Maxime Godfroy

Mahmoud Khalil

Claude Niebel

Thibaut Jarrosson

Dominique Foix

Valérie Flaud

Françoise Serein-Spirau

Romain Viennois

Michel Granier
email: michel.granier@umontpellier.fr

Mickaël Beaudhuin
email: mickael.beaudhuin@umontpellier.fr

Transition metal silicide surface grafting by multiple functional groups and green optimization by mecanochemistry

Chromium disilicide (CrSi2) particles were synthesized by arc melting furnace followed by mechanical milling. XRD and DLS analyses show that aggregates of around 3 µm containing about 10 nm sized crystallites were obtained. These aggregates were functionalized in solution by coupling agents with different anchoring groups (silane, phosphonic acid, alkene and thiol) in order to disperse them into an organic polymer. Dodecene was used to modify CrSi2 surface during mecano-synthesis in a grinding bowl with quite little solvent quantity and the optimization step allowed to reduce the aggregates size to 500 nm. A thermoelectric composite was then made between alkene CrSi2 grafted samples and poly(pphénylène-2,6-benzobisoxazole). This study opens the route of new surface grafting of intermetallic silicides for applications linked to electronic and/or energy.

INTRODUCTION

Nowadays, 55 % of the electricity produced by heat engines and turbines is wasted into lost thermal energy 1 . Thermoelectric materials are a promising solution to convert this waste-heat into electricity. Indeed, they exhibit the advantage to have no moving parts as they are solidstate devices. Their efficiency is evaluated by the dimensionless thermoelectric figure of merit ZT which is defined as ZT = α²σT/λ where α, σ, T and λ are the Seebeck coefficient, the electrical conductivity, the absolute temperature and the thermal conductivity respectively. The widely used devices are built from inorganic compounds that offer high electrical conductivity and high Seebeck coefficient. Nevertheless, good ZT of about 1 are only obtained by expensive, toxic and hard to process compounds such as Bi2Te3 (200 -500K) and PbTe (500 -800K) [START_REF] Rowe | Thermoelectric Handbook -Macro to Nano[END_REF][START_REF] Rowe | Thermoelectrics and its energy harvesting -Materials, preparation, and characterization in thermoelectrics[END_REF] . In order to improve the competitiveness of thermoelectric materials, it is necessary to develop new performant and cheap inorganic compounds. Silicides are really competitive in terms of cost that is less than 5 $/kg compared to more than 100 $/kg for Bi2Te3 [START_REF] Leblanc | [END_REF] . Among them, we have chosen to work with CrSi2 which presents the main advantage to have a power factor (PF = α²σ) similar to Bi2Te3 and PbTe. Nevertheless, it possesses a relatively high thermal conductivity between 11 and 16 W.m -1 .K -1 at room temperature and between 7 and 12 W.m -1 .K -1 at 900 K conducting to a ZT of 0.025 at room temperature and more than 0.25 at 900 K 5 . Different strategies are possible to reduce the thermal conductivity of thermoelectric materials: alloying with heavy elements [6][7][8] , nanostructuring [9][10][11] or combining nano-particles with organic polymers that are known to have low thermal conductivities below 1 W.m -1 .K - 1 12, 13 can be noticed. In addition, using organic polymers is relevant because they are low-cost raw materials, easy to shape and their electrical properties can be easily modulated by changing their structure or the doping according to different ways by chemical or electrochemical processes 14,15 . Up to now, the most famous organic polymer is the PEDOT:PSS that becomes competitive when it is combined with inorganic materials exhibiting electrical conductivity that can achieve 5400 S.cm -1 14 and a ZT as high as 0.42 12 . A recently developed strategy to increase the figure of merit ZT is to take the advantages of inorganic materials (high Seebeck coefficient and electrical conductivities) and organic polymers (low thermal conductivities). Some examples of inorganic materials/organic polymers composites have recently been tested 16,17 but only a part of them show a synergistic effect of the composite compared to the separated elements such as PEDOT:PSS passivated Te nanorods 18 . However, some papers pointed out its low moisture and thermal stability associated to the PSS doping 19,20 . To answer to this problem, we decided to choose the most thermally stable organic polymer known in the literature under nitrogen atmosphere (>900K), the poly(pphenylene-benzobisoxazole) (PBO) 21 to develop our composite films. We will see later in this paper that the composite films have poor mechanical properties even at low concentrations of CrSi2. It was then necessary to investigate the functionalization of silicides CrSi2 particles before their dispersion in the organic matrix and this was done with different coupling agents. We believe that the inorganic nanomaterials functionalization can enhance their interaction with organic polymers by Van der Waals interactions and this could permit to explore high inorganic/organic ratios. Furthermore, it is possible to graft coupling agents with terminal hanging groups that could react for example by click-chemistry with functional moieties carried by the organic polymers 22 .

Many functional groups could be used to functionalize CrSi2 surfaces due to the presence of two different atoms. Phosphonic acids are largely used in the functionalization of metal oxide surfaces such as TiO2, ZrO2 23 , ZnO 24 , ITO (Indium Tin Oxide) 25 , SnO2 26 and one example is reported for phosphonic acid group grafting on Co-Cr alloy oxide layer 27 . The interest of silane groups is well established as they are used in many applications such as chromatography, liquid crystal displays and microelectronics. The silane groups can functionalize a wide variety of substrates from carbon black to silica. On inorganic oxides (Fe2O3, TiO2, Cr2O3) and on silicon 22, 28 the effectiveness of the grafting ranges from poor to excellent effectiveness 22,28 . The thiol group is more acknowledged to be efficiently grafted on gold nanoobjects 29,30 and also on silver 31, 32 ; it was recently attempted to be grafted on silicon nanocrystals but a poor stability towards water or oxygen was recorded 33 . Finally, alkene moieties were largely used to functionalize silicon surfaces to prevent from the formation of a native oxide surface especially in the domain of electronics 34,35 . Thus, long dodecane hydrophobic chains bearing a phosphonic acid, triethoxysilane, thiol or alkene functionalities were chosen to modify CrSi2 particles surface with the aim to make them more compatible with soluble organic materials soluble in organic solvent.

Interestingly, the four-studied different functional groups have been successfully grafted on the inorganic particles. These newly grafted objects have been characterized by XPS and FTIR and hydrophobicity tests were realized to show the grafting efficiency. In the following, the work will be focused on the alkene moiety coupling agents and on its possible direct grafting in a grinding bowl. This enables to reduce the experiments time and to take benefit from the heat delivered by the milling balls to favour the coating. Finally, we show that grafting with coupling agent improves significantly the mechanical properties of PBO/CrSi2 composites.

RESULTS AND DISCUSSION

Synthesis of CrSi2 particles

The obtained particles were characterized by XRD that showed a pure hexagonal CrSi2 phase (Figure 1-left). The Rietveld refinements were performed on the XRD patterns in the 2θ-range of 20-110°.

The estimated grains size ranges between 10-16 nm according to the Williamson-Hall formula for the mechanical milled nanocrystallites. These sizes were in agreement with our previous works 7,9 . Moreover, the mechanical milled nanocrystallites have been characterized by BET that informed us about the rather low specific surface of 2.67 m²/g that could be explained by the presence of large aggregates as observed by SEM (Figure 1-right) with sizes between 200 nm and more than 2 microns. These aggregates were confirmed by DLS with an average size of around 3 microns. TEM images showed us that the aggregates were mainly composed of nanocrystallites with a size comprised between 6 and 10 nm, in adequacy with XRD analysis (see S.I. Fig. S1).

To improve further interactions between inorganic particles and organic materials different coupling agents were investigated.

Functionalization of particles with coupling agents and their characterization

As we introduced, the CrSi2 particles functionalization was attempted with long hydrophobic dodecane chains with a phosphonic acid, triethoxysilane, thiol or alkene anchoring group. Indeed, each coupling agent was expected or not to be grafted either on chromium or on silicon with a passivated oxide surface (Scheme 1).

Scheme 1 Functionalization of CrSi2 by different coupling agents: dodecyltriethoxysilane, dodecylphosphonic acid, 1-dodecene and 1-dodecanethiol.

Before XPS and FTIR characterization on the coated particles, a hydrophobic test was carried out. It consisted in introducing a small quantity of nanocrystallites in a hemolysis tube subsequently filled with distilled water. The results are given in Figure 2 and it is observed that no-grafted CrSi2 particles sink whereas grafted CrSi2 particles float on water. This is due to the oxide surface that gives a hydrophilic character to these particles.

In all attempts of grafting, CrSi2 particles stayed at the surface and no crystallites sank. This can be explained by the hydrophobicity of dodecyl chains that enable particles to float on water. To check the stability of the grafting, hemolysis tubes stirring was vertically done for ten seconds. The only particles which sank into the water were the ones grafted with dodecanethiol ones, (Figure 2b,3). Similar behavior has been observed by Yu et al. 33 after only 20 seconds in distilled water, silicon nanocrystals grafted with dodecanethiol lost emissive properties that the authors explained as the result of the surface oxidation. Thus, the water induces the loss of the thiol-functionalized chains from the silicon surface. One also notices that the functionalized chains of alkene, phosphonic acid and silane are stable for a long period either in water or MeOH/water mixtures (see below). Indeed, after more than 12 months in-hemolysis tubes, the CrSi2 particles are still floating.

In addition to this simple hydrophobic test, the functionalized nanocrystallites were introduced in hemolysis tubes filled with different ratios of MeOH/water which is generally referred to the methanol number test (Figure 3).

The more the particles stay at the surface for higher MeOH/water ratios, the more the particles are highly coated by hydrophobic coupling agents. phosphonic acid, 3) silane and 4) alkene coupling agent were introduced in MeOH/water mixtures with 40%, 50%, 60% and 70% of methanol.

Grafting agent

No The methanol number is defined by the percentage of methanol in methanol/water mixture where no particles sank. Thus, the methanol number of the particles functionalized with phosphonic acid is ~50%, ~50% with silane and ~60% with alkene. This indicated that the alkene-coated CrSi2 particles are the most densely packed functionalized nanocrystallites.

FTIR characterizations are given in figure 4, no signal of any C-H bonds was observed in the 2800-3000 cm -1 region for the ungrafted CrSi2 while Csp3 CH2 symmetrical stretching vibration around 2855 cm -1 and Csp3 CH2 asymmetrical stretching vibration between 2920 and 2925 cm -1

were well revealed on the grafted CrSi2 nanocrystallites. Furthermore, the Csp3 CH3 unsymmetrical stretching vibration around 2960 cm -1 could be seen for silane, alkene and phosphonic acid-grafted CrSi2 nanoobjects. As these measurements were done several weeks after the grafting of CrSi2 particles, the low signal observed for thiol-functionalized CrSi2 can be explain by its instability under air.

In table 1, the atomic percentage of the elements presents in CrSi2 powders are calculated from XPS spectra and showed the presence of high C and O amount on no-grafted sample, whereas C come from external contamination, O found at the surface of the powder signifies that the surface is oxidized. It is known that CrSi2 is stable in air up to 973K 36,37 due to the formation of a protective layer either of Cr2O3 or SiO2 which limits the oxygen diffusion. The peaks at 103 eV and, 577 and 587 eV attributed to oxidized silicon and oxidized chromium respectively confirm the presence of this oxide layer. It is then interesting to notice that the oxygen concentration decreases after grafting meaning that the grafting agent protects, at least partially, the surface from oxidation. These results also showed clearly the presence of phosphorus bonding P2p at 134.1 eV (see S.I. Fig. S2) in the attempt of functionalization of CrSi2 particles with n-dodecylphosphonic acid. The ratio of carbon in grafted nanoparticules with dodecene (43 %), with dodecylphosphonic acid (37 %)

and with dodecyltriethoxysilane (45 %), were all higher than those of the no-grafted reference (33 %), which confirmed the long carbon chains coatings.

To highlight the efficiency of the grafting process, it has then been also decided to graft a molecule containing a heteroatom clearly visible via XPS such as the fluorine. This heteroatom has a very clear XPS signature and has the advantage to be missing from the raw material surface.

The chosen grafted molecules are the same as the ones described in scheme 1, but all the -CH2/-CH3 groups are replaced by -CF2/-CF3 ones. The XPS analyses show that this fluorine element is present on the four treated surfaces (S.I. Fig. S3 andS4). For the thiol-grafting, the amount of detected fluorine is very low. These surface analyses allow to conclude that the grafting process is efficient for the three fluorine molecules, in total agreement with the water immersion tests.

On both families of coupling agents (carbon or fluorine chain), XPS measurements did not highlight the presence of the sulfur on thiol-functionalized CrSi2 nanocrystallites, which confirms the coupling agent removing from the surface under ambient atmosphere containing water. All the investigations performed on narrow-range C1s spectra did not permit to determine the type of bondings between CrSi2 and the grafting agent due to the high amount of C-C bondings.

Optimization of the alkene functionality grafting coupling agent

For thermoelectric properties, it is necessary to have a material as conductive as possible, oxide surface must therefore be avoided due to their inherent insulating properties.

As phosphonic acid and silane groups are known to react with oxidized surfaces and thiol group did not permit to obtain stable coatings, the best coupling agent to graft CrSi2 appears to be alkene moiety since it reacts with non-oxidized hydrogen-terminated silicon surface.

Nevertheless, the induced hydrosilylation reaction mostly takes place at 150 -200 °C in the presence of pure alkene or alkene high concentrated solutions for several hours. Hallmann et al.

have described the high-energy ball milling as a simple method to functionalize silicon nanoparticles with different alkene and alkyne groups 38 .

We first studied, for reference, the effect of milling time and of the presence of solvent on the average crystallites size and on the average aggregates size in Table 2, entries 1 to 4. Then we attempted to graft our CrSi2 nanocrystallites directly in the grinding ball in Table 2 entries 5 to 8 and, as expected, all of our carried-out experiments showed an efficient recovering. From these experiments we found that a decrease of the aggregates size also leads to a strong decrease of the polydispersity.

In entries 1 and 5, the arc melted button is milled for 4h respectively with or without grafting agent. The average aggregate size strongly decreases from 3000 to 400 nm whereas the nanocrystallite size increases from 10 to 80 nm. This result can be explained by the possible lubrication property of the dodecyl chain.

So, although this method permits to strongly decrease the average aggregates size, in order to maintain a small average crystallite size, it is important to firstly mill the arc melted button in order to obtain crystallite size already about 10 nm. Then, pure dodecene can be added in the vessel, with or without solvent, and sealed under argon (all the process was performed in a glovebox), followed by 4 h milling (entry 6 to 8).

This is what has been performed in entries 2 and 6, we found that the crystallite size is similar whereas the aggregate size decreases from about 2800 to 500 nm. This result indicates here again that the alkene solution has a strong impact on the average aggregates size.

Then, as we observed in entries 3 and 4 that the use of solvent lead to a strong decrease of the average aggregates size, we attempted to reduce again the aggregates size by using a solvent with dodecene (entries 7 and 8), and to look for a cumulative effect. We found that the average crystallite size is not modified nor the average aggregates size, however, our XRD pattern are much less intense as if the coverage was denser and that the quantity of alkyl chains hides the signal of CrSi2. This was also observed by Hessel et al. in the case of alkene-functionalized silicon nanoparticles 39 . In conclusion, the functionalization is important to break the aggregates of nanocrystallites but this could be only obtained on previously milled buttons. In figure 6-a, it is observed that the not grafted CrSi2 particles composite film is heterogeneous with two different regions, the bottom part of the composite is composed with CrSi2 particles and a low amount of PBO whereas the upper part is mainly composed of PBO and a low amount of CrSi2 particles. At contrary, in figure 6-b the composite film is homogeneous and the alkene grafted CrSi2 particles are well dispersed with the PBO. We believe that it can be explained by the presence of long alkyl chains on CrSi2 particles which bring a better interaction with the organic polymer, increasing its dispersion. The electrical resistivity of the grafted films at room temperature is about 1kΩ.cm which is 1000 times higher than pure CrSi2 however, the Seebeck coefficient is similar to that of pure CrSi2 with 80 𝜇V/K. This led to a relatively low power factor compared to bulk CrSi2. Consequently, although the thermal conductivity is expected to be quite close to the polymer thermal conductivity, the figure of merit is somewhat negligible. These results were expected as the alkyl chains are not electrically conductive and because the HOMO-LUMO of the polymer has not been adjusted with the inorganic compound. The expected percolation of CrSi2 particles, even at large CrSi2 amount, was consequently not reached.

CONCLUSIONS

We have seen that it is possible to obtain pure CrSi2 nanocristallites by combining arc-melting and mechanical alloying. The as-obtained powder is made of aggregates (~3 µm) of nanocristallites (~10 nm). Two methods to functionalize the chromium disilicide powders were reported, in solution or in a grinding bowl. In solution, the successful functionalization of CrSi2 particles has been described with 3 different coupling agents: alkene, silane and phosphonic acid grafting was confirmed by XPS and FTIR. The stability of the covalent interactions involved were also evaluated. A second optimized procedure involving the direct grafting of an alkene coupling agent in the grinding bowl enabled to save time and energy compared to the more conventional solution processing. Furthermore, this mechanochemical procedure enables to reduce the size of the aggregates from 3 µm down to 500 nm together with being greener due to the strong decrease of the amount of solvent used. These new coated CrSi2 particles have been used in a mixture with organic polymers to realize inorganic-organic composites for thermoelectric applications. The obtained composites have a better mechanical behavior with grafted CrSi2 which open the routes to new high temperature thermoelectric applications using thermoelectric composites. Their limited electronic properties coming from the long alkyl chain could be compensated by working with pi-conjugated chain and by adjusting the HOMO-LUMO of the polymer with the inorganic alloys. These results could be easily extended to other transition metal silicides for applications not only limited to thermoelectric applications but also to electronic, photovoltaic.

EXPERIMENTAL SECTION Materials

Chromium pieces (3-8 mm, 99,99%) and silicon lump (99,9999%) were purchased from Alfa

Aesar. 1-Dodecene (95%) was purchased from Janssen Chimica. n-Heptane (99%) was purchased from Sigma Aldrich. N-N-dimethylallylamine (98%) was purchased from Acros Organics. n-dodecylphosphonic acid from Sikemia. Dodecyltriethoxysilane and 1-dodecanethiol from Sigma Aldrich. All reactants and solvents were used as received.

Synthesis of CrSi2 particles

In order to avoid external contamination either during the sample preparation or the milling, all the process described below is performed under argon atmosphere. CrSi2 alloys were prepared by arc-melting technique under argon atmosphere using high purity Cr pieces (99.99%) and very high purity Si lumps (99.9999%) in a button shape (≈2.7g each). Sample homogeneity was improved by melting the samples four times with a turnaround between each melting. The buttons were crushed and grinded in an agate mortar pestle before being introduced into a 45 mL Si3N4 grinding bowl with 5 Si3N4 balls (15 mm diameter). Vessels were sealed under argon atmosphere in a glovebox. The milling was carried out with a ''Fritsh Pulverisette 7 classic line'' planetary micromill. The speed of the supporting disc and of the grinding bowl was, respectively, fixed to 650 rpm and 1300 rpm for all the experiments corresponding to a ball acceleration of about 147 m.s -2 . The ball to powder ratio was fixed to 10:1 for all the experiments.

Functionalization of CrSi2 particles in Schlenk tubes

Schlenk tubes were filled in a glovebox under argon atmosphere with 400 mg of freshly synthesized CrSi2 particles. Schlenk tubes were sealed and were taken off from the glovebox.

 Procedure for n-dodecylphosphonic acid, dodecyltriethoxysilane and 1-dodecanethiol grafting: 10 mL of a 10 -2 M solution of the coupling agent in THF or EtOH (for ndodecylphosphonic acid) were transferred into the Schlenk tube and the solution was stirred The procedure is similar for coupling agent with fluorine chain.

Functionalization of CrSi2 particles by mechano-chemistry with 1-dodecene

Freshly synthesized CrSi2 particles (2.7 g) were transferred into a glovebox. 1-Dodecene (700 µmol) was introduced in the Si3N4 vessel in presence or in absence of heptane (different volumes). The vessel was sealed in the glovebox and was introduced in the planetary mill for different times mentioned in the main text in Table 2. The treatment of the suspension of particles was identical to the one previously mentioned for the functionalization in solution.

Fig. 1 (

 1 Fig. 1 (Left) Powder X-Ray diffraction of CrSi2 alloys obtained by mechanical alloying and

Fig. 2

 2 Fig. 2 Tests of CrSi2 particles in water, a) Before stirring. b) After stirring. 1) No-grafted

Fig. 3

 3 Fig. 3 Tests of CrSi2 particles in methanol/water mixtures. The different functionalized particles with 2)

Fig. 4 FTIR

 4 Fig.4 FTIR spectra of ungrafted (crude) compared to grafted CrSi2 nanocrystallites.

Fig. 4

 4 Fig. 4 FTIR spectra of ungrafted (crude) compared to grafted CrSi2 nanocrystallites.

Fig. 5

 5 Fig. 5 Not grafted CrSi2 particles and alkene grafted CrSi2 particles for two volume ratio of

Fig. 6

 6 Fig. 6 SEM images obtained with secondary electron of 75/25 CrSi2/PBO volume ratio for a)

4

  days at room temperature. The suspension was centrifuged at 20 000 rpm for 15 minutes and the supernatant was removed. Modified CrSi2 particles were washed three times with CHCl3 under sonication for 5 minutes. Particles were separated from the washing solvent by centrifugation at 20 000 rpm for 15 minutes. The resulting powders were dried under vacuum for one hour. Procedure for 1-dodecene grafting: 5 mL of 1-dodecene was added in the Schlenk tube and the solution was stirred 4 days at 150 °C. After cooling at room temperature, the suspension was centrifuged at 20 000 rpm for 15 minutes and the supernatant was removed. Modified CrSi2 particles were washed three times with CHCl3 under sonication for 5 minutes. Particles were separated from the washing solvent by centrifugation at 20 000 rpm for 15 minutes. The resulting powders were dried under vacuum for one hour.

grafted Alkene Phosphonic acid Silane

	C1s	33.26	42.73	37.34	44.53
	Cr2p	5.02	3.34	3.22	2.66
	Si2p	25.01	29.34	26.56	21.61
	O1s	36.71	24.58	31.77	31.20
	P2p			1.11

Table 1 .

 1 Atomic percentage of the elements present in CrSi2 powders calculated from XPS spectra, no grafted or grafted with either alkene, phosphonic acid or silane.

Acknowledgements

This work was supported by the CheMISyst labex ANR-10-LABX-05-01 and University of Montpellier and the authors would also like to thank Bertrand Rebière and Didier Cot (IEM, France) for SEM-EDX characterization, Jean-Olivier Durand for his help with the DLS.

Notes and references

Thermoelectric composite

In figure 5, the inorganic-organic composites are presented for two different volume ratio CrSi2/PBO (50/50 and 75/25) after heat treatment at 400°C for 1h. One notice that not grafted CrSi2 dispersed in the PBO lead to films with numerous cracks and by an exfoliation of the composite from the glass substrate. At the contrary, CrSi2 grafted particles dispersed in the PBO polymer gives relatively uniform films with no cracks and good adhesion with the substrate.

Synthesis of poly(p-phénylène-2,6-benzobisoxazole)

The highly thermal resistive poly(p-phenylene-2,6-benzobisoxazole) (PBO) films were prepared following the optimized procedure described by Fukumaru et al. 21 . A TBS-prePBO precursor, soluble in most organic solvents, was obtained in two steps from commercially available 4,6diaminoresorcinol. Films of this soluble PPBO precursor could then be easily spin-coated on glass substrates, and a subsequent thermal treatment at 400°C for 1h provided the PPBO films, which were characterized by 1 H NMR, IR spectroscopy and by thermogravimetric analysis (see

Thermoelectric composite

The soluble precursor TBS-prePBO was dissolved at a concentration of 50 mg.mL -1 in N,Ndimethylacetamide. The CrSi2 nanoparticles were added to give the expected ratio in percentage by volume and the mixture was sonicated for 30 min being used. Glass substrates of 12.5 x 12.5 mm were successively sonicated in acetone and in isopropanol for 10 minutes in each case, before being dried with dry air. The mixture of CrSi2/TBS-prePBO was drop-casted (50 µL) on glass substrates and the substrates were progressively heated at 400 °C with a ramp of 10 °C per minute then were let at 400 °C for one hour in a furnace under air. The resulting film were characterized electrically by Van der Paw technic with an homemade apparatus after deposition of gold plot. The Seebeck coefficient has been determined with an homemade apparatus.

Powder X-ray Diffraction

The powders were analyzed by using an X-Ray diffraction apparatus (Philips X'PERT, Cu-K radiation 1.5406 Å with an accelerated detector PW3050/60 at 45 kV and 30 mA settings).

Rietveld refinement with Fullprof software [START_REF] Rodriguez-Carvajal | Satellite meeting on powder diffraction of the XV congress of the IUCr[END_REF] was used for structural analysis of each sample.

Brunauer-Emmett-Teller (BET)

N2 sorption analyses were performed at 77 K using a Micromeritics Tristar 3000, equipped with automated surface area and pore size analyzer. Before analysis, samples were degassed at 250°C for 7 h using a Micromeritics VacPrep 061 degasser. BET surface areas were determined in the relative pressure range P/P0 from 0.01 to 0.03.

Dynamic Light Scattering (DLS)

DLS measurements were performed on a Vasco Particle Size Analyzer DL135 from Cordouan

Technologies and analyzed with nanoQ software.

Fourier Transform InfraRed (FT-IR) Spectroscopy. ATR infrared spectra were recorded on a FTIR Perkin-Elmer 2000 spectrometer equipped with narrow band liquid nitrogen cooled MCT detector and a multireflexion ATR accessory. The sample compartment was purged with dry air.

All the spectra were recorded at a resolution of 4 cm -1 , and 128 scans were accumulated. The size of the silicon ATR crystal was 70x10x1 and 35 reflections were used.

X-ray Photoelectron Spectroscopy

XPS analysis was performed using a ESCALAB 250 Thermo Electron apparatus with Al Kα (1486.6 eV) as an excitation source and with a Kratos Axis Ultra spectrometer, using focused monochromatic Al K radiation (hν = 1486.6 eV). For the ESCALAB 250 Thermo Electron apparatus, a surface of 400 μm in diameter was analyzed. The XPS spectra were calibrated to the energy of C1s of the C-C bond (284.8 eV). Concerning the Kratos Axis ultra spectrometer, the analyzed area of the samples was 300 × 700 μm 2 . Peaks were recorded with constant pass energy of 20 eV. For the Ag 3d5/2 line the full width at half-maximum (FWHM) was 0.58 eV under the recording conditions. The pressure in the analysis chamber was around 5 × 10 -9 mbar.

Short acquisition time spectra were recorded before and after each normal experiment to check that the samples did not suffer from degradation during the measurements. The binding energy scale was calibrated using the C 1s peak at 285.0 eV from the hydrocarbon contamination invariably present. Core peaks were analyzed using a nonlinear Shirley-type background [START_REF] Shirley | [END_REF] . The peak positions and areas were optimized by a weighted least-squares fitting method using 70%

Gaussian, 30% Lorentzian line shapes. Quantification was performed on the basis of Scofield's relative sensitivity factors 42 . The curves fit for core peaks were obtained using a minimum number of components in order to fit the experimental curves.

Electron Microscopy

The chemical homogeneity and composition of the broken buttons were checked using an Energy Dispersive Analysis of X-Ray (EDX) Hitachi S4800 mounted on a Hitachi S2600N scanning electron microscope as well as aliquot of the powder after ball grinding. Powders, dispersed on carbon film, were analyzed on a Jeol 1200 EXII TEM at 100 kV, equipped with a Quemesa camera (SIS Olympus) with 11 Mpixel CCD detector.

Conflicts of interest

There are no conflicts to declare.


Katharina Fietz

Elena Trofimenko

Pierre-Edouard Guerin

Véronique Arnal

Montserrat Torres-Oliva

Stéphane Lobréaux

Angel Pérez-Ruzafa

Stéphanie Manel

Oscar Puebla

Angel Pérez-Ruzafa

Stephanie Manel
email: stephanie.manel@ephe.psl.eu

Stephanie Manel

New genomic resources for three exploited Mediterranean fishes

Keywords: Mediterranean, fishes, genomes, assembly, annotation, RAD sequencing

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

Introduction

Extensive fishing has led to the decline of Mediterranean fish stocks over the last decades [START_REF] Colloca | Recent Trends and Impacts of Fisheries Exploitation on Mediterranean Stocks and Ecosystems[END_REF][START_REF] Vasilakopoulos | The Alarming Decline of Mediterranean Fish Stocks[END_REF]). Yet the identification of stocks is often problematic due to generally low levels of population genetic structure [START_REF] Calo | Spatial genetic structure in the saddled sea bream (Oblada melanura Linnaeus, 1758 ) suggests multi-scaled patterns of connectivity between protected and unprotected areas in the Western Mediterranean Sea[END_REF][START_REF] Gkagkavouzis | The genetic population structure and temporal genetic stability of gilthead sea bream Sparus aurata populations in the Aegean and Ionian Seas, using microsatellite DNA markers[END_REF][START_REF] Viret | Absence of spatial genetic structure in common dentex (Dentex dentex Linnaeus, 1758) in the Mediterranean Sea as evidenced by nuclear and mitochondrial molecular markers[END_REF]. In this situation, a large number of genetic markers is required to detect fine-scale population structure [START_REF] Carreras | Population genomics of an endemic Mediterranean fish: differentiation by fine scale dispersal and adaptation[END_REF][START_REF] Dibattista | Seascape genomics reveals fine-scale patterns of dispersal for a reef fish along the ecologically divergent coast of Northwestern Australia[END_REF], assign catches to genetic populations [START_REF] Benestan | RAD genotyping reveals fine-scale genetic structuring and provides powerful population assignment in a widely distributed marine species, the American lobster (Homarus americanus)[END_REF] and assess levels of genetic and demographic connectivity [START_REF] Waples | Separating the wheat from the chaff: Patterns of genetic differentiation in high gene flow species[END_REF]. A large number of genetic markers can also contribute to evaluate the effect of marine protected areas (MPAs) on fished areas and optimize the efficiency of MPA networks [START_REF] Xuereb | Marine Conservation and Marine Protected Areas[END_REF], since Marine Protected Areas (MPAs) tend to be a reservoir of genetic richness (Pérez-Ruzafa, González-Wangüemert, [START_REF] Pérez-Ruzafa | Effects of fishing protection on the genetic structure of fish populations[END_REF]. When genetic markers are mapped to an annotated reference genome of the same or a closely related species, they also provide the opportunity to characterize adaptive divergence [START_REF] Ellegren | Genome sequencing and population genomics in non-model organisms[END_REF]. This aspect is particularly relevant in the Mediterranean Sea, which is a hotspot in terms of bothsubmitted to strong anthropogenic pressures [START_REF] Ramirez | Spatial congruence between multiple stressors in the Mediterranean Sea may reduce its resilience to climate impacts[END_REF]Stock, Crowder, Halpern, & Micheli, 2018) and including global warming [START_REF] Giorgi | Climate change hotspots[END_REF][START_REF] Parry | Assessment of potential effects and adaptations for climate change in Europe: The Europe ACACIA project[END_REF].

Restriction-site Associated DNA (RAD) sequencing [START_REF] Etter | SNP discovery and genotyping for evolutionary genetics using RAD sequencing[END_REF] and related reduced-representation approaches [START_REF] Toonen | ezRAD: a simplified method for genomic genotyping in nonmodel organisms[END_REF][START_REF] Van Orsouw | Complexity Reduction of Polymorphic Sequences (CRoPS (TM)): A Novel Approach for Large-Scale Polymorphism Discovery in Complex Genomes[END_REF][START_REF] Wang | 2b-RAD: a simple and flexible method for genome-wide genotyping[END_REF] have become methods of choice to generate large numbers of Single Nucleotide Polymorphism (SNP) markers. Due to its applicability to non-model organisms, RAD sequencing has revolutionized the fields of ecological and conservation genomics [START_REF] Andrews | Harnessing the power of RADseq for ecological and evolutionary genomics[END_REF]).

Yet while the utility of RAD sequencing is well recognized, procedures for library preparation, sequencing and filtering sometimes lack details that are critical to assess the quality of the data and the robustness of the results. For example, PCR clones generated during library preparation can represent a significant proportion of the data and thereby bias allele frequencies if not identified and filtered [START_REF] Andrews | Trade-offs and utility of alternative RADseq methods: Reply to Puritz et al[END_REF]. This is particularly true when the number of PCR cycles is increased, which is often needed when the starting DNA is degraded or in low concentrations. The number of RAD markers needs to be known in order to adjust the sequencing effort, yet this number is difficult to predict in the absence of a previous study or reference genome. This often results in a sub-optimal sequencing effort, i.e. too low or too high coverage. The availability of a reference genome allows to estimate the number of RAD markers generated by different restrictions enzymes [START_REF] Lepais | SimRAD: an R package for simulation-based prediction of the number of loci expected in RADseq and similar genotyping by sequencing approaches[END_REF] and can greatly improve genotyping quality by providing a template to call SNP markers [START_REF] Manel | Genomic resources and their influence on the detection of the signal of positive selection in genome scans[END_REF]. A reference genome also allows to assess physical linkage among markers and consider population genetic statistics along scaffolds as opposed to a SNP-by-SNP basis [START_REF] Catchen | Stacks: an analysis tool set for population genomics[END_REF]). Yet the process of genome assembly and annotation is complex and computationally intensive. It requires high-molecular-weight DNA with high purity and structural integrity, especially when long-read technologies are used [START_REF] Del Angel | Ten steps to get started in Genome Assembly and Annotation [version 1; referees: 2 approved[END_REF]. Finally, stringent filtering of SNP markers with respect to sequencing coverage, missing data, minimum allele frequency, and linkage is often required for downstream population genomic analyses.

Here, we present annotated genome assemblies of three exploited Mediterranean fish species from three families, the white seabream (Diplodus sargus (Linnaeus, 1758), Sparidae), the striped red mullet (Mullus surmuletus (Linnaeus, 1758), Mullidae), and the comber (Serranus cabrilla (Linnaeus, 1758), Serranidae). These three species are exploited in the Mediterranean Sea [START_REF] Goni | Spillover from six western Mediterranean marine protected areas: evidence from artisanal fisheries[END_REF][START_REF] Lloret | A comparative analysis between recreational and artisanal fisheries in a Mediterranean coastal area[END_REF], and previous studies have found weak to no population genetic structure for all of them (D. sargus: González-Wanguemert, Pérez-Ruzafa, Canovas, [START_REF] González-Wanguemert | Temporal genetic variation in populations of Diplodus sargus from the SW Mediterranean Sea[END_REF]; González-Wanguemert, Pérez-Ruzafa, García-Charton, and Marcos (2006); [START_REF] Lenfant | Temporal genetic changes between cohorts in a natural population of a marine fish, Diplodus sargus[END_REF]; M. surmuletus: [START_REF] Dalongeville | Geographic isolation and larval dispersal shape seascape genetic patterns differently according to spatial scale[END_REF]; [START_REF] Mamuris | Intraspecific genetic variation of striped red mullet (Mullus surmuletus L.) in the Mediterranean Sea assessed by allozyme and random amplified polymorphic DNA (RAPD) analysis[END_REF]; S. cabrilla: [START_REF] Schunter | Matching genetics with oceanography: directional gene flow in a Mediterranean fish species[END_REF]). We use these nuclear assemblies as references to map RAD markers and characterize SNPs for the three species, which we filter stringently with respect to PCR clones, coverage, missing data, minimum allele frequency and linkage.

Results

Genome assemblies

Whole-genome sequencing of D. sargus, M. surmuletus and S. cabrilla with the Illumina HiSeq 4000 platform produced 651, 649 and 755 million paired-end 150 bp reads, respectively. After quality filtering and trimming, 609, 588 and 730 million reads were kept, respectively, and used to assemble each genome with the Platanus assembler [START_REF] Kajitani | Efficient de novo assembly of highly heterozygous genomes from wholegenome shotgun short reads[END_REF] ( cabrilla, which represents 72%, 103% and 79% of estimated genome size based on C-value [START_REF] Dolezel | Nuclear DNA content and genome size of trout and human[END_REF], respectively. Summary statistics of several fish genome assemblies, including our study species and the best currently available fish assembly of D. labrax, are presented in Table S1.

The search for BUSCOs showed the high completeness of the three genome assemblies.

From the set of 978 metazoan BUSCOs, the D. sargus assembly contains 97.5%, the M. surmuletus assembly 92.5% and the S. cabrilla assembly 96.7% (Fig. 1A, Table S2). From the set of 4,584 Actinopterygii BUSCOs, the D. sargus assembly contains 96.6%, the M. surmuletus assembly 89.9% and the S. cabrilla assembly 95.3% (Fig. 1C, Table S3). These results show that the D. sargus assembly is not only the most contiguous, but also the most complete assembly.

The mitochondrial sequences assembled into circular sequences with a length of 16,513bp -16,620bp. The mtDNA comprised 37 genes, including 13 protein-coding genes (COX1, COX2, ATP8, ATP6, COX3, ND3, ND4L, ND4, ND5, ND6, CYTB, ND1), 22 transfer RNA genes (tRNA), two ribosomal RNA genes (rRNA) (12S rrn and 16S rrn) and the control region (Fig. S1).

Gene annotation and Ortholog gene analysis

The number of predicted genes totaled 31,055 for M. surmuletus, 32,375 for D. sargus and 27,222 for S. cabrilla. To assess these annotations, we compared the percentage of BUSCOs present in the assemblies and investigated how many we could recover as annotated gene models. The D. sargus annotation contained 96.0% of complete BUSCOs, which is close to the 97.5% found in the genome assembly. Similar results were obtained for the other two species:

the S. cabrilla and M. surmuletus annotations contained 95.6% and 90.8% metazoan BUSCOs, respectively (Fig. 1B, Table S4). Of the Actinopterygii BUSCOs, the D. sargus annotation contained 90.2%, the S. cabrilla annotation 87.6% and the M. surmuletus annotation 80.3% (Fig. 1D, Table S5).

Using OrthoMCL analysis, we identified genes that are conserved across our focal species and the D. rerio reference genome, as well as genes that are unique to our fish species (Fig. 2).

Out of the total of 16,432 1:1 orthologs identified, 6,446 genes (39%) were shared among all 4 species and 3,577 genes (21%) were shared by our 3 target species. respectively. The SNPs were spread evenly across the genomes (Fig. 3 A,C,E), with a mean number of 9.81 SNPs per 400,000 bp window in scaffolds larger than this size. The mean sequencing coverage across individuals was comparable among the three species, with 38X, 45X

and 48X for D. sargus, M. surmuletus and S. cabrilla, respectively (Fig. 3 B,D,F). Of all SNPs filtered, 15%, 18% and 17% were located in exons for D. sargus, M. surmuletus and S. cabrilla, respectively (Table 2).

Discussion

We presented annotated nuclear and mitochondrial genome assemblies of three exploited Mediterranean fishes from three different families, the striped red mullet (M. surmuletus, Mullidae), the white seabream (D. sargus, Sparidae) and the comber (S. cabrilla, Serranidae).

To our knowledge, these genome assemblies represent the first genomes for these species.

The quality of a genome assembly in terms of both completeness and continuity greatly influences its usefulness for both genome-wide marker development and gene model prediction [START_REF] Yang | Chromosome-level genome assembly of Triplophysa tibetana, a fish adapted to the harsh high-altitude environment of the Tibetan Plateau[END_REF]. The quality of our three nuclear genome assemblies is attested by the almost complete gene content (89.9 -96.6% of Actinopterygii BUSCOs) and by the fact that the sizes of our assemblies are in the expected range based on C-value. The difficulty in genome assembly generally increases with repeat content [START_REF] Sedlazeck | Piercing the dark matter: bioinformatics of long-range sequencing and mapping[END_REF]. Therefore, discrepancies between expected genome size and assembly size from short-read sequencing technologies are still common. In a meta-study of avian genomes, [START_REF] Peona | How complete are "complete" genome assemblies?-An avian perspective[END_REF] note that regions highly enriched in repetitive DNA or with strong deviations in nucleotide composition are often underrepresented in assemblies.

The presence of such GC-rich or repeat-rich genome regions is a possible explanation for the ca. 20-30% gap between assembly sizes and estimated genome sizes for D. sargus and S.

cabrilla in the present study. Comparing contiguity, we found the D. sargus genome to be more contiguous (higher scaffold N50, Table 1) than the M. surmuletus and S. cabrilla genomes. Possible explanations could be higher molecular weight of DNA or a higher homozygosity of D. sargus in comparison to M. surmuletus and S. cabrilla [START_REF] Kajitani | Efficient de novo assembly of highly heterozygous genomes from wholegenome shotgun short reads[END_REF].

The mitochondrial genome is essential to eukaryote life and highly conserved across vertebrate species. Our mitochondrial genome assemblies match those of other fishes and vertebrates in terms of size (mean length = 16kb), the presence of 37 genes (13 protein coding, 22 tRNA, and 2 rRNA genes) and the non-coding control region [START_REF] Satoh | Structure and variation of the mitochondrial genome of fishes[END_REF]. In all three genomes, the annotation has identified genes that are highly conserved across metazoans with great accuracy (Fig. 1). For the benefit of providing a resource as flexible as possible, we did not filter annotated gene lists with respect to the presence of a starting codon.

For comparison, filtered G. aculeatus and D. rerio genome annotations contain 20,787 and 26,152 protein-coding genes, respectively, which are fewer compared to our unfiltered output (Howe et al., 2013;Jones et al., 2012). To note is that a significant percentage of Actinopterygii specific orthologs are fragmented (8%, 13% and 9.8% in D. sargus, M.

surmuletus and S. cabrilla, respectively). This is probably due to the lack of RNA-seq data for our focal species, which could have allowed the training of specific gene prediction models.

However, we can confirm that the three genome annotations are exhaustive and almost complete, as the percentage of missing BUSCOs is low and almost the same in the assembly and annotation (Fig. 1A andB, Tables S2 andS4). The OrthoMCL output revealed that D.

sargus and S. cabrilla share more ortholog genes than the two other species pairs. This is consistent with the phylogeny of the Perciformes, which shows that Mullidae have diverged during the early Lower Cretaceous (LC), while Sparidae and Serranidae have forked during the late LC [START_REF] Meynard | A Phylogenetic Perspective on the Evolution of Mediterranean Teleost Fishes[END_REF]. As such, D. sargus and S.

cabrilla are more closely related to each other than to M. surmuletus [START_REF] Meynard | A Phylogenetic Perspective on the Evolution of Mediterranean Teleost Fishes[END_REF], which is also supported by phylogenetic findings of Albouy et al. (2015). We used the reference genomes to generate rigorously filtered SNP datasets for the three species. Our approach with a single restriction enzyme (SbfI) recovered between 82% and 88% of the total number of RAD markers predicted by in silico digestion. These RAD markers provided between 31,000 and 47,000 SNPs pre-filtering and between 15,000 and 21,000 SNPs post-filtering that are evenly distributed across the genome. Besides providing a reference to align markers, this exemplifies that we also provide the expected number of markers. This allows knowing exactly what sequencing effort is needed to attain a given coverage. The number of high-quality markers generated here provides strong statistical power for future population genetic analyses. They can for instance be used for stock identification, investigations of population connectivity, and assignment studies. In addition, between 2,908 and 3,589 of our filtered SNPs lie in exonic regions. These markers may be used to start investigating functional variation (see e.g. [START_REF] Guo | Population genomic evidence for adaptive differentiation in Baltic Sea three-spined sticklebacks[END_REF], DiBattista et al. ( 2017)).

This study provides the first genomic resources for three economically important fish species in the Mediterranean Sea and as such lays a solid foundation for future population and conservation genomic and adaptive studies.

Materials and Methods

Genome sequencing

An individual of each species was sampled in the Western Mediterranean Sea (Table S6). Fin tissues of M. surmuletus and D. sargus were preserved in 96% ethanol at 4°C prior to DNA extraction, which was done within less than 24 hours. Tissues were cut into ~2 mm 2 pieces, dried at 70°C for 20 minutes, lysed in proteinase K at 56°C for 18 hours and incubated in RNAse A solution for 10 minutes at ambient temperature. DNA was extracted with a Macherey-Nagel Nucleospin® kit. For S. cabrilla, DNA extraction was conducted directly upon sampling. Tissues were dried out with filter paper and either flash-frozen in liquid nitrogen and crushed or cut into ~2 mm 2 pieces. The fragmented tissues were lysed in proteinase K at 56°C for 60 minutes and incubated in RNAse A for 10 minutes at ambient temperature. DNA was extracted using a Qiagen MagAttract HMW DNA kit.

For each genome, two paired-end libraries with insert sizes of 350 bp and 550 bp were generated from 1-2 µg of double-stranded DNA, as well as two mate-pair libraries with insert sizes of 3 kbp and 5 kbp from 4 µg of DNA. Libraries were sequenced on an Illumina HiSeq 4000 platform (150 bp paired-end reads). Library preparation and sequencing was conducted by FASTERIS (https://www.fasteris.com/dna).

Genome assemblies

Nuclear and mitochondrial genomes were assembled using three computing clusters, the Montpellier Bioinformatics Biodiversity platform (MBB: 64 cores, 512 Gb RAM), the High Performance Computing Platform of Occitanie / Pyrénées-Méditerranée Region of the Montpellier Mediterranean Metropole (MESO@LR: 80 cores, 1 Tb RAM), and CIMENT infrastructure in Grenoble (https://ciment.ujf-grenoble.fr, Froggy: 32 cores, 512 Gb RAM). The entire bioinformatics workflow for genome assembly is described in Appendix S1. Reads with < 50% bp with a phred quality > 20 were filtered out. Adapter sequences were also filtered out and the 3' extremities of the retained reads were trimmed with ngsShoRT (C. Chen, Khaleel, [START_REF] Chen | NgsShoRT: a software for preprocessing Illumina short read sequences for de novo genome assembly[END_REF]. Finally, reads shorter than 90 bp were removed. Nuclear genomes were assembled using the Platanus assembler [START_REF] Kajitani | Efficient de novo assembly of highly heterozygous genomes from wholegenome shotgun short reads[END_REF] (Fig. S2). Platanus was selected due to its excellent performance with highly heterozygous genomes [START_REF] Kajitani | Efficient de novo assembly of highly heterozygous genomes from wholegenome shotgun short reads[END_REF], as well as with simulated datasets that we produced (data not shown). The paired-end libraries were used to assemble reads into contigs, and both the paired-end and matepair libraries were used for scaffolding and gap closing. Mitochondrial genomes were assembled and annotated using MitoZ [START_REF] Meng | MitoZ: a toolkit for animal mitochondrial genome assembly, annotation and visualization[END_REF]. Five million sequences were randomly selected as a subset of the full paired-end sequence set. Mitochondrial sequences were then identified from this subset using a ranking method based on a Hidden Markov Model profile of known mitochondrial sequences from 2,413 chordate species. Mitochondrial sequences were then used to assemble the mitochondrial genome.

Gene annotation

Each fish genome was annotated using the ab initio gene predictor Augustus v3.2.3 [START_REF] Stanke | Uncertainty analysis and robust areas of high and low modeled human impact on the global oceans[END_REF]) and homology-based extrinsic hints. Each genome was first repeat-masked using RepeatMasker v4.0.8 [START_REF] Smit | RepeatMasker Open-4[END_REF]. Zebrafish (Danio rerio) and stickleback (Gasterosteus aculeatus) annotated protein sequences were downloaded from the Ensembl website (versions GRCz11 and BROADS1, respectively) and aligned to each repeat-masked fish genome using Exonerate [START_REF] Slater | Automated generation of heuristics for biological sequence comparison[END_REF].

Untranslated regions (UTRs) and alternative isoforms were not predicted due to the lack of species-specific RNA-seq data. Therefore, in each focal fish species, Augustus was run with the options "--species=zebrafish --UTR=off --alternatives-from-evidence=false" and with the respective Exonerate alignments as extrinsic hints. All reviewed metazoan proteins were downloaded from UniProt [START_REF] Bateman | UniProt: a worldwide hub of protein knowledge[END_REF] and used as database to run a search in Blast+ v2.2.30 [START_REF] Camacho | BLAST plus : architecture and applications[END_REF]. The highest scoring hit was selected as the putative gene name for each gene model. To functionally annotate the predicted genes, InterProScan v5.19 [START_REF] Jones | InterProScan 5: genome-scale protein function classification[END_REF] was run with options "-appl Pfam -b interpro -iprlookup -goterms" and functional information was added to the final annotation dataset using Annie v1.0 [START_REF] Tate | Annie the functional annotator-initial release[END_REF]. To identify ortholog gene families and species-specific genes in each Mediterranean fish genome, the OrthoMCL pipeline (L. [START_REF] Li | OrthoMCL: Identification of ortholog groups for eukaryotic genomes[END_REF] was used on the three annotated protein datasets along with the D. rerio protein dataset. Results were visualized with the venndiagram R package (H. [START_REF] Chen | VennDiagram: a package for the generation of highlycustomizable Venn and Euler diagrams in R[END_REF]. Finally, mitochondrial assemblies were annotated using BLAST family alignments on known protein coding genes, transfer RNA genes and rRNA genes. Quality of the nuclear genome assemblies and annotations were validated against the Metazoan and Actinopterygii Benchmarking Universal Single-Copy Orthologs (BUSCOs) with BUSCO v3.0.2 [START_REF] Simao | BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs[END_REF].

RAD markers prediction

SimRAD [START_REF] Lepais | SimRAD: an R package for simulation-based prediction of the number of loci expected in RADseq and similar genotyping by sequencing approaches[END_REF] was used to perform in silico digestion of the three genome assemblies with SbfI to predict the number of restriction sites and RAD markers in the three species.

RAD sequencing

A total of 90 samples (30 per species) from the Western Mediterranean were provided by local artisanal fishermen (Table S7, Fig. S3) and preserved in 96% ethanol. RADseq libraries were prepared using 1 µg of genomic DNA per sample in 50 µl reaction volume. Libraries were prepared following the protocol described in [START_REF] Etter | SNP discovery and genotyping for evolutionary genetics using RAD sequencing[END_REF] with a few modifications. At step 3.1 (restriction enzyme digestion), DNA was digested with 3 µL of the restriction enzyme SbfI-HF (New England Biolabs Inc., USA) in a 50 µL reaction volume. At step 3.2 (P1 adapter ligation), we used 2 µL of barcoded P1 adapters (100nM) in a 60 µL reaction volume and incubated the samples at room temperature for 1.5 h. Forty-eight samples were pooled per library. At steps 3.4 and 3.5, NEB Next® Ultra TM II DNA Library Prep Kit for Illumina (New England Biolabs Inc., USA) was used following the manufacturer's instructions to combine DNA end repair, 3´-dA overhang addition and P2 adapter ligation, followed by purification with a Qiagen QIAQuick PCR Purification Kit (Qiagen N.V., Netherlands). Finally, step 3.6 (PCR amplification) was run with the following settings: 30 s 98°C, 18x (10 s 98°C, 30 s 68°C, 30 s 72°C), 5 min 72°C, hold 4°C. P1 and P2 adapter sequences as well as PCR primer sequences are provided in Table S8. Each library was sequenced on one lane of a HiSeq 4000 Illumina Sequencer (paired-end, 2x 150bp) at the Institute of Clinical Molecular Biology, Kiel University, Germany.

SNP calling, genotyping and filtering

PhiX174 sequences that were used for quality control and calibration of the sequencing run were filtered out using BBMap v38.06 [START_REF] Bushnell | BBMerge -Accurate paired shotgun read merging via overlap[END_REF]. Raw sequences were demultiplexed and filtered using the process_radtags pipeline in STACKS v2.2 [START_REF] Catchen | Stacks: building and genotyping loci de novo from short-read sequences[END_REF][START_REF] Catchen | Stacks: an analysis tool set for population genomics[END_REF]. This included keeping only individuals with > 1,000,000 reads at this step, the removal of reads with more than one mismatch in the barcode sequence, and the removal of low-quality reads (with an average raw phred-score <20 within a 0.2 sliding window). In addition, reads were trimmed to a final length of 139bp due to a drop in read quality towards the end of the read. Taking advantage of paired-end information, clone_filter was used to remove pairs of paired-end reads that matched exactly, as the vast majority of these are expected to be PCR clones. Paired-end read sequences were subsequently aligned with BWA (H. [START_REF] Li | Fast and accurate short read alignment with Burrows-Wheeler transform[END_REF] to the reference genomes of M.

surmuletus, D. sargus, and S. cabrilla, thereby improving the reliability of stacks building.

Aligned reads were sorted using SAMTOOLS 1.9 (H. Li et al., 2009) and loci were built with gstacks providing genotype calls.

In order to retain only high-quality biallelic SNPs for population genetic analysis, called genotypes were further filtered with the populations pipeline and vcftools v0.1.16 [START_REF] Danecek | The variant call format and VCFtools[END_REF]. Only the first SNP was retained per RAD marker, and a SNP was retained only if present in at least 85% of individuals with a minimum minor allele frequency (MAF) of 1%. In order to reduce linkage among markers, only one locus was retained for all pairs of loci that were closer than 5000 bp or that had an r 2 value >0.8. Finally, individuals with >30% missing data were filtered out. 365 366 Table 1: Summary statistics of the genome assembly for each species using Platanus [START_REF] Kajitani | Efficient de novo assembly of highly heterozygous genomes from wholegenome shotgun short reads[END_REF] S2); B) percentage of conserved Metazoan genes found in our gene annotations, compared to the annotations of the D. rerio and G. aculeatus reference genomes used to train the Augustus gene prediction model (Table S4); C) percentage of conserved Actinopterygii genes found in our Platanus genome assemblies (Table S3); D) percentage of conserved Actinopterygii genes found in our gene annotations compared to the annotations the D. rerio and G. aculeatus reference genomes used to train the Augustus gene prediction model (Table S5). BUSCO stands for Benchmarking Universal Single Copy Ortholog genes.

ID

Figure 1 :

 1 Figure 1: A) Percentage of conserved Metazoan genes (BUSCOs) found in our Platanus genome assemblies (TableS2); B) percentage of conserved Metazoan genes found in our gene annotations, compared to the annotations of the D. rerio and G. aculeatus reference genomes used to train the Augustus gene prediction model (TableS4); C) percentage of conserved Actinopterygii genes found in our Platanus genome assemblies (TableS3); D) percentage of conserved Actinopterygii genes found in our gene annotations compared to the annotations the D. rerio and G. aculeatus reference genomes used to train the Augustus gene prediction model (TableS5). BUSCO stands for Benchmarking Universal Single Copy Ortholog genes.

Figure 2 :

 2 Figure 2: Four-set Venn diagram of 1:1 orthologous genes shared by M. surmuletus, D. sargus, S. cabrilla and D. rerio. Each ellipse shows the total number of genes specific to each species. Intersections indicate orthologous genes.

Figure 3 :

 3 Figure 3: RADseq coverage along the D. sargus (A, B), M. surmuletus (C, D) and S. cabrilla (E, F) genomes. A), C), E) Number of SNPs per 400,000 bp sliding window along the genome; B), D), F) Coverage per SNP per individual. Each blue dot represents the coverage of one SNP in one individual and the black line represents mean coverage in 400,000 bp sliding windows. Grey and white rectangles represent the assembly scaffolds.

Figure S1 :

 S1 Figure S1: Visualization of circular mtDNA for (a) D. sargus (16,620 bp), (b) M. surmuletus (16,577 bp) and (c) S. cabrilla(16,513 bp). The innermost circle shows the GC content, which is calculated with a sliding-window method. The middle circle shows the coverage depth distribution with the dark green outline. Parts of the genome that have a coverage lower than 20X are shaded in red, whereas parts of genome that have coverage larger than the upper quartile are shaded in dark green. The outer circle shows the gene annotations and is shaded in orange for rRNA, in red for tRNA, and in blue for CDS.

Figure S2 :Figure S3 .

 S2S3 Figure S2: Schematic workflow of DNA extraction, genome assembly and annotation.

Table

Table 2 :

 2 Summary statistics for the SNP markers generated by RAD sequencing for each species.

	371 2 MBB is 64 cores and 512Gb RAM	370 1 MESO@LR is 80 cores and 1Tb RAM	369	insert size	S. cabrilla MBB 2 3kbp & 5kbp	mate-pair	insert size,	M. surmuletus MESO@LR 1 550bp	350bp &	D. sargus MESO@LR Paired-end	Species Computing platform Library	368	367 sizes larger than 4 kbp.
					627			613		785	(Mbp)	Total size
					2,169,385 1,135			3,146,055 384		2,408,078 1,101	# of Contig N50 contigs (kbp )
					2,190			2,940		2,344	scaffolds	# of
					0.61 352 2.76 63X			0.49 317 3.28 74X		3.37 58 2.27 57X	Scaffold Scaffold N50 L50 (Mbp) Length of largest scaffold (Mbp) Coverage		. All statistics are based on contig

Table S1 :

 S1 Summary statistics of several fish genomes, including the ones assembled in this study and one of the the best currently available fish genome (D. labrax). Sources: AlMomin et al. (2016) 1 , Tine et al. (2014) 2 , Domingos, Zenger, and Jerry (2015) 3 , Xu et al. (2016) 4, Shin et al. (2014) 5 , Nakamura et al. (2013) 6

						N50	N50
						contig	scaffold
	Species	Year Assembler Library	Coverage	(kbp)	(kbp)
	Pampus	2016 SOAP	Paired-end 500bp insert size 58X	0.5	1.5
	argenteus 1		de novo
	Dicentrachus	2015 Celera	Paired-end 300bp & 500bp	30X	53	5100
	labrax 2		assembler	& 1kbp
				insert size,
				Mate-pair 8kbp & 20 kbp
				insert size
	Lates	2015 Velvet	Paired-end 330bp insert size 46X	15
	calcarifer 3
	Miichthys	2016 AllPaths-	Paired-end 180bp to 800bp	158X	81	1150
	miiuy 4		LG	insert size,
				Mate-pair 3kbp & 8kbp &
				20kbp
				insert size
	Notothenia	2014 Celera /	Paired-end 150bp & 300bp	38X	17
	coriiceps 5		Pbjelly	& 500bp
				& 600bp insert size,
				Mate-pair 3kbp & 5kbp &
				8kbp
				& 20kbp insert size
	Thunnus	2013 Newbler	Paired-end 300bp & 600bp	43X	82
	orientalis 6			insert size,
				Mate-pair 3kbp & 20kbp
				insert size
	Diplodus	This	Platanus	Paired-end 350bp & 550bp	57X	1.1	3371
	sargus	study		insert size,
	Mullus	This	Platanus	Mate-pair 3kbp & 5kbp	74X	0.38
	surmuletus	study		insert size
	Serranus	This	Platanus		63X	1.1
	cabrilla	study

Table S2 :

 S2 Percentage of conserved Metazoan genes (BUSCOs) found in the genome assemblies of D. sargus, M. surmuletus and S. cabrilla.

		D. sargus	M. surmuletus	S. cabrilla
	Complete BUSCOs	97.5%	92.5%	96.7%
	Complete and single-copy BUSCOs 93.5%	89.1%	93.9%
	Complete and duplicated BUSCOs 4.0%	3.4%	2.8%
	Fragmented BUSCOs	0.8%	2.2%	0.6%
	Missing BUSCOs	1.7%	5.3%	2.7%

Table S3 :

 S3 Percentage of conserved Actinopterygii genes (BUSCOs), found in the genome assemblies of D. sargus, M. surmuletus and S. cabrilla.

		D. sargus	M. surmuletus	S. cabrilla
	Complete BUSCOs	96.6%	89.9%	95.3%
	Complete and single-copy BUSCOs 94.4%	87.2%	92.6%
	Complete and duplicated BUSCOs 2.2%	2.7%	2.7%
	Fragmented BUSCOs	1.4%	4.3%	2.0%
	Missing BUSCOs	2.0%	5.8%	2.7%

Table S4 :

 S4 Percentage of conserved Metazoan genes (BUSCOs) found in the gene annotations of D. sargus, M. surmuletus and S. cabrilla.

		D. sargus	M. surmuletus	S. cabrilla
	Complete BUSCOs	96.0%	90.8%	95.6%
	Complete and single-copy BUSCOs 92.3%	87.3%	92.3%
	Complete and duplicated BUSCOs 3.7%	3.5%	3.3%
	Fragmented BUSCOs	3.4%	5.9%	3.6%
	Missing BUSCOs	0.6%	3.3%	0.8%

Table S5 :

 S5 Percentage of conserved Actinopterygii genes (BUSCOs), found in the gene annotations of D. sargus, M. surmuletus and S. cabrilla.

		D. sargus	M. surmuletus	S. cabrilla
	Complete BUSCOs	90.2%	80.3%	87.6%
	Complete and single-copy BUSCOs 86.9%	77.1%	84.1%
	Complete and duplicated BUSCOs 3.3%	3.2%	3.5%
	Fragmented BUSCOs	8.0%	13.3%	9.8%
	Missing BUSCOs	1.8%	6.4%	2.6%

Table S6 :

 S6 Tissue collection site, date and depth for the individuals used to assemble the reference genomes.

				Latitude	Longitude
	Species	Date	Location	(decimal degree)	(decimal degree) Depth (m)
	M. surmuletus 22.02.17 Mallorca, Spain 39.911	3.132	24
	D. sargus	15.07.16 Corsica, France 42.323	8.567	14
	S. cabrilla	08.06.18 Banyuls, France 42.472	3.157

Table S7 :

 S7 Tissue collection site, date and depth of the samples used for RAD sequencing.

		26.09.2017	39.797	3.201	Can Picafort	27
		30.03.2018	39.756	3.222	Can Picafort	14.2
	sp. Label 1 2 3 4 5 6 7 8 9 1 2 Mullus surmuletus 3 4 5 6 7 8 9 1 Serranus cabrilla 2 3 4 5 6 7 8 Diplodus sargus 9	Date 25.10.2017 25.10.2017 10.07.2017 06.07.2017 12.07.2017 06.07.2017 12.07.2017 06.07.2017 12.07.2017 08.08.2017 12.07.2017 09.08.2017 12.07.2017 10.08.2017 10.10.2017 13.09.2017 27.09.2017 10.10.2017 27.09.2017 20.10.2017 25.09.2017 02.11.2017 05.10.2017 26.07.2017 13.11.2017 26.07.2017 19.03.2018 06.09.2017 18.08.2017 05.10.2017 04.12.2017 26.10.2017 20.04.2018 30.08.2017 27.07.2017 17.10.2017 18.09.2017 17.10.2017 07.09.2017 04.09.2017 28.05.2017 04.09.2017 04.06.2017 04.09.2017 09.08.2017 21.09.2017 06.09.2017 12.07.2017 17.10.2017 12.07.2017 26.06.2017 16.10.2017 31.08.2017 05.09.2017 01.12.2017 23.05.2017 31.05.2017 12.07.2017 23.10.2017 15.11.2017 27.10.2017 02.11.2017 20.11.2017 11.10.2017 12.07.2017 20.07.2017 12.07.2017 31.08.2017 12.07.2017 06.09.2017 28.04.2018 20.10.2017 27.10.2017 31.08.2017 05.10.2017 05.09.2017 05.09.2017 05.08.2017 20.11.2017 08.08.2017 05.12.2017 13.09.2017	Latitude (decimal degree) 43.277 43.277 41.964 42.323 42.469 42.323 42.469 42.323 42.472 39.894 42.472 39.889 42.472 39.894 37.554 38.281 42.285 37.550 42.285 37.753 39.605 37.702 39.638 41.801 36.892 41.801 39.707 42.323 37.079 39.325 39.280 40.034 40.073 41.857 42.239 41.113 37.931 39.874 38.303 38.161 39.829 38.161 41.202 38.161 36.864 38.317 38.220 42.479 39.880 42.493 42.521 37.583 37.031 38.181 38.144 39.276 41.027 42.514 37.585 36.892 39.824 39.650 39.804 37.591 42.515 40.076 42.515 37.184 42.515 42.490 39.756 39.652 39.776 37.197 39.792 38.172 38.172 39.852 39.764 39.905 39.786 39.831	Longitude (decimal 3.517 3.517 degree) 3.228 3.310 3.163 3.310 3.163 3.310 3.157 0.687 3.157 0.667 3.157 0.687 -0.917 -0.505 3.297 -0.981 3.297 -0.744 3.413 -0.752 3.450 3.071 -1.935 3.071 3.468 3.310 -1.842 2.970 2.945 4.187 4.080 3.149 3.181 1.302 -0.672 3.259 -0.464 -0.528 0.708 -0.528 2.142 -0.528 -1.980 -0.447 -0.459 3.148 3.160 3.134 3.148 -0.794 -1.867 -0.444 -0.613 -0.137 1.377 3.138 -0.782 -1.935 3.471 3.607 3.380 -0.725 3.139 4.085 3.139 -1.807 3.139 3.135 3.250 3.474 3.414 -1.766 3.734 -0.448 -0.448 0.682 3.405 0.632 3.440 4.345	Cap d'Agde Location Cap d'Agde Aiguafreda Cap de Creus Banyuls Cap de Creus Banyuls Cap de Creus Banyuls Columbretes Banyuls Columbretes Banyuls Columbretes Cabo del Agua El Altet Cadaques Escombreras Cadaques La Manga Cala Bona La Manga Cala Bona Palamos Cala de San Pedro Palamos Cala Ratjada Cap de Creus Castillo Macenas Colònia Sant Jordi Colònia Sant Jordi Fornells Illa Bledes Palamos Roses Tarragone Torrevieja Alcudia Alicante Alicante Islas Columbretes Alicante L'Hospitalet Alicante Las Negras Alicante Monte Faro Banyuls Pollença Banyuls Port Vendres Cabo Negro Río Alias Santa Pola Santa Pola Sueca Tarragona Banyuls Cabo Negro Cala de San Pedro Cala Ratjada Cala Ratjada Cala Ratjada Calblanque Banyuls Cap de Cavalleria Banyuls Garrucha Banyuls Banyuls Betlem Cala Ratjada Cala Ratjada Garrucha Holanda Isla de Tabarca Isla de Tabarca Islas Columbretes Cala Ratjada La Calç Cala Ratjada Maó	Depth 28.4 (m) 28.6 15.4 23.3 15.4 12.4 15.6 11.7 7 11.5 3 10.2 7 10.2 8 32.4 59.4 16.8 1.8 17 3.6 21 17.1 28 17.1 63 18.7 13.2 32.4 22 56 27.7 23.4 21.4 14.2 36 56 21.5 18 9.8 18 94 18 693 25 55.8 17.8 21.5 17 26 21.6 10 36 21 13.5 64.5 129.8 21.6 63 53.1 76.6 44.6 25.2 19.5 18 24.5 27 24.5 24.5 21.7 23.8 39.7 72 116 19 19 39.1 32.5 47.3 32.2 36.5 65.8

Table S8 :

 S8 RADseq adapter sequences, F=Forward, R=Reverse. Concentrations of P1 adapters are 25nmole, P1 adapter treatment = standard desalting, Phosphorothioate linkage between bases marked with * in forward P1 adapters, P2 reverse adapter and PCR primers, 5' Phosphorylation as indicated in reverse P1 and forward P2 adapters. Concentrations of P2 adapters and PCR primers are 100 nmole, P2 adapter and PCR primer treatment = HPLC purification,

Acknowledgements

This research was funded through the 2015-2016 BiodivERsA COFUND call for research proposals, with the national funders ANR (France), Formas (Sweden), DLR (Germany), AEI (Spain) and the CNRS for the PICS SEACONNECT.

Data Accessibility Statement

The three reference genomes have been uploaded in the European Nucleotide Archive (ENA, project accession number PRJEB38135 at https://www.ebi.ac.uk/ena/data/view/PRJEB38135), the RAD data a git repository: https://gitlab.mbb.univ-montp2.fr/reservebenefit/subset_30_samples_vcf and the scripts in a git repository: https://gitlab.mbb.univ-montp2.fr/reservebenefit/genomic_resources_for_med_fishes.

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Author contributions

VA processed samples for genome sequencing. KF processed samples for RAD sequencing, and contributed to write the manuscript. ET processed samples for RAD sequencing and wrote a first draft of the manuscript. SL performed genome assemblies. PEG performed genome assemblies, processed samples for RAD sequencing, and contributed to write the manuscript. MTO performed gene annotation. APR contributed to the sampling design and sampling. SM and OP designed the research, supervised the project and contributed to write the manuscript.

Animal experiments

All samples were obtained from fishermen. No experiments were conducted.

Author statement

Véronique Arnal processed samples for genome sequencing. Katharina Fietz processed samples for RAD sequencing, and contributed to write the manuscript. Elena Trofimenko processed samples for RAD sequencing. Stéphane Lobreau performed genome assemblies.

Pierre-Edouard Guerin performed genome assemblies, processed samples for RAD sequencing, and contributed to write the manuscript. Montserrat Torres-Oliva performed gene annotation.

Angel Pérez-Ruzafa contributed to the sampling design and sampling. Stéphanie Manel and Oscar Puebla: Funding acquisition, designed the research, supervised the project and contributed to write the manuscript.

Supplementary Information

Katharina Fietz, Elena Trofimenko, Pierre-Edouard Guerin, Véronique Arnal, Montserrat Torres-Oliva, Stéphane Lobréaux, Angel Pérez-Ruzafa, Stephanie Manel, Oscar Puebla (see main text for affiliations)


Marco Longhin

Mahmoud Khalil

Linda Abbassi

Mickael Beaudhuin

Philippe Papet
email: philippe.papet@umontpellier.fr

Romain Viennois
email: romain.viennois@umontpellier.fr

Enhanced thermoelectric properties in Polypyrrole composites with silicide fillers

Keywords: Composite materials, electrical properties, Intermetallic alloys and compounds, Polymers

I. Introduction

The thermoelectric properties of polymers are still low compared to the state-of-the-art inorganic thermoelectric materials [START_REF] Zhang | Organic Thermoelectric Materials: Emerging Green Energy Materials Converting Heat to Electricity Directly and Efficiently[END_REF][START_REF] Bharti | Conductive polymers for thermoelctric power generation[END_REF][START_REF] Kim | Engineered doping of organic semiconductors for enhanced thermoelectric efficiency[END_REF] however, their low cost, easy shaping for large-scale production, flexibility and light weight are strong assets to make them promising for thermoelectric applications. Their thermoelectric properties are characterized by the figure of merit ZT =  2 T/ with  the Seebeck coefficient,  the electrical conductivity, T the temperature and  the thermal conductivity [START_REF] Zhang | Organic Thermoelectric Materials: Emerging Green Energy Materials Converting Heat to Electricity Directly and Efficiently[END_REF]. To improve their performances, one approach was to incorporate inorganic thermoelectric materials into the host organic matrix [START_REF] Du | Research progress on polymer-inorganic thermoelectric nanocomposite materials[END_REF]. Polymerbased thermoelectric benefits from the advantages of the polymers stated above, and those of the inorganic fillers in term of performances. Several authors have studied organic/inorganic thermoelectric composites [START_REF] Bharti | Conductive polymers for thermoelctric power generation[END_REF][START_REF] Du | Research progress on polymer-inorganic thermoelectric nanocomposite materials[END_REF][START_REF] Liu | Structure and electronic transport properties of polyaniline/NaFe4P12 composite[END_REF][START_REF] Zhang | Promosing thermoelectric properties of commercial PEDOT:PSS materials and their Bi2Te3 powder composites[END_REF][START_REF] Yong | Synthesis and characterization of Bi2Te3/polyaniline composites[END_REF][START_REF] Ao | Synthesis and characterization of polythiophene/ Bi2Te3 nanocomposite thermoelectric material[END_REF][START_REF] Ail | Room temperature synthesis of transition metal silicide-conducting polymer micro-composites for thermoelectric applications[END_REF]. However, if some improvements of the thermoelectric properties of the polymers were observed, no significant enhancement was reported compared to state-of-the-art materials. Among them one find the intensively studied Pedot:PSS with a ZT up to 0.4 at room temperature [START_REF] Bharti | Conductive polymers for thermoelctric power generation[END_REF][START_REF] Kim | Engineered doping of organic semiconductors for enhanced thermoelectric efficiency[END_REF], however their stability is low. There are also several other pi-conjugated polymers which have been investigated for thermoelectric applications such as [START_REF] Zhang | Organic Thermoelectric Materials: Emerging Green Energy Materials Converting Heat to Electricity Directly and Efficiently[END_REF][START_REF] Bharti | Conductive polymers for thermoelctric power generation[END_REF]: Poly-anyline (PANI), Polypyrrole (PPy), Polythiophene (PT), Poly(phenylenevinylene (PPV), poly(2,7-carbazolylenevinylene). Among them PPy has attracted much interest as a promising TE candidate as it has no toxicity, it is easy to synthesize it and it has a rather good electrical conductivity and low thermal conductivity [START_REF] Zhang | Organic Thermoelectric Materials: Emerging Green Energy Materials Converting Heat to Electricity Directly and Efficiently[END_REF][START_REF] Bharti | Conductive polymers for thermoelctric power generation[END_REF][START_REF] Du | Research progress on polymer-inorganic thermoelectric nanocomposite materials[END_REF]. As for the inorganic load it is possible to use low toxicity materials such as silicides like CoSi-based or CrSi2-based compounds with ZT max of about 0.2-0.3 [START_REF] Burkov | Silicide Thermoelectrics: Materials for Energy Harvesting[END_REF][START_REF] Longhin | Nanostructuration of CoSi by mechanical milling and mechanical alloying[END_REF][START_REF] Longhin | Nanostructured CoSi Obtained by Spark Plasma Sintering[END_REF][START_REF] Khalil | A modeling approach for new CrSi2 based alloys : Application to metastable Cr1-xZrxSi2 as a potential thermoelectric material[END_REF][14]. To date, there is only one study of organic/silicide composite namely Pedot:PSS/CrSi2 investigated by the Ail et al. [START_REF] Ail | Room temperature synthesis of transition metal silicide-conducting polymer micro-composites for thermoelectric applications[END_REF]. However, its thermoelectric performances were lower than for its components taken independently. In the present study, we have investigated the enhancement of the thermoelectric properties of p-type PPy with the use of p-type silicides fillers such as the semi-metallic Co0.85Fe0.15Si or the semiconductor CrSi2 alloys.

II. Materials and methods

Powder of Polypyrrole (PPy) doped with organic acid (not specified) were purchased to Sigma Aldrich company. Co0.85Fe0.15Si and CrSi2 alloys were prepared by arc melting technique using high purity Co slug (99,95%, Alfa Aesar), Cr granules (99.995%, Alfa Aesar), Fe (99.99%, Alfa Aesar) and Si lumps (99.999%, Alfa Aesar). The drops were milled with a commercial Fritsch "Pulverisette 7" planetary micromill with silicon nitride container (45ml) and five 15 mm diameter balls with a ball to powder mass ratio fixed to 10:1 for both alloys. The speeds of the supporting disc and of the grinding ball acceleration were respectively 575 RPM and 1150 RPM. All experiments were done under argon atmosphere for 24h. The composites were made by mixing the powders of the inorganic phase (volume fraction ϕi) and of the PPy (volume fraction ϕp). We both mix and mill the powders together in a polystyrene tube filled with ZrO2 balls with a ball to powder ratio of 10:1 by using a roller mixer at 30 rpm. The shaping of the powder was investigated by cold-pressing using a uniaxial pressure of 35 MPa applied for 5 min.

The crystal structure of the alloys was determined by XRD with Cu K wavelength and refined with Fullprof software [START_REF] Rodríguez-Carvajal | Recent advances in magnetic structure determination by neutron powder diffraction[END_REF] while the grain size was determined using the Williamson-Hall formula [START_REF] Longhin | Nanostructuration of CoSi by mechanical milling and mechanical alloying[END_REF][14][START_REF] Rodríguez-Carvajal | Recent advances in magnetic structure determination by neutron powder diffraction[END_REF][START_REF] Williamson | X-ray line broadening from filed aluminium and wolfram[END_REF]. The thermoelectric properties were measured using homemade Van der Paw technic and hot probe Seebeck equipment as described in ref. [START_REF] Khalil | A modeling approach for new CrSi2 based alloys : Application to metastable Cr1-xZrxSi2 as a potential thermoelectric material[END_REF].

III. Results and discussion

As shown in our previous studies using the same synthesis process [START_REF] Longhin | Nanostructuration of CoSi by mechanical milling and mechanical alloying[END_REF][START_REF] Longhin | Nanostructured CoSi Obtained by Spark Plasma Sintering[END_REF][START_REF] Khalil | A modeling approach for new CrSi2 based alloys : Application to metastable Cr1-xZrxSi2 as a potential thermoelectric material[END_REF][14], CrSi2 and Co0.85Fe0.15Si compounds have a nanocrystallite size about 10-20 nm (raw XRD data are given in the S. I. Figs. S2 andS3). From Figure 1 (a), increasing the content of the filler 𝜙 𝑖 with Co0.85Fe0.15Si leads, as expected, to an increase of the electrical conductivity from a percolation threshold above 𝜙 𝑖 = 25 %. The higher the content of the inorganic phase is, the larger the electrical conductivity is. Similar behavior is observed for the Seebeck coefficient. In Figure 1 (b) the electrical conductivity is normalized to its value at 60°C (chosen for better comparison), and is given as a function of the temperature. One observes that the electrical conductivity of pure PPy increases with the temperature, which is typical of a semiconductor. On the contrary, the electrical conductivity of pure Co0.85Fe0.15Si decreases with temperature which is typical of thermoelectric properties than doped PPy. The properties of these PPy-based composite are better than the best PPy-based composite from the literature which contain 2D reduced oxide graphene with power factor of 8.56 µW/ m.K 2 [START_REF] Bharti | Conductive polymers for thermoelctric power generation[END_REF].

III. Conclusion

In the present work, we have reported the design and thermoelectric properties of green organic/inorganic composites made of PPy and of two different silicide compounds (Co0.85Fe0.15Si and CrSi2). In the case of the PPy/Co0.85Fe0.15Si composite, both the electrical conductivity and the Seebeck coefficient increase above a percolation threshold of ~25%. In the case of the PPy/CrSi2 composite, although no clear behavior has been observed on the electrical conductivity, we find that the Seebeck coefficient strongly increase above a percolation of ~30%. We also found that the CrSi2/PPy composite has better thermoelectric properties than CrSi2/Pedot:PSS and the best PPy-based composite [START_REF] Bharti | Conductive polymers for thermoelctric power generation[END_REF]. This can be explained by the higher power factor of CrSi2 compared to CoSi at room temperature. Moreover, it is easy to manage for large scale applications.

FiguresFigure 1

 1 Figures

Figure 2 a

 2 Figure 2 a) Thermoelectric properties of the composite PPy/CrSi2 as a function of the volume

Figure 3

 3 Figure 3 Evolution of the power factor ( 2 ) of the composite PPy/Co0.85Fe0.15Si and PPy/CrSi2

Acknowledgement

We thank Fabien Perrin for his help during this work, the University of Montpellier and the Institute Carnot Chimie Balard Cirimat for their funding.

a metallic behavior. This is not surprising given that undoped and doped CoSi are known to have this behavior [START_REF] Burkov | Silicide Thermoelectrics: Materials for Energy Harvesting[END_REF][START_REF] Longhin | Nanostructuration of CoSi by mechanical milling and mechanical alloying[END_REF][START_REF] Longhin | Nanostructured CoSi Obtained by Spark Plasma Sintering[END_REF][START_REF] Viennois | Stability and Thermoelectric Properties of Transition-Metal Silicides[END_REF]. When considering the evolution of the electrical conductivity with the volume fraction of the inorganic phase, for the sample with 𝜙 𝑖 = 10%, the electrical conductivity increases with the temperature faster than the PPy. With increasing the temperature, electrical charges from the isolated inorganic particles can be injected into the matrix PPy. The inorganic phase does not appear to strongly contribute to the electrical conductivity of the composite which is expected as it does not percolate. In contrast, for large values of 𝜙 𝑖 ( 50 %) the 𝜎/𝜎 60°𝐶 decreases rapidly with temperature which is consistent with an inorganic content above the percolation limit.

In the case of the PPy/CrSi2 composite, we have also changed the ratio between organic and inorganic phase in order to follow the evolution of the thermoelectric properties.

The Seebeck coefficient (Figure 2) strongly increases with increasing 𝜙 𝑖 to that of the inorganic phase. However, there is no clear trend for the electrical conductivity. Thus in the present PPy/CrSi2 composite, one can see that the percolation threshold ϕi for the Seebeck coefficient is above 30%, value very similar to the case of PPy/Co0.85Fe0.15Si composite shown previously.

Despite its semiconducting nature, CrSi2 has metallic-like behavior close to room temperature which is due to its extrinsic transport regime [START_REF] Khalil | A modeling approach for new CrSi2 based alloys : Application to metastable Cr1-xZrxSi2 as a potential thermoelectric material[END_REF]14].

From these data, one can determine the power factor ( 2 ) (See Fig. 3) which is proportional to the ZT. In the case of the composite PPy/Co0.85Fe0.15Si, one finds a power factor of 0.8 µW/m.K 2 for 𝜙 𝑖 = 50 % and of 2 µW/m.K 2 for 𝜙 𝑖 = 90 % which is two orders higher than for the PPy. However, in the case of the PPy/CrSi2 composite, we find a power factor of 3.37 µW/m.K 2 for 𝜙 𝑖 = 50 % and of 9.7 µW/m.K 2 for 𝜙 𝑖 = 90 %, much larger than for PPy/ Co0.85Fe0.15Si composite and more than two orders times larger than in PPy. These values are also respectively, about 10 times and 2 times larger than the values of the power factor found for a Pedot-PSS/CrSi2 composite [START_REF] Ail | Room temperature synthesis of transition metal silicide-conducting polymer micro-composites for thermoelectric applications[END_REF] despite that doped Pedot-PSS has much better