm. Clearly the homogeneous 2D layer present on previous films seems to be completely modified. Same kind of morphology has been reported by Polop et al [START_REF] Polop | The STM view of the initial stages of polycrystalline Ag film formation[END_REF] in their study of Ag growth on amorphous silicon in the same range of thickness (16 nm). This could be explained by the unwetting of the previously formed 2D layer. It has been reported that, in some cases, this 2D layer becomes unstable when the 3D islands start to grow. This has been explained by the fact that some of the Ag atoms leave the 2D layer and go into the 3D islands, exposing area of the original substrate [START_REF] Glueckstein | Surface unwetting during growth of Ag on Si(001)[END_REF]. In the 33 nm thick film (Fig. 1d), the 2D layer does not exist anymore, the clusters exhibit an intermediate size and form a structure typically obtained when pure 3D island growth occurs like, for example, in the case of Pd growth on silicon [START_REF] Brault | Percolative growth of palladium ultrathin films deposited by plasma sputtering[END_REF].

It is important to note that the 2D layer observed at low Ag content seems not to be related to chemical reactivity at the interface since 1) the Si wafer is covered by a stable 2 nm thick native oxide and 2) Ag is known to have no chemical affinity with Si [START_REF] Polop | The STM view of the initial stages of polycrystalline Ag film formation[END_REF].

Complete study of the growth mode of Ag is out of the scope of this paper, the aim in the present work being to evidence the morphology of Ag films depending on the thickness and correlate them with the wetting property. Comparison with literature shows that Ag growth occurs in an already reported "pseudo-Stranski-Krastanov" mode. This mode could be very interesting for the targeted property since different morphologies could be obtained: continuous 2D layer or 3D islands. However, analysis of AFM images reveals that the roughness does not vary in a very large range (0.6 to 2.2 nm). In an attempt to enlarge this range, depositions have been carried out in various deposition conditions.

Figure 2 shows SEM and AFM images of a 6 nm thick Ag film grown at 400°C. The obtained morphology is completely different from what has been seen at room temperature: clusters of various sizes ranging from 10 to 80 nm are present and substrate surface is visible between them, especially on SEM images. Thus, it seems that when the substrate is heated during the growth, 2D layer is not able to be formed or, that the unwetting phenomenon starts early, i.e. at lower deposited thickness. Deposition of 33 nm thick Ag film was performed at 230 and 400°C. Results are presented in Figure 3. Large 3D aggregates are formed, which size increases and density decreases with temperature. It is important to note, however, that very small clusters are also visible in addition to big ones: the cluster size distribution appears to be bimodal.

The tendency of Ag films to unwet the substrate and form large 3D islands when heated has already been reported [START_REF] Jing | Stranski-Krastanov-like growth of an Ag film on a metallic glass[END_REF]. Kim et al has also evidenced this behavior in their study on the thermal stability of Ag thin films [START_REF] Kim | Thickness dependence on the thermal stability of silver thin films[END_REF]. They investigated the resistivity of Ag films with thicknesses of 35, 69 and 85 nm. They showed that unwetting of the film, i.e. formation of 3D clusters by agglomeration, which induces a sharp increase of the resistivity, occurred at higher temperature for thickest films. They found an agglomeration temperature of 102°C for the 35 nm thick film. This is in quite good agreement with our results, since large aggregates are observed on 33 nm thick film from 230°C deposition temperature (see Fig. 3a). However, in the present work, Ag clusters of very small sizes are also present, forming a kind of background on SEM pictures. This is especially visible on the 33 nm thick film deposited at 400°C (see Fig. 3b). This difference with Kim's results may be due to the fact that our work does not deal with an annealing process, but with an increase of the deposition temperature. In Kim's case the thermal stability of the film is involved, in ours, the growth mechanism itself is affected. The main result regarding the present study is that very different morphologies were obtained and larger roughnesses were reached.

Contact angle measurements were performed on all synthesized Ag films. Results are presented on the graph of Figure 4 in function of the roughness measured on AFM images. When the roughness is very low, i.e. at small thicknesses, high values are measured, typical of hydrophobic films. A clear trend observed on the figure is a decrease of the contact angle as the roughness rises.

3.2.Characterization of W layers

The growth of W on Si by magnetron sputter deposition has widely been investigated and the film microstructure and density are known to vary a lot [START_REF] Wang | Density measurement of W thin films coating by combination of ion beam analysis and scanning electron microscopy[END_REF][START_REF] Sun | Microstructure and Mechanical Properties of Nanocrystalline Tungsten Thin Films[END_REF][START_REF] Khedir | Morphology control of tungsten nanorods grown by glancing angle RF magnetron sputtering under variable argon pressure and flow rate[END_REF]. The kinetic energy brought by W atoms at the substrate has been evidenced as the major parameter determining the morphology, for a fixed input power [START_REF] Wang | Density measurement of W thin films coating by combination of ion beam analysis and scanning electron microscopy[END_REF]. Sputtered W atoms lose their kinetic energy by collision with Ar gas atoms in their travel towards the substrate. The number of collisions between Ar and W atoms depends on both the pressure and the distance between the target and the substrate, thus variation of the P*d parameter allows to synthesize W films with various morphologies. Table 1 presents the experimental parameters used in this work to develop three typical W films: WA, WB and WC on Si wafer. SEM plane views and cross sections are given in Figure 5 for typical W films grown at low and high P*d. At low values, for WA layer (Fig. 5a), kinetic energy of the W atoms is higher, and thus surface diffusion occurs which leads to a dense close-packed structure exhibiting a smooth surface with a roughness value equal to 2.1 nm. On the contrary, at high values (WB and WC layers), when the deposited kinetic energy is low, a porous columnar film is formed, composed of sharp grains at the surface which join together to form kind of islands separated by a deeper void frontier (see Fig. 5b and Fig. 5c).

AFM analysis was conducted on these W layers, but images have only been successfully obtained on WA (see Fig. 6). It is thought that the kind of needles made of hard W (see Fig. 5c) present on the other W layers could damage the AFM tip and, consequently, prevent from obtaining an image of the surface. This wear mechanism is expected to be less critical on the smooth morphology of WA. On this last layer, lamellar nature of the grains grown at low P*d evidenced by SEM is well observed and AFM images too (Fig. 6).

XRD performed on WA and WB layers (see Fig. 7) shows that the -W phase is present in both cases, in contrast to a small amount of the -W phase which is nevertheless only detected on WA synthesized at the lowest P*d value. Appearance of both phases in sputtered W thin films has been studied by many authors. -W phase is a defect phase, usually obtained at high pressure and stabilized by the presence of O contamination [START_REF] Shen | Influences of oxygen on the formation and stability of A15 -W thin films[END_REF][START_REF] Djerdj | XRD line profile analysis of tungsten thin films[END_REF][START_REF] O'keefe | Phase transformation of sputter deposited tungsten thin films with A15 structure[END_REF]. It has also been evidenced in W nanorods elaborated at grazing incidence and high P*d values [START_REF] Karabacak | -phase tungsten nanorod formation by oblique-angle sputter deposition[END_REF][START_REF] Khedir | Morphology control of tungsten nanorods grown by glancing angle RF magnetron sputtering under variable argon pressure and flow rate[END_REF]. On the contrary, -W phase formation is furthered at low pressure, in agreement with its presence in the WA layer. On the XRD patterns, it is interesting to note that the FWHM of the peaks are broader in the case of WC. The average size of the crystallites in WA layer calculated using Scherer formula, is equal to 35 and 29 nm corresponding respectively to the first two reflections at around 35° and 40°. For WC layer, the obtained values are lower: about 7 and 11 nm. Even if diffracting domains cannot be determined from SEM observations, the better crystallinity of WA evidenced by XRD can be correlated to the denser layer composed of larger grains seen on SEM images (Fig. 5).

Contact angle measurements performed on these W layers on silicon have revealed a highly hydrophilic behavior, whatever the morphology (see Fig. 9). On some of them, water was so spread that no angle could be measured. In some cases, when the surface was tilted, the water was seen to slide very easily until it was removed from the surface. This sliding behavior could be interesting for cleaning applications. As reported by some authors for hydrophobic surfaces, when a water droplet slides, in addition to roll off, the removal of dirt and soil is promoted [START_REF] Boinovich | Hydrophobic materials and coatings: principles of design, properties and applications[END_REF]. If water completely spread the surface before sliding, the cleaning effect could be enhanced.

Ag deposition onto W layers

Deposition of Ag at room temperature

Table 2 presents the notation used to describe the different Ag layers deposited on W/Si substrates according to their thickness and temperature. In this first part, Ag3_RT, Ag6_RT and Ag33_RT were deposited onto WA, WB and WC layers. SEM and AFM images for Ag3_RT and Ag33_RT layers are given in Figure 8. One has to keep in mind that a wetting 2D layer was obtained on Si wafer for a thickness of 3 nm. On SEM image of the Ag3_RT on WA sample (Fig. 8a), the fine lamellar structure of the bare WA (see Fig. 5a) is not visible anymore, whereas the main general aspect is still apparent. The same conclusion can be made on the AFM image: lamellae that were clearly evidenced on the original WA layer (see Fig. 6a), appear now covered by particles (Fig. 8a) that are attributed to the Ag deposit. On the two other Ag3_RT samples (Fig. 8b and Fig. 8c), the structure of the bare W layer is still visible on SEM images, but a close look indicates that the W structure is partly coated by Ag (compare Fig. 8b with Fig. 5b and Fig. 8c with Fig. 5c). On AFM images (Fig. 8b and8c), the general appearance of the surface is the same as evidenced by SEM. The islands present on bare WC (Fig. 5c) are particularly well evidenced. It appears that the morphology of WB and WC layers impedes the formation of a completely wetting Ag layer, in opposite to what happens on the "flat" WA.

On 33 nm thick Ag film different conclusions can be drawn (see Fig. 8 d-f). First on Ag33_RT on WA, the morphology of the W bare layer has completely disappeared, very fine structure is observed on both SEM and AFM images. Ag film, also evidenced on SEM cross section, seems to hide the W morphology. On Ag33_RT on WB and Ag33_RT on WC images, the general appearance remains, but the W sharp grains cannot be recognized anymore, they are now decorated by Ag. On the Ag33_RT deposit the obtained morphologies are thus a mixture of Ag and W film features: a two-scale morphology has been built. Contact angle and roughness measurements were performed on all these Ag layers on W/Si samples. On the graph of Figure 9 histograms present contact angles for these samples as well as for bare W and Ag layers on Si samples. Tungsten is intrinsically hydrophilic, the smooth WA layer exhibits a contact angle of 65°. When the roughness increases, the hydrophilic character of W is enhanced (see values for WB and WC), as expected, since it is known that roughness tends to further the wetting property of a chemical element [START_REF] Raibeck | Investigating environmental burdens and benefits of biologically inspired self-cleaning surfaces[END_REF]. It is seen on this graph that when the Ag film is thick (Ag33_RT), the wetting property is mainly governed by Ag element: high contact angles are measured. As mentioned above, Ag on Si samples are hydrophobic (see Fig. 4). Another result visible on the histograms is that for the same Ag film thickness, the contact angle value is the lowest when the W layer is the roughest. For instance Ag3_RT layers, contact angle values turn from 93° on WA, 55° on WB to 32° on WC. It can be deduced that the wetting property of the thinnest Ag deposits (Ag3-RT and Ag6_RT) on W/Si is clearly a combination of Ag films and W layers intrinsic properties.

Deposition of Ag at high temperature

6nm and 33nm thick Ag films (Ag6_400 and Ag33_400 layers) have also been deposited at 400°C onto various W layers. SEM and AFM images are shown on Figure 10. From SEM cross-sections given on Figure 11, it can be checked that the underlying W layer does not evolve during the deposition step at this temperature.

On WA layer the thinnest Ag film (Fig. 10 a) exhibits morphology similar to that obtained on Si wafer, which is not surprising since, as mentioned above, this flat WA layer does not affect the Ag growth mode. Morphology of the Ag6_400 on WC (Fig. 10b) on the contrary, is completely different. The general appearance of the bare W layer is preserved, but, again, sharp grains seem to be decorated by Ag. Some larger clusters are also visible locally. AFM analysis gave clear images and no wear process of the tip took place. This is an indication that W grains are covered by Ag. Comparison of Fig. 10a and 10b clearly shows that rough WC under-layer induces the growth of smaller grains. Similar trends can be drawn from the images of the thick Ag film: the morphology on WA layer is close to that on silicon (see Fig. 3b), whereas the texture of the WC under-layer impedes the formation of big Ag grains (Fig. 10c and Fig. 10d).

The contact angles of all the Ag on W/Si samples are put together in the graph given Figure 12. The same trend is observed than on Si wafer, i.e. the contact angle is the highest when the roughness is the lowest. But on these Ag on W/Si samples the range of contact angle is larger than on Ag on Si ones, starting from 106° down to 17°, in a limited roughness interval. On the Figure 12, green squares correspond to Ag deposits performed at 400°C. Working at this temperature allows reaching higher roughness values and very low contact angle values. An interesting result is that some of the Ag films deposited onto rough W layers exhibit the already described sliding property that could be very useful for cleaning application.

Conclusion

In this work, Ag thin films were deposited onto flat Si wafers and W layers of various morphologies. Variation of the growth mode and of the final roughness on these textured under-layers was investigated by SEM and AFM. Wetting property of Ag deposit on W/Si samples was compared to that of the bare layers (Ag on Si and W on Si) and correlated to the roughness measured on AFM images.

Ag on Si films were found hydrophobic, they exhibit contact angles larger than 90°. It was shown that the sharp column structure furthers the intrinsic hydrophilic property of tungsten. On the roughest W layers a sliding behavior was evidenced.

Depositing a non-continuous Ag film on W layers leads to a wetting property that is a combination of the surface roughness driven by the W sublayer, and a chemical effect (role of the Ag upper layer). This allows, for the same amount of deposited Ag, to turn from a hydrophobic film to a highly hydrophilic one, by playing with the W sublayer morphology.

On the smoothest W layer, the growth of Ag was found similar to that on Si.

No super-hydrophobic neither super-hydrophilic films were obtained in this work, partly because the variation of roughness and size of the patterns remains in a limited nano-scale range. The aim of this work was thus not to synthesize such surfaces, but to find a compromise between desired properties. We have shown that the stacking of Ag and W layers allowed to modify the intrinsic wetting property of silver and to synthesize highly hydrophilic sliding Ag surfaces that would further the interaction with bacteria and promote the removal of soil under a water flow.

Figure 1 :

 1 Figure 1: SEM and AFM images of Ag films with various thicknesses deposited on silicon: a) 3 nm, b) 6 nm, c) 16 nm, d) 33 nm. Morphology of the 3 nm thick film is to fine to be observed by SEM.

Figure 2 :

 2 Figure 2: SEM and AFM images of a 6 nm thick Ag film grown on Si at 400°C.

Figure 3 :

 3 Figure 3: SEM and AFM images of a 33 nm thick Ag film deposited onto Si at a) 230°C and b) 400°C.

Figure 4 :

 4 Figure 4: Water contact angle measurements (dots) obtained on Ag on Si films as function of the roughness measured on AFM images. The modeled line (dotted line) is only to guide the eyes.

Figure 5 :

 5 Figure 5: SEM plane views and cross sections of W layers used as substrates in the present work: a) WA, b) WB and c) WC.

Figure 6 :

 6 Figure 6: AFM images of WA layer: a) 1 µm  1 µm, b) 500 nm  500 nm.

Figure 7 :

 7 Figure 7: -2 XRD diffractograms of WA and WC layers.

Figure 8 :

 8 Figure 8: SEM and AFM images of Ag films deposited at room temperature on W layers: a) Ag3_RT on WA, b) Ag3_RT on WB, c) Ag3_RT on WC, d) Ag33_RT on WA, e) Ag33_RT on WB, f) Ag33_RT on WC.

Figure 9 :

 9 Figure 9: Water contact angles measured on Ag films deposited on Si wafers and various W layers. Values obtained on bare W layers are also given.

Figure 10 :

 10 Figure 10: SEM and AFM images of Ag films deposited at 400°C on W layers a) Ag6_400 on WA, b) Ag6_400 on WC, c) Ag33_400 on WA and d) Ag33_400 on WC.

Figure 11 :

 11 Figure 11: SEM plane views and cross-sections a) and b) before and c) and d) after deposition of Ag at 400°C.

Figure 12 :

 12 Figure 12: Evolution of the water contact angle with respect to the RMS roughness evaluated by AFM on Ag on W/Si samples.

Figure 1 Figure 2

 12 Figure 1

Figure 3

 3 Figure 3

Figure 4

 4 Figure 4

Figure 5

 5 Figure 5

Figure 6

 6 Figure 6

Figure 7

 7 Figure 7

Figure 8

 8 Figure 8

Figure 9

 9 Figure 9

Figure 10

 10 Figure 10

Figure 11

 11 Figure 11

Figure 12

 12 Figure 12

Table 1 :

 1 Experimental parameters of the typical W films: WA, WB and WC.

	W layers on Si	Experimental conditions Power (W) Temperature Pressure* distance (Pa.cm) Thickness (nm)

Table 2 :

 2 Experimental conditions and notations for Ag layers deposited on W layers.

	Ag layers on W	Experimental conditions Thickness (nm) Temperature (°C)
	Ag3_RT	3	RT
	Ag6_RT	6	RT
	Ag6_400	6	400
	Ag33_RT	33	RT
	Ag33_400	33	400

Acknowledgements

The authors want to thank Patrick Laffez for XRD analyses and discussions on results. This work was supported by La Région Centre and Europe via European regional development fund (FEDER).

Sputtered Ag thin films with modified morphologies: influence on wetting property P. Dutheil a* , A.L. Thomann a , T. Lecas a , P. Brault a , M.Vayer b a GREMI, UMR 7344-Université D'Orléans, 14 rue d'Issoudun, BP 6744, Orléans 45067, France b CRMD, 1b rue de la Férollerie, 45071 Orléans, France.


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Adriana Lopes

Santos

Ian Probert

Daniel Vaulot

Colomban De Vargas

Richard Christen
email: richard.christen@unice.fr

PhytoREF: a reference database of the plastidial 16S rRNA gene of photosynthetic eukaryotes with curated taxonomy

Keywords: plastidial 16S rRNA gene, photosynthesis, high-throughput sequencing, metabarcoding, phytoplankton, protists

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

Introduction

Eukaryotes that acquired photosynthesis through endosymbiosis with cyanobacteria or plastidbearing eukaryotes are distributed across most eukaryotic super-groups and exhibit a bewildering morphological diversity across more than eight orders of magnitude in organism size [START_REF] Archibald | The evolution of algae by secondary and tertiary endosymbiosis[END_REF]Not et al. 2012). Most photosynthetic eukaryotes are unicellular (referred to as protists), but a few lineages, essentially macroalgae (e.g. the rhodophyte class Florideophyceae or the chlorophyte class Ulvophyceae) and the embryophyte land plants, have evolved into multicellular forms. The radiation of photosynthetic marine protists during the Neoproterozoic arguably led to a major oxidation event in the history of the Earth system [START_REF] Knoll | Paleobiological perspectives on early eukaryotic evolution[END_REF]. Today, eukaryotic microalgae are key players in aquatic food webs and global biogeochemical processes. In the marine ecosystem, they are the major contributors to primary production through their capacity to perform oxygenic photosynthesis [START_REF] Falkowski | The evolution of modern eukaryotic phytoplankton[END_REF][START_REF] Worden | Assessing the dynamics and ecology of marine picophytoplankton: the importance of the eukaryotic component[END_REF][START_REF] Jardillier | Significant CO2 fixation by small prymnesiophytes in the subtropical and tropical northeast Atlantic Ocean[END_REF], and to export and sequester organic carbon to the deep ocean and sediments [START_REF] Richardson | Small phytoplankton and carbon export from the surface ocean[END_REF]. In addition, evidence is growing that many eukaryotic microalgal taxa are mixotrophs, being able to both photosynthesize and feed on various microbial prey (McKie-Krisberg & Sanders 2014; [START_REF] Unrein | Mixotrophic haptophytes are key bacterial grazers in oligotrophic coastal waters[END_REF]. Their contribution to bacterivory can even exceed that of strict heterotrophs in oceanic waters [START_REF] Zubkov | High bacterivory by the smallest phytoplankton in the North Atlantic Ocean[END_REF][START_REF] Hartmann | Mixotrophic basis of Atlantic oligotrophic ecosystems[END_REF]. In coastal areas, some microalgal species can be toxic and/or form harmful blooms, which can be highly detrimental to marine life and human activities such as

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This article is protected by copyright. All rights reserved. fisheries, aquaculture and tourism [START_REF] Zingone | Harmful algal blooms: keys to the understanding of the phytoplankton ecology[END_REF][START_REF] Chambouvet | Control of toxic marine dinoflagellate blooms by serial parasitic killers[END_REF][START_REF] Anderson | Progress in understanding harmful algal blooms: paradigm shifts and new technologies for research, monitoring, and management[END_REF].

Despite their ecological and economic importance, it remains difficult to assess the total diversity of photosynthetic eukaryotes in the natural environment using classical microscopy-based techniques. For most taxa, taxonomic identification is greatly hindered by their minute size (as small as 0.8 µm for the prasinophyte Ostreococcus; [START_REF] Courties | Smallest eukaryotic organism[END_REF][START_REF] Vaulot | The diversity of small eukaryotic phytoplankton (≤3 μm) in marine ecosystems[END_REF], lack of distinctive morphological features, and fragility when classical fixatives are used [START_REF] Vaulot | A simple method to preserve oceanic phytoplankton for flow cytometric analyses[END_REF]. The complex life cycles of many microalgal species are additional obstacles that render detection in the environment very difficult with traditional microscopy. Many taxa undergo a succession of morphologically distinct forms (e.g. sexual morphotypes, resting cysts; [START_REF] Montresor | Phases, stages, and shifts in the life cycles of marine phytoplankton[END_REF][START_REF] Gaebler-Schwarz | A new cell stage in the haploid-diploid life cycle of the colony-forming haptophyte Phaeocystis antarctica and its ecological implications[END_REF] or can be "hidden" within a host cell as a parasitic or mutualistic symbiont [START_REF] Skovgaard | The parasitic dinoflagellates Blastodinium spp. inhabiting the gut of marine, planktonic copepods: morphology, ecology and unrecognized species diversity[END_REF][START_REF] Decelle | An original mode of symbiosis in open ocean plankton[END_REF]. In this context, environmental DNA metabarcoding (high-throughput sequencing of DNA markers), which has unveiled a vast and unsuspected diversity of microorganisms in recent years, provides a powerful new tool to assess the composition and ecological function of microalgal communities [START_REF] Bik | Sequencing our way towards understanding global eukaryotic biodiversity[END_REF][START_REF] Bittner | Diversity patterns of uncultured Haptophytes unravelled by pyrosequencing in Naples Bay[END_REF]. Environmental metabarcoding approaches have also been proposed for bio-assessment and bio-monitoring of sentinel or indicator species, including microalgae [START_REF] Taberlet | Towards next-generation biodiversity assessment using DNA metabarcoding[END_REF][START_REF] Kermarrec | Next-generation sequencing to inventory taxonomic diversity in eukaryotic communities: a test for freshwater diatoms[END_REF][START_REF] Pawlowski | Environmental monitoring through protist next-generation sequencing metabarcoding: assessing the impact of fish farming on benthic foraminifera communities[END_REF], and the study of diet regimes in predators [START_REF] Pompanon | Who is eating what: diet assessment using next generation sequencing[END_REF][START_REF] Piñol | A pragmatic approach to the analysis of diets of generalist predators: the use of next-generation sequencing with no blocking probes[END_REF]. For marine protists, variable regions of the nuclear ribosomal RNA genes (particularly the small subunit, 18S rRNA) are traditionally used as "universal" markers in environmental surveys [START_REF] Stoeck | Multiple marker parallel tag environmental DNA sequencing reveals a highly complex eukaryotic community in marine anoxic water[END_REF][START_REF] Logares | Diversity patterns and activity of uncultured marine heterotrophic flagellates unveiled with pyrosequencing[END_REF][START_REF] Logares | Patterns of rare and abundant marine microbial eukaryotes[END_REF]). However, several drawbacks limit the use of these nuclear markers to assess the biodiversity of photosynthetic eukaryotes: (i) some 18S rDNA clone library-based surveys

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have been shown to be biased towards heterotrophic eukaryotes, and consequently tend to overlook phototrophs in complex community assemblages [START_REF] Vaulot | Are autotrophs less diverse than heterotrophs in marine picoplankton[END_REF][START_REF] Kirkham | Basinscale distribution patterns of photosynthetic picoeukaryotes along an Atlantic Meridional Transect[END_REF]); (ii) ribosomal DNA of large protist cells (mainly heterotrophs and potentially multinucleated) or metazoans tends to be preferentially PCR-amplified because of the relatively higher copy number of ribosomal genes in these organisms [START_REF] Zhu | Mapping of picoeucaryotes in marine ecosystems with quantitative PCR of the 18S rRNA gene[END_REF][START_REF] Godhe | Quantification of diatom and dinoflagellate biomasses in coastal marine seawater samples by real-time PCR[END_REF];

(iii) distinction between phototrophic and heterotrophic taxa is very often not possible in complex multi-functional protistan groups, such as dinoflagellates. In addition, given the extreme genetic diversity of eukaryotes, "universal" DNA markers cannot detect all lineages with a high taxonomic resolution (CBOL Protist: [START_REF] Pawlowski | CBOL Protist Working Group: Barcoding eukaryotic richness beyond the animal, plant, and fungal kingdoms[END_REF]. Therefore, barcoding systems with narrower taxonomic and/or functional focus need to be developed to provide a better picture of the taxonomic and functional composition of eukaryotes in complex ecosystems.

In order to focus on the phototrophic compartment of eukaryotic communities, the photosynthetic protein-coding psbA (protein D1 of photosysytem-II reaction center) and rbcL genes (large subunit of the Ribulose-l,5-diphosphate carboxylase/oxygenase, RuBisCO) have been used as markers for phytoplankton communities [START_REF] Paul | Micro-and macrodiversity in rbcL sequences in ambient phytoplankton populations from the southeastern Gulf of Mexico[END_REF][START_REF] Zeidner | Molecular diversity among marine picophytoplankton as revealed by psbA analyses[END_REF]Man-Aharonovich et al. 2010). However, the primers targeted essentially cyanobacteria and cyanophages (viruses), and to a lesser extent photosynthetic eukaryotes, and the same species can have different sequence types (e.g. forms IA, IB for rbcL). By contrast, the plastidial 16S rRNA gene has been successfully employed in several marine surveys since it contains sufficiently conserved regions to use generalist primers to target all plastid-bearing eukaryotes and can distinguish major eukaryotic lineages with a relatively good taxonomic resolution (Fuller et al. 2006;MacDonald et al. 2007;[START_REF] Lepère | Photosynthetic picoeukaryote community structure in the South East Pacific Ocean encompassing the most oligotrophic waters on Earth[END_REF][START_REF] Shi | Plastid 16S rRNA gene diversity among eukaryotic picophytoplankton sorted by flow cytometry from the South Pacific Ocean[END_REF][START_REF] Kirkham | Basinscale distribution patterns of photosynthetic picoeukaryotes along an Atlantic Meridional Transect[END_REF][START_REF] Kirkham | A global perspective on marine photosynthetic picoeukaryote community structure[END_REF]. However, annotation and interpretation of the plastidial 16S rDNA clone libraries

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This article is protected by copyright. All rights reserved. obtained in these studies have been hindered by the lack of reference sequences of taxonomically well-identified organisms. Although a number of curated reference databases are publicly available for ribosomal RNA genes of eukaryotes and prokaryotes, such as the Protist Ribosomal Reference Database (PR2; Guillou et al. 2013), SILVA [START_REF] Pruesse | SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB[END_REF]), Ribosomal Database Project (Cole et al. 2005), and Greengenes (DeSantis et al. 2006), no reference database exists for the plastidial 16S rRNA gene of photosynthetic eukaryotes. Here, we describe an extensive reference database of the plastidial 16S rRNA gene including sequences from all major lineages of photosynthetic eukaryotes, comprising terrestrial, freshwater and marine organisms. This database, named PhytoREF, has been built through the compilation of all of the publicly available plastidial 16S rDNA sequences (amplicons and sequences extracted from plastidial genomes), as well as novel Sanger amplicons that we obtained from a wide taxonomic spectrum of cultured microalgal strains. PhytoREF is not only a new resource to explore, evaluate and monitor the diversity of photosynthetic eukaryotes in aquatic and terrestrial ecosystems, but is also useful to taxonomically identify new plastidial 16S rDNA sequences and design primers and probes to target specific lineages of photosynthetic eukaryotes. PhytoREF will pave the way for a range of applications in bio-monitoring photosynthetic eukaryotes in various habitats (e.g. water, sediments and ice), paleoecological studies of primary producers in past environments, and dietary studies in unicellular and multicellular herbivores.

Data sources

Retrieval of plastidial 16S rDNA sequences from public databases

Plastidial 16S rDNA sequences were first retrieved from the International Nucleotide Sequence Database Collaboration (INSDC: http://www.insdc.org) using various keywords (e.g. plastidial,

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This article is protected by copyright. All rights reserved. plastid, chloroplast, 16S, small subunit), and BLAST searches with different query sequences of distinct photosynthetic eukaryotic lineages. Additional sequences were retrieved from the PR2 database (May 2014) (Guillou et al. 2013; http://ssu-rrna.org/). When available in GenBank (release 201), the source literature for each sequence was searched to compile and/or verify their specific features (e.g. taxon names, culture strains), resulting in a bibliographic database of 565 source publications. 16S rDNA sequences were also extracted from all plastidial genomes available at http://www.ncbi.nlm.nih.gov/genomes/GenomesGroup.cgi?taxid=2759&opt=plastid.

All plastidial 16S rDNA sequences that originated from an identified organism (e.g. culture strain or isolated organism) were defined as reference sequences in the PhytoREF database.

Environmental Sanger 16S rDNA sequences obtained in clone libraries were also retrieved from INSDC and included in PhytoREF. Those sequences that lacked taxonomic identification were assigned at the class level based on sequence similarity scores with the references sequences of PhytoREF. Finally, all of the 16S rDNA sequences of Cyanobacteria were extracted from SILVA (release 115, [START_REF] Quast | The SILVA ribosomal RNA gene database project: improved data processing and web-based tools[END_REF]) in order to root the phylogenetic trees and unambiguously annotate and classify eukaryotic sequences. The cyanobacterial sequences are available as separate files at http://phytoref.org.

Newly generated 16S rDNA sequences from microalgal cultures

We generated 411 novel plastidial 16S rDNA sequences from eukaryotic microalgal strains from the Roscoff Culture Collection (RCC, http://roscoff-culture-collection.org/), the NCMA (formerly CCMP; https://ncma.bigelow.org/), and the culture collection of the Stazione Zoologica Anton Dohrn of Naples (Table S1). Cultured cells were harvested in exponential growth phase and concentrated by centrifugation. Total nucleic acids were extracted using the

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Nucleospin RNA II kit (Macherey-Nagel) and quantified using a Nanodrop ND-1000 Spectrophotometer (Labtech International). An 850 bp fragment was PCR-amplified with a generalist photosynthetic eukaryote primer set biased against cyanobacteria: PLA491F: 5'-GAG GAA TAA GCA TCG GCT AA -3′ (Fuller et al. 2006), and OXY1313R: 5′-CTT CAY GYA GGC GAG TTG CAG C -3′ [START_REF] West | Closely related Prochlorococcus genotypes show remarkably different depth distributions in two oceanic regions as revealed by in situ hybridization using 16S rRNA-targeted oligonucleotides[END_REF]. PCR amplifications were performed with the Phusion high-fidelity DNA polymerase (Finnzymes) in a 25-μL reaction volume, using the following PCR parameters: 30 s at 98°C; followed by 35 cycles of 10 s denaturation at 98°C, 30 s annealing at 60°C, and 30 s extension at 72 °C; with a final elongation step of 10 min at 72 °C.

PCR products were purified by either EXOSAP-IT (GE Healthcare Bio-Sciences Corp.) or the NucleoSpin® Extract II kit (Macherey-Nagel, Hoerdt, France), and sequenced in both forward and reverse directions using the ABI-PRISM Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems). Raw Sanger sequences were edited and assembled with ChromasPro v1.7.5 (Gene Codes), and primer sequences were trimmed off. The new plastidial 16S rDNA sequences were deposited in GenBank under the accession numbers LN735194 to LN735532 (Table S1), and can also be retrieved on the PhytoREF web interface at http://phytoref.org.

Construction of the PhytoREF database

The core content of the database is composed of the reference plastidial 16S rDNA sequences from public databases with unambiguous taxonomic assignation, and the novel sequences obtained from duly identified cultures. Each reference sequence, including taxonomic affiliation, was validated and filtered following different steps: (i) sequences shorter than 400 bp from cultures, and shorter than 800 bp from public sequences (including environmental sequences) were removed; (ii) sequences with more than 10 consecutive non-ACGT characters were also

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This article is protected by copyright. All rights reserved. discarded; (iii) sequence alignments were performed for different well-defined taxonomic groups (e.g. at the class level) using MAFFT v6. 953b with default options [START_REF] Katoh | MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform[END_REF], and visualized to verify the presence of introns or putative chimeric sequences; (iv) poorly aligned or difficult-to-align nucleotide positions were removed for subsequent phylogenetic analyses using the program trimAl v1.4 program (with a -gt value of 0.8, and -st value of 0.001; Capella-Gutierrez 2009); (v) phylogenetic trees were constructed separately for each taxonomic group (i.e. generally at the class level) using FastTree v.2.1.1, a fast and accurate approximate maximum-likelihood method using the GTR model [START_REF] Price | FastTree 2 -Approximately maximum-likelihood trees for large alignments[END_REF], in order to identify mislabeled sequences and other possible conflicts, and to build up the taxonomic framework (see below).

Additional publicly available plastidial 16S rDNA sequences with uncertain taxonomic status were subsequently added to this validated core dataset. These sequences were assigned to a given phylum using a similarity threshold based on global pair-wise alignments (using a Needleman-Wunsch algorithm) against the reference sequences. Sequences of each phylum were then aligned based on conserved 2D structures and sequences of the archaeal 16S, bacterial 16S and eukaryotic 18S small subunit ribosomal RNA using the SSU-align program and Infernal software package, which generate large-scale alignments of up to millions of sequences [START_REF] Nawrocki | Infernal 1.0: Inference of RNA Alignments[END_REF]). 2D-based alignments allowed us to verify whether the new sequences corresponded to the 16S rRNA gene or other ribosomal genes. Phylogenetic trees were then built using BioNJ as implemented in Seaview v.4 [START_REF] Gouy | SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building[END_REF], and visualized using TreeDyn [START_REF] Chevenet | TreeDyn: towards dynamic graphics and annotations for analyses of trees[END_REF]. Functions implemented in TreeDyn as well as specific Python scripts allowed us to determine the taxonomic level of each sequence (e.g at the "Family" level). All sequences

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This article is protected by copyright. All rights reserved. included in PhytoREF have two unique identifiers, the GenBank accession number and a PhytoREF ID number.

The taxonomic framework of PhytoREF

For every new validated sequence, we established a standardized and ranked taxonomy with 10 levels: 1-Domain; 2-Super-group; 3-Phylum; 4-Class; 5-Subclass; 6-Order; 7-Sub-order; 8-Family; 9-Genus; and 10-Species. For the "Super-group", "Phylum" and "Class" levels, the taxonomic framework of PhytoREF was derived from the PR2 database (http://ssu-rrna.org/; Guillou et al. 2013), which mainly follows a comprehensive recent classification framework of eukaryotes [START_REF] Adl | The revised classification of eukaryotes[END_REF]. The "Family" and "Order" levels of terrestrial, marine and freshwater micro-and macroalgae were based on the taxonomic classification system of the AlgaeBase database (Guiry and Guiry 2014;Guiry et al. 2014; http://www.algaebase.org/). For the taxa that were not present in AlgaeBase and PR2 (mostly embryophytes), the taxonomic classification of NCBI (May 2014) was followed (taxdump.nodes and taxdump.names files at http://www.nlm.nih.gov/research/umls/sourcereleasedocs/current/NCBI/metarepresentation.html ). Overall, the standardized taxonomic framework established in PhytoREF was designed to assist in the analysis of large datasets of environmental plastidial 16S rDNA amplicons generated by high-throughput environmental metabarcoding.

For some 16S rDNA sequences, it was not possible to define an accurate and/or complete taxonomic identity because the taxonomic description of the corresponding organism is not fully resolved, e.g. only at the "Family" or "Genus" level. In these cases, the sequence was labeled as described for the PR2 database. For instance, the taxonomic path of a sequence identified up to the "Family" level would be: Family, Family_X (for the "Genus" level), and Family_XX (for the

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"Species" level). Moreover, some key groups of microalgae have only been classified into informal clades and sub-clades based on published phylogenetic analyses without morphologybased taxonomy (e.g. prasinophyte clades VII, IX; Apicomplexa-related lineages I -V). For the PhytoREF database, information about the molecular clade was verified through specific phylogenetic analyses with the 16S rRNA gene and indicated at different taxonomic ranks. For instance, prasinophytes belonging to clade VII and sub-clade A1 are annotated: clade 7 ("Order" level), clade_7A ("Family" level), clade_7A1 ("Genus" level) and clade_7A1+sp ("Species" level). A confidence level for taxonomic assignation (named Refseq) was given to each PhytoREF sequence, indicating the level at which a given sequence is unambiguously assigned (RefSeq=1: Eukaryota; RefSeq=2: super-group; RefSeq=3: Phylum; RefSeq=4: Class; RefSeq=5:Order; RefSeq=6:Family; RefSeq=7:Genus). Finally, we also included 16S rDNA sequences originating from symbiotic microalgae or kleptoplastids found in hosts. The origin of these sequences that are generally incorrectly assigned to the host in public databases was modified and marked as "symbiont" or "kleptoplastid" in the PhytoREF database.

Results and Discussion

Overview of PhytoREF database

The PhytoREF database (release 1) currently contains 6,490 partial and complete plastidial 16S rDNA sequences (of which 6,051 sequences are > 800 bp long). In total, 411 novel sequences from marine microalgal strains were produced in this study and 6,079 sequences retrieved from public databases (5,200 amplicons from organisms and environmental samples, and 879 sequences extracted from plastidial genomes, Fig. 1). 2D alignments combined with BLAST analyses allowed us to determine that 52 sequences (mostly from streptophytes) considered as

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16S rDNA in GenBank were actually nuclear 18S rDNA, and were therefore excluded from PhytoREF. In addition to sequences from identified plastid-bearing organisms, PhytoREF contains 1,867 environmental Sanger sequences from clone libraries, which have been assigned to known eukaryotic lineages based on sequence similarity (combining a Needleman-Wunsch algorithm and phylogenetic analyses). Every PhytoREF sequence was quality-checked, phylogenetically-analyzed and classified following our standardized taxonomy. In addition to the taxonomic path, all sequences were associated to a suite of descriptors, such as the organism, molecular origin (amplicon or extracted from genomes), GenBank accession number, cultured strain, and original publication. Additional categories indicated whether sequences are environmental or belong to morphologically identified organisms, and if they correspond to kleptoplastids, parasitic or mutualistic microalgae (in such cases, the taxonomic name of the host is also provided).

Taxonomic composition of PhytoREF

All of the known major lineages of photosynthetic eukaryotes from terrestrial, freshwater and marine environments are represented in PhytoREF. At the super-group level, the composition of the database is as follows: Archaeplastida (3,834 sequences), Stramenopila (1,704 sequences), Alveolata (144 sequences), Hacrobia (501 sequences), Excavata (288 sequences), and Rhizaria (20 sequences) (Figs. 2 and3). Although our effort while building PhytoREF (in particular in producing novel plastidial reference sequences) mainly focused on marine microalgal taxa, reference sequences from streptophytes (i.e. from mosses and ferns to gymnosperms and angiosperms), and marine and freshwater macroalgae (e.g., Rhodophyceae, Phaeophyceae, Ulvophyceae) were also included in the final reference database of all known photosynthetic

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eukaryotes. Thus, PhytoREF can be used in metabarcoding surveys to study communities of microalgae in different marine and freshwater habitats (e.g. seawater, estuaries, brackish waters, lakes), as well as to detect the presence of macroalgae and streptophytes in aquatic systems as reproductive stages (gametes, pollen) or in the digestive tracts of herbivores.

Land plants (streptophytes) are numerically the dominant group in PhytoREF with 2,973 sequences, representing 373 families and 796 genera. The macroalgae from the classes Rhodophyceae (61 genera), Phaeophyceae (7 genera), and Ulvophyceae (18 genera) are represented by 161, 46, and 82 sequences, respectively (Fig. 3). Diatoms (Bacillariophyta), green algae (Chlorophyta) and Haptophyta (coccolithophores and their relatives) are numerically the most important microalgae in the database with 1094, 653 and 369 sequences, respectively, covering a wide taxonomic diversity with 109, 79, and 34 described genera and 268, 113, and 67 described species, respectively (Fig. 3). Within the Haptophyta, one environmental sequence found in the Pacific Ocean, named "S25_1200" (EF574856), was included as it has been identified to form a novel photosynthetic lineage (Janouškovec et al. 2011). For the green algae, freshwater taxa are less represented than their marine relatives and represent an obvious target for future reference sequencing. Amongst the Hacrobia, there are 126 sequences of cryptophytes covering 36 described species from marine (e.g. Rhodomonas sp.), brackish (e.g. Chroomonas sp. and Geminigera sp.) and fresh (e.g. Cryptomonas sp.) waters. Because genetic diversity does not correspond well with taxonomic features and life-stages are likely to be complex, the systematics of cryptophytes are still under revision, except for the genera Cryptomonas and Hemiselmis that have been relatively well delineated [START_REF] Hoef-Emden | Revision of the genus Cryptomonas (Cryptophyceae): a combination of molecular phylogeny and morphology provides insights into a long-hidden dimorphism[END_REF][START_REF] Hoef-Emden | Multiple independent losses of photosynthesis in the genus Cryptomonas (Cryptophyceae) -combined phylogenetic analyses of DNA sequences of the nuclear and the nucleomorph ribosomal operons[END_REF]. The euglenozoans from the super-group Excavata are also well represented in PhytoREF with 115 described species from freshwater and marine habitats, such as species of

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Euglena, Monomorphina and Trachelomonas. Of note, PhytoREF also contains 6 sequences of the recently discovered rappemonads (Hacrobia), an uncultured microalgal group widely distributed in marine and fresh waters, but taxonomically undescribed [START_REF] Kim | Newly identified and diverse plastid-bearing branch on the eukaryotic tree of life[END_REF]. Since no nuclear ribosomal (18S rRNA gene) and genomic sequences are available for the rappemonads, the plastidial 16S rRNA gene is currently the only genetic marker available for evolutionary and environmental studies of this lineage.

During the course of evolution, photosynthesis has been lost in several lineages of plants and single-celled eukaryotes, but a vestigial plastid containing a 16S rRNA gene has been retained in some taxa [START_REF] Williams | Cryptic organelles in parasitic protists and fungi[END_REF]. Some of these non-photosynthetic organisms present in PhytoREF are very often parasites, such as the holoparasitic angiosperm Epifagus virginiana, the heterotrophic euglenid Euglena longa, and the green alga Helicosporidum. In particular, 33 sequences correspond to the non-photosynthetic alveolate apicomplexans (e.g. Plasmodium, Toxoplasma, Babesia), which are obligate intracellular parasites of metazoans and protists, but which have kept a relict plastid, known as the apicoplast [START_REF] Lim | The evolution, metabolism and functions of the apicoplast[END_REF][START_REF] Macfadden | Apicoplast[END_REF]. Apicomplexan-related lineages, called ARLs (class Colpodellid), which include the microalgae Chromera [START_REF] Moore | A photosynthetic alveolate closely related to apicomplexan parasites[END_REF] and Vitrella [START_REF] Oborník | Morphology, ultrastructure and life cycle of Vitrella brassicaformis n. sp., n. gen., a novel chromerid from the Great Barrier Reef[END_REF], are also represented in PhytoREF by 77 sequences (mostly environmental), and classified according to the framework proposed by Janouškovec et al. (2011) i.e. ARL I, II etc.

As in apicomplexans and ARLs, the plastidial 16S rRNA gene of photosynthetic dinoflagellates is rapidly-evolving and their sequences are very difficult to align. This may be related to the unique genomic organization of plastid genes in dinoflagellates that can be found separately in small minicircles [START_REF] Zhang | Evolution of dinoflagellate unigenic minicircles and the partially concerted divergence of their putative replicon origins[END_REF][START_REF] Green | Chloroplast genomes of photosynthetic eukaryotes[END_REF]. This extreme genetic divergence may explain the very low PCR amplification success rates we obtained during the present study on different

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This article is protected by copyright. All rights reserved. cultures of photosynthetic dinoflagellates. Consequently, one shortcoming of PhytoREF is the limited number of dinoflagellate sequences (34 sequences representing 15 genera), a caveat to consider when interpreting metabarcoding datasets using PhytoREF. It is important to note that several plastidial 16S rDNA sequences from GenBank were re-assigned in the database because they were mislabeled as "dinoflagellate" when in fact they correspond to plastids of photosynthetic eukaryotes "stolen" by dinoflagellate hosts (kleptoplastids). For instance, the dinoflagellate Dinophysis can sequester plastids of different microalgal prey, such as cryptophytes, raphidophytes and chlorophytes [START_REF] Kim | The marine dinoflagellate genus Dinophysis can retain plastids of multiple algal origins at the same time[END_REF]. This issue was also found in all other organisms present in PhytoREF that can either establish kleptoplastidy (e.g. the Ciliata Mesodinium rubrum and benthic Foraminifera), or photosymbiosis with microalgal cells (e.g. the katablepharid Hatena arenicola). Re-assignment of these sequences was necessary to avoid biases in annotation of the metabarcoding reads. Finally, 2700 16S rDNA cyanobacterial sequences have also been included in PhytoREF as separate files to avoid any ambiguities in the taxonomic assignation of query sequences. These sequences were clustered at different similarity levels (from 98 to 80%) and the longest sequences of each cluster are available for download at http://phytoref.org/.

PhytoREF: a new tool to explore the ecology of photosynthetic eukaryotes

To date, PhytoREF is the only tool in molecular ecology specifically designed to explore the total diversity of photosynthetic eukaryotes from complex marine and terrestrial ecosystems using metabarcoding or metagenomics approaches. Although the taxonomic resolution of the plastidial 16S rDNA barcode is not as high as that of established barcodes like the mitochondrial cytochrome c oxydase I gene for animals [START_REF] Herbert | Biological identifications through DNA barcodes[END_REF] and the large subunit of ribulose

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1,5-bisphosphate carboxylase gene (rbcL) for plants [START_REF] Plant | A DNA barcode for land plants[END_REF], it can recover and distinguish all photosynthetic eukaryotes at the class level, family level (e.g. Cryptophyta; [START_REF] Stern | Diversity and plastid types in Dinophysis acuminata complex (Dinophyceae) in Scottish waters[END_REF], and down to the genus and sometimes species level for most major lineages, such as the haptophytes [START_REF] Edvardsen | Ribosomal DNA phylogenies and a morphological revision provide the basis for a revised taxonomy of the Prymnesiales (Haptophyta)[END_REF], euglenozoans [START_REF] Linton | Reconstructing euglenoid evolutionary relationships using three genes: nuclear SSU and LSU, and chloroplast SSU rDNA sequences and the description of Euglenaria gen. nov. (Euglenophyta)[END_REF][START_REF] Na | Molecular identification and culture trials of Eutreptiella gymnastica (Eutreptiales, Euglenophyceae)[END_REF], and diatoms [START_REF] Pillet | Molecular identification of sequestered diatom chloroplasts and kleptoplastidy in Foraminifera[END_REF]. As proposed by the CBoL Protist Working Group for the 18S rRNA [START_REF] Pawlowski | CBOL Protist Working Group: Barcoding eukaryotic richness beyond the animal, plant, and fungal kingdoms[END_REF], the 16S rRNA gene can be used as a "pre-barcode" to explore the diversity of photosynthetic eukaryotes in the environment.

In this study, we found that the copy number of the 16S rRNA gene in plastid genomes can range from 1 to 10 (e.g. 4 and 6 copies in the euglenophyte Euglena gracilis and the prasinophyte Pedinomonas minor, respectively). However, in about 80% of the plastid genomes of eukaryotes (mainly streptophytes) sequenced so far, only 2 copies of the 16S rRNA were found (Table S2), which is in accordance with the plastid genome structure with two inverted repeats that duplicate ribosomal RNA genes [START_REF] Green | Chloroplast genomes of photosynthetic eukaryotes[END_REF]. The copy number variation of the plastidial 16S rRNA gene seems therefore to be much less important than that of the nuclear 18S rRNA gene, which correlates with genome size, cell size and biovolume and can vary by up to four orders of magnitude (e.g., the green algae Prasinococcus sp. and Ostreococcus sp. have 2 and 4 copies of the 18S RNA gene, respectively, while the diatoms Ditylum sp. and Coscinodiscus sp. have >30,000 copies; [START_REF] Zhu | Mapping of picoeucaryotes in marine ecosystems with quantitative PCR of the 18S rRNA gene[END_REF][START_REF] Godhe | Quantification of diatom and dinoflagellate biomasses in coastal marine seawater samples by real-time PCR[END_REF]. Thus, the plastidial 16S rRNA gene has the potential to be a suitable proxy in metabarcoding studies for assessing the relative abundance of eukaryotic phototrophs in the environment. Nevertheless, one has to consider that biological biases may also occur with the plastidial 16S rRNA gene. The number of plastids can vary (hence the number of 16S copies per individual) not only within one cell among eukaryotes, but also throughout the life cycle of a species (e.g. before and after cytokinesis). Although most

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This article is protected by copyright. All rights reserved. species in many microalgal groups (e.g. haptophytes, cryptophytes, chlorophytes, pennate diatoms) have only one or a few plastids, some taxa can harbour more than 100 plastids (e.g. centric diatoms). Less is known about the number of plastid genome copies in microalgal species, which can also alter the 16S rDNA copy number per individual. Photosynthetic eukaryotes typically maintain 50-100 copies of the plastid genomes per plastid. This number varies greatly in land plants from tens to hundreds during the plant development [START_REF] Oldenburg | Changes in the structure of DNA molecules and theamount of DNA per plastid during chloroplast development in maize[END_REF]. In microalgae, the plastid of the chlorophyte Chlamydomonas reinhardtii contains about 75 genome copies [START_REF] Armbrust | Uniparental inheritance of chloroplast genomes[END_REF]), but continuous replication and accumulation of plastid DNA throughout the cell cycle has been shown for this taxon and for the dinoflagellate Amphidinium operculatum and the chrysophyte Ochromonas [START_REF] Coleman | Temporal and spatial coordination of cells with their plastid component[END_REF][START_REF] Hiramatsu | Morphological changes in mitochondrial and chloroplast nucleoids and mitochondria during the Chlamydomonas reinhardtii (Chlorophyceae) cell cycle[END_REF]Koumandou & Howe 2007).

Description of the PhytoREF web interface

The PhytoREF web interface provides easy and rapid access to all reference plastidial 16S rDNA sequences, and allows users to explore the database with interactive graphs (e.g. Krona pie charts) and perform different search options. Sequences can be retrieved in Fasta format either by taxonomic rank (e.g phylum, genus, species) using a taxonomy browser, or through specific identifiers such as the GenBank accession number or the culture strain code (e.g. AY702161 or RCC393). Information associated with each sequence can be also downloaded as a tab-separated file, and different formats of the database are proposed to be used by the QUIIME, Mothur or TreeDyn programs. In addition, for each sequence, publication metadata such as title, authors and abstract are available on the web site. Web links to the Roscoff Culture Collection and GenBank database provide more information about the taxonomy and the origin of each

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This article is protected by copyright. All rights reserved. plastidial 16S rDNA sequence. Finally, a BLAST interface is available on the web site allowing users to identify individual or multiple plastidial 16S rDNA sequences against all PhytoREF reference sequences, and download selected hit sequences. In order to improve future releases of PhytoREF, users are encouraged to indicate errors and suggest better taxonomic placements for reference sequences in a dedicated page.

Conclusion and perspectives

PhytoREF is the first resource allowing exploration of the total diversity of photosynthetic eukaryotes in any given ecosystem. It can be used for a range of purposes, such as: (i) annotation and classification of new plastidial 16S rDNA sequences; (ii) taxonomic assignation of environmental sequences from massive metabarcoding and metagenomic datasets; and (iii) design of primers and probes to target any group of photosynthetic eukaryotes. All of the main eukaryotic lineages that have a functional or relict plastid are represented in PhytoREF, including free-living, mutualistic or parasitic organisms from aquatic and terrestrial habitats. Some organisms are under-represented in the current PhytoREF version, such as dinoflagellates and freshwater microalgae, which may lead to coarse taxonomic assignations. PhytoREF has therefore the potential to be used for many applications from biomonitoring of photosynthetic eukaryotes in past and present environments (water, sediment, ice) to feeding selectivity studies.

Updates of the database will be performed every six months by adding new reference sequences, expert validation of new public sequences from GenBank, and inclusion of novel taxonomic features from the literature, such as the description of novel algal classes..

Figures and Legends

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This article is protected by copyright. All rights reserved. (Burki & Keeling 2014). Each plastid-containing eukaryotic lineage is highlighted in green, and the number of plastidial 16S rDNA sequences available in the PhytoREF database is indicated in small grey circles.

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Figure 1 :

 1 Figure 1: Treemap and histograms showing the origin and number (A), and length (B) of the plastidial 16S rDNA sequences compiled into the PhytoREF database. A: PhytoREF is composed of 3,333 amplicons from identified organisms, 1,867 environmental amplicons produced from Sanger clone libraries, 879 sequences extracted from plastidial genomes, and 411 novel amplicons that have been generated in this study from cultures of marine microalgae. B: Most 16S rDNA sequences in PhytoREF are distributed in two peaks: the one with 700-900 bplong sequences containing the novel amplicons obtained here from cultured microalgal strains, and the other one with full-length (ca. 1500 bp) sequences from public databases.

Figure 2 :

 2 Figure2: Distribution and number of PhytoREF plastidial 16S rDNA sequences in the tree of eukaryotic life. The schematic phylogenetic tree is based on up-to-date phylogenomics and morphological evidence(Burki & Keeling 2014). Each plastid-containing eukaryotic lineage is highlighted in green, and the number of plastidial 16S rDNA sequences available in the PhytoREF database is indicated in small grey circles.

Figure 3 :

 3 Figure 3: Taxonomic composition of the PhytoREF database at the class level. Bar charts represent the number of PhytoREF plastidial 16S rDNA sequences and taxonomically-described families, genera and species that are present in a given class. Several key groups of microalgae lack full taxonomic description, such as the prasinophytes (clade VII) and the rappemonads. Streptophytes (land plants) that are represented by 2,973 sequences (373 families and 796 genera) were not considered here for a better clarity.

Acknowledgments

This work was supported by the OCEANOMICS project, funded by the French Government and managed by the Agence Nationale de la Recherche, under the grant agreement "Investissements d'Avenir" ANR-11-BTBR-0008. Financial support for this work was also provided by the European Union programs MicroB3 (UE-contract-287589) and MaCuMBA (FP7-KBBE-2012-6-311975) and the French "Investissements d'Avenir" program EMBRC-France. This work was granted access to the HPC and visualization resources of "Centre de Calcul Interactif" hosted by "Université Nice Sophia Antipolis". We are grateful to Ales Horák, Jan Janouškovec, Chris Jackson, Akira Kuwata, Camille Poirier and Daphné Grulois for providing 16S rDNA sequences and microalgal strains.

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Data Accessibility

All of the plastidial 16S rDNA sequences in the PhytoREF database can be downloaded via the web interface http://phytoref.org/, and the accession numbers of the newly produced reference 16S rDNA sequences are provided in Table S1.

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Supporting Information

Table S1: List of the eukaryotic microalgal strains from which the plastidial 16S rDNA sequence have been obtained in this study by DNA extraction and PCR.

Table S2. Number of 16S rRNA copies found in public plastidial genomes in photosynthetic eukaryotes (essentially land plants).

Author Contributions

JD, SR and CdV conceived and designed the research, and wrote the paper. JD, SR, RS, MB, AZ, FG, PG, ALS performed the experiments. JD, SR, DV, RC analyzed data. MG, LG, IP provided resources and databases. RC and DT designed the web site platform of PhytoREF.