Yaqiong Wang

Zouhour Hsine

Helene Sauriat-Dorizon

Rym Mlika

Hafsa Korri-Youssoufi
email: hafsa.korri-youssoufi@universite-paris-saclay.fr

Structural and electrochemical studies of functionalization of reduced graphene oxide with alkoxyphenylporphyrin mono-and tetra-carboxylic acid: application to DNA sensors

Keywords:

Association of graphene with conjugated organic macromolecules presents an interest in various applications. Here, we study the chemical and electrical properties of nanomaterials which combine the features of chemical reduced graphene oxide (CRGO) and its modification with tetrabutyloxyphenylporphyrins bearing a mono-or tetra-carboxylic functions. The hybrid nanomaterials are characterized through different techniques including UV-visible, FT-IR, Raman spectroscopy, MEB and SEM. Their electrochemical properties are studied by cyclic voltammetry (CV) with inner sphere and outer sphere redox markers as well as electrochemical impedance spectroscopy (EIS). The overall results support the formation of strong interactions between porphyrins and CRGO. Furthermore, we highlight the relationship of structural and electronic properties of porphyrins on the resulted composite. Thus, we demonstrate that modified porphyrins with alkoxy and carboxyl groups improve the dispersion of CRGO and have positive effects on their electronic properties in particular the electron transfer ability. These nanocomposites open new opportunities for their application in biosensors devices and the detection of DNA of M. Tuberculosis was demonstrated with LOD at attomolar range and an ability to discrimination of M. tuberculosis strand from mutated in PCR sample.

Introduction

Since 2004 [1] , graphene has known a growing interest in numerous field of materials science. Indeed, its remarkable thermal, mechanical and electronic properties makes it a potential material for nano-electronic devices such as field effect transistors [2] , memory device [3] or ultrafast laser [4] .

Besides, graphene possesses also a high surface area (up to 2630 m 2 /g), easy bio-functionalization [5] and a good biocompatibility [6] . These properties afford it a real potential for biotechnology applications, including biosensing device [7] , photothermal therapy [8] , drug delivery [9] , nanomedicine [10] , and cancer targeting [11] . To date, there are several known methods for preparing graphene [12] , including mechanical cleavage [13] and chemical vapor deposition [14] . Chemically exfoliation of graphite gives graphene oxide generally in acidic [15] or basic [16] conditions. The chemical formation of reduced graphene oxide (CRGO) is the best method for large-scale production [17] and the obtained nanomaterial possesses a large surface area compared to other forms of graphene [18] . The removal of oxygen and the recovery of aromatic double-bonded in chemical reduced graphene oxide restore the conductivity by several orders of magnitude [19] as well as its electrochemical properties [20] .

Reduced graphene oxide (RGO) is hydrophobic and tends to agglomerate in water through strong π-π k g v W [21] . As a consequence, considerable efforts have been made to improve its dispersion [22] . Chemical modification of RGO sheets or the entrapment of functional molecules has been performed for this purpose. For example, covalent functionalization of graphene sheets with various groups such alkyl azides [23] , porphyrins [24] allowed a better dispersibility in organic solvents [23] . However, introduction of functional groups by covalent modifications creates sp 3 carbon sites, compromising the sp 2 structure of graphene and then the loss of its electronic properties. Noncovalent f z g π-π v W f p f ' π-conjugated structure and the electronic properties of graphene. Furthermore, this strategy allows the dispersion of the composite and affords news properties for further applications [25] . Various ionic [26] and (or) aromatic molecules [27] were investigated for their association with reduced graphene oxide such as poly(styrene sulfonate) [28][29] ,

quinoline [30] , pyrene [31] , anthracene [32] and bis(3,4-dihydroxybenzylidene)-1,2-diamino benzene Schiff base ligand [33] .

Porphyrins, due to their numerous chemical, electronic and optical properties, have been associated with conjugated nanomaterials such as graphene without covalent attachment [34] . Moreover, porphyrins can be easily modified with numerous groups, in various positions of the macrocycle, endowing additional properties, such as anionic or cationic form [35] . Numerous papers have described the formation of nanocomposite porphyrin/graphene [36] . The structure and electronic properties of the nanocomposite were demonstrated to be controlled by functional groups attached to the porphyrins [37] ,

the metal ion [38] as well as the structural properties of graphene [39] .

For example, tetra phenyl porphyrin bearing sulfonic acid was demonstrated to have a good association with reduced graphene and enhanced the dispersion due to the repulsive forces between the two materials [34] . However, cationic porphyrins such as 5,10,15,20-tetrakis(1-methyl-4-pyridinio) porphyrin could forms flattering complex through electrostatic interactions with chemically negatively charged graphene and leads to electronic modifications of such complex [40] . It was also demonstrated that elect π-π k g s between charged porphyrins and graphene are controlled by functional groups on the surface of graphene and edge structure [41] . Therefore, the chemical structure of the porphyrin controls the mechanism of its association with the graphene nanosheets though, hydrogen bonding, ionic or π-π staking interactions. Thus, the nature of the chemical structure of porphyrin rules the approach between the porphyrin plane and aromatic rings-based graphene as well as its association in the peripheries or the basal plane of graphene.

Nanocomposites formed between graphene and porphyrins have been demonstrated to bring new properties. They could be applied in various applications such as catalysis [42] , photocatalysis [43][44] ,

electrocatalytic CO 2 conversion [38] , sodium ion batteries [45] , sensors for various molecules and ions such as, explosive [46] , dopamine [47] , and immunosensors [48] .

Tetraphenylporphyrins, bearing carboxylic acid, have the advantage to introduce other molecules or biomolecules through chemical link. Their association with graphene allows the covalent attachment of DNA and their application in DNA sensing [49] . Thus, association of CRGO with such macromolecules bearing carboxylic acid constitutes an interest in biosensor devices. Here, we synthetized two modified teraphenylporphyrins (butyloxyphenylporphyrin) where phenyls were substituted in para position with one or four (carboxybutyloxy groups as donor substituents, to give a mono-or a tetra-carboxylic molecule (H 2 TPP-1CP and H 2 TPP-4CP, respectively, schema 1). We studied their association with chemically reduced graphene oxide (CRGO) nanomaterial through UVvisible, FT-IR, Raman spectroscopy and MEB, SEM images to highlight the effect of specific side group on the structure of the nanocomposite. Electrochemical properties of the nanocomposites were also performed within inner sphere and outer sphere redox markers. The effect of chemical structure of the carboxyporphyrins in the mechanism of association with CRGO and thus their electrochemical properties was studied. We investigated this two nanomaterials H2TPP-nCP(n=1,2)/CRGO into DNA sensor and demonstrated their sensitivity and selectivity in PCR sample from M. Tuberculosis and resistant strand.

Schema 1. Schematic representation of modified material

Results and discussions

Characterization of the nanocomposites by imaging

The morphologies of the hybrid nanomaterials were characterized by SEM and AFM images (Figure 1). SEM images show that CRGO exhibits compact sheet with irregular layer and folding structure, where nano-sheets with many wrinkles and corrugation are observed (Figure 1A, image a).

In the case of the hybrid nanomaterials, nano-sheet layers are observed with edge domains and thin and semitransparent flakes with less wrinkled textures compared to CRGO, (Figure 1A, images b, c).

This demonstrates that anchoring the porphyrin to graphene leads to spacing CRGO nanosheets and form nano-sheets domain. The flakes of H 2 TPP-1CP/CRGO fold together with ordered of 20-30 μ range and present crinkly sheets with more aggregation, compared to that of H 2 TPP-4CP/CRGO with 5 10 μ g . T s demonstrated that, the dispersion of CRGO was obtained with both porphyrins and was improved in the case of the association of H 2 TPP-4CP with CRGO (Figure 1A, image c), due to the presence of a highest number of carboxylic functions. and compact structure with a thickness of 6 nm. This value is in the same order than that of RGO nanosheets (of 1 nm) [50] . The reduction of the GO favors the multi-layer formation of sheets on the surface through π-stack. In the case of hybrid nanomaterials, dispersed nano-sheets of graphene were observed (Figure 1B, images b, c). The roughness factor measured of hybrid nanomaterial H 2 TPP-1CP/CRGO is up to 250 nm, because of the formation of small nano-sheet domains and edge plane (Figure 1B, image b). In the case of nano-hybrid H 2 TPP-4CP/CRGO, the roughness factor is in the same order of magnitude than CRGO (15 nm) and differences between the edges of the sheets and the interior regions are also observed (Figure 1B,image c). This result suggests different associations behavior of the porphyrins with the surface of the reductive graphene oxide. Indeed, the two porphyrins interact with the basal plane of graphene through -interaction due to their great conjugated structure similar to graphene. But in the case of H 2 TPP-4CP porphyrin where the number of carboxylic groups are highest, hydrogen bonds with oxygen functions present on the peripheries of graphene could also be favored.

Spectroscopic characterization

To underline the molecular and electronic properties of the nanocomposites, different techniques FT-IR, UV-Visible, Raman spectroscopies and XPS were performed.

The interactions between the porphyrins and graphene were analyzed by UV-visible experiments by successive addition of CRGO to constant solution of modified porphyrins (Figure 2 A,B). After addition of CRGO, an increase of the Soret band intensity was observed at 417 nm resulting from the formation of the complex. This increase of absorbance is related to a better solubility of porphyrin after their attachment to CRGO. In fact, porphyrins bearing carboxylic group form aggregates due to the hydrogen bonding and when adsorbed on graphene their solubility increase enhancing the absorbance. A same behavior was observed after the interaction of the meso-tetra (4-carboxyphenyl) porphyrin with graphene [51] . The absence of red shift of Soret band underlines that any modification of porphyrin structure such as flattening was obtained as observed in some modified porphyrins [39] . The interaction of porphyrin with CRGO was analyzed using the Benesi-Hildebrand [52] approach to determine the association constant. This model is generally used in the case of soluble or dispersed solution.

k H 2 TPP-n =1,4 C + CRGO CRGO/[H 2 TPP-n =1,4 C] k
where k is the number of porphyrins associated to CRGO graphene.

The association constant could be calculated following the equation equation ( 1):

(

where K a pp ε 417 is the molar absorption coefficient of complex porphyrin/CRGO and C H2TPPC the porphyrin concentration. Inverse of the difference absorbance at 417 nm (1/D O ) was plotted versus inverse of the CRGO concentration (1/C CRGO ) (Figure 3C,D). The apparent association constant of H 2 TPP-1CP/CRGO and H 2 TPP-4CP/CRGO was calculated to be (2.92 x10 4 M -1 and 8.93 x 10 4 M -1 ), respectively. These values are in the same order of magnitude of those demonstrated previous in the case of association of a cationic porphyrin, meso-tetrakis(4-N-methylpyridiniumyl)porphyrin, with RGO 3.1x10 4 M -1 where stable dispersed complex are formed [53] .

The difference of a factor of three between the mono and tetra substituted porphyrin can be attributed to the presence of highest number of carboxylic groups in H 2 TPP-4CP promoting the strongest hydrogen bonds association. This result demonstrates the strong interaction of H 2 TPP-1CP and H 2 TPP-4CP to CRGO and the well dispersion properties of the composite. The FT-IR of the composites were performed to analyze the mechanism of interaction between porphyrins and CRGO. FT-IR spectra (Figure 3A and 3B) exhibit two stretching vibration bands at around 1720 cm -1 and 1580 cm -1 corresponding to C=O and C=C bound, respectively. In comparison with the spectrum of porphyrin alone, a red shift of C=O band by 20 cm -1 is noted in the both composites. This demonstrates hydrogen bonding formation between the porphyrin and graphene due to the presence of carboxylic function. In the case C=C vibrations a shift of about 34 cm -1 (from 1611 to 1577 cm -1 ) is observed for H 2 TPP-1CP/CRGO composite and 15 cm -1 (from 1602 cm -1 to 1587 cm - 1 ) for H 2 TPP-4CP/CRGO. This difference suggests that the aromatic carbon structure of H 2 TPP-1CP is more electronic influenced by the conjugated plane of CRGO than H 2 TPP-4CP. Furthermore, the presence of only one functional group in H 2 TPP-1CP favors the interactions with the basal plane of the g p g π-π due to some less steric effects. The distance between basal plane and macrocycle could be then lower compared to the H 2 TPP-4CP (see, SI 2, Table S2).

Raman spectroscopy shows significant charge-transfer effects, through -interactions between aromatic structure and the graphene basal plane. First, the variation of the intensity ratio of I D / I G gives a good information of molecules association (where the D-band corresponds to sp 3 -hybridized carbon and the G-band to the vibration of sp 2 -bonded carbon atoms of graphene) (Figure 3C,a). The interaction of CRGO with H 2 TPP-1CP or H 2 TPP-4CP rises the intensity ratio of I D / I G suggesting an increase of sp 3 domain upon association of porphyrin. In addition, a downshift of the graphene G-band at 1600 cm -1 in the case of H 2 TPP-1CP/CRGO and at 1596 cm -1 with H 2 TPP-4 P/ G gg πinteractions between porphyrin and CRGO. It is known that the G-band shifts to lower-frequencies or higher frequencies depending on the electronic properties of the aromatic molecules as donor or acceptor. Indeed, Müllen and Feng [27] observed a downshift by 5 cm -1 of the G-band of graphene sheets after the attachment of electron-donor pyrene-1-sulfonic acid and an upshift after anchoring the electron acceptor perylene tetracarboxylic diimide bis-benzenesulfonic acid. In our case, the downshift observed for the G bands in nano-hybrids is related to electron donor properties of the two porphyrins due to the presence of alkoxy groups attached on the porphyrin macrocycles.

Figure. 3. FT-IR spectra of (a) CRGO, (b) H 2 TPP-nCP, and (c) H 2 TPP-nCP/CRGO [(A) H 2 TPP-1CP and (B) H 2 TPP-4CP] (C) Raman spectra of (a) CRGO, (b) H 2 TPP-1CP/CRGO and (c) H 2 TPP-4CP/CRGO of powder

The modification of the fermi level of graphene could be also observed by XPS in C1s position. It was demonstrated that depending on the electron-donating or withdrawing groups, charge transfer leads to respectively upshifts or downshifts in C=C peaks [54] . The devolution of C1s bands reveals mainly three components presented in CRGO films: C=C at 284.6 eV, corresponding to sp 2 carbon in the basal plane of graphene sheets, C-O at 285.9 eV and C=O at 289.3 eV, coming from remained oxygen groups present in CRGO. Association with modified alkoxyphenyl-porphyrin with four carboxylic groups leads to upshifts in C=C by 0.4 eV (see SI 2.2, Figure S4). This variation corresponds to the charge transfer between the aromatic molecules and the graphene sheets and confirm the Raman results. The results obtained by spectroscopic analysis highlight that both porphyrins interact with graphene and have an effect in electronic properties of graphene.

Electrochemical characterization is powerful methods to demonstrates such effect.

Electrochemical characterization

Characterization with external redox marker by cyclic voltammetry

To check the electrochemical and surface properties of composite compared to CRGO, the electron transfer was analysis through redox properties of various external redox couples: [Fe(CN) 6 ] 3- /4-, [Ru(NH 3 ) 6 ] 2+/3+ and benzoquinone/hydroquinone. Inner-sphere redox marker [Fe(CN) 6 ] 3-/4-, was used to inform of the chemical surface properties and the nanostructure of graphene. Ruthenium hexaamine, [Ru(NH 3 ) 6 ] 2+/3+ is an outer-sphere redox system, which is surface insensitive, and its redox properties and its electron transfer ability are influenced more likely by the density of electronics state (DOS) of the nanomaterials. Benzoquinone was also selected as a redox marker, because it is sensitive to proton transfer and informs on the ionic properties of the surface.

The electron transfer of the film of nanomaterials deposit onto GCE, was analyzed by cyclic voltammetry (CV) in a solution containing the specific redox couple.

The electrochemical behavior of the composites was firstly performed with inner-sphere [Fe(CN) 6 ] 3-/4- (Figure 4A). T V w f Δ p of the redox marker of 117 mV and 517 mV for respectively composites associated H 2 TPP-1CP and H 2 TPP-4CP compared to CRGO. The redox process of the negatively charged redox marker becomes more difficult with the electrode surface due to the increase of the negative charges on the composite surface (from H 2 TPP-1CP/CRGO to H 2 TPP-4CP/CRGO) that promotes electrostatic repulsions. Besides, to evaluate the effects of the proton exchange provided by the attached porphyrins onto the graphene surface, pbenzoquinone/hydroquinone redox couple was employed (Figure 4B). Small shift of oxidation potential is observed that demonstrated the lower effect of proton transfer from acidic porphyrins.

Indeed, at a neutral pH range, the porphyrins are mostly in anionic forms.

When the outer sphere redox marker hexamine ruthenium, was employed, a remarkably higher electrochemical process and high reversibility is observed, particularly, for the hybrid H 2 TPP-4CP/RGO nanomaterial (Figure 4C). Indeed, the ruthenium complex is sensitive to the density of electronics state of the graphene. This result underlines the electronic effect related to the alkoxy substituents attached to the porphyrins as electron-donating groups, and it is in good agreement of results obtained in Raman spectroscopic measurements and XPS. The kinetic of electron transfer (k s ) with various redox markers could be measured following Nicholson method by measuring the peak-to-peak variation at various scan rate. This was achieved for all composites with the different redox probes to underline the relationships of structural effect on electronic properties (See SI 3.1 and SI 3.2 for details). The apparent coefficient diffusion (Do) of various electrolytes was also calculated to shows the ionic transfer on the different nanomaterials. The data are summarized in Table 1.

Table 1: Electrochemical parameters obtained for electrodes of various modifications

In the case of inner-sphere redox marker Fe(CN) 6 3-/4-, the k s values were calculated to be 0.22 cm s -1

for CRGO, which is close to the value obtained for RGO [55] . The value decreased drastically with the two composites H 2 TPP-1CP/CRGO and H 2 TPP-4CP/CRGO to respectively 0.002 and 0.0003 cm. s -1 .

This underlines the effect of negative charged coming from the carboxylate groups of the both porphyrins. The k s value, determined with benzoquinone/hydroquinone, shows small variation p g p p v p g ' k p b w porphyrins and redox marker.

In the case of inner-sphere redox marker [Ru(NH 3 ) 6 ] 2+/3+ , a small variation of ks was obtained for H 2 TPP-1CP/CRGO composite compared to CRGO. However, an increase was observed for composite with H 2 TPP-4CP/CRGO varying from 0.3 to 0.95 cm.s -1 . This result demonstrates clearly that the association of H 2 TPP-4CP porphyrin with graphene has an electronic effect on the CRGO due to the presence of four donating alkoxy groups. This behavior supports the data obtained in Raman measurement and proves that the association of porphyrins with donating effect increases the density of electronic states of composite and then the fermi level.

The calculated diffusion coefficient remains the same with the composite H 2 TPP-1CP/CRGO for all electrolyte and is much lower for the composite H 2 TPP-4CP/CRGO (Table 1). This highlights that the permeability of the redox complex is impeded by the number of the functional group attached in lateral position of tetraphenyl porphyrin.

Characterization through electrochemical impedance spectroscopy

Electrochemical impedance measurement (EIS) was performed in PBS free of redox markers to underline the electrical properties of the composites. Results are shown in Figure 5 (see SI. S3).

This indicates that the formation of hybrid nano-materials, with the association of H 2 TPP, enhances the charge transfer process. Comparing the variation of R ct value between CRGO and H 2 TPP-4CP/ CRGO, a small variation was observed. This can probably be attributed to less connection of dispersed nanosheets of nanohybrids. The values of capacitance were calculated from CPE (constant phase element)

and could be directly related to the thickness of the nanocomposites (See detail of calculus in SI.3.3).

The thickness of CRGO was evaluated of 0.7 nm which is in agreement with compact structure.

However, the value was calculated as 60 nm and 75 nm, respectively for H 2 TPP-1CP/CRGO and H 2 TPP-4CP/CRGO. From all these results, we can conclude that the electron transfer between the surface and the electrode is enhanced when these two porphyrins are attached to CRGO. In addition, attachment of porphyrins increase the nano-sheets domains factor compared to CRGO. These results underline also

f p p w G ' f w f b p p . H 2 TPP-1 P G g π-π b w p g p π-
conjugated CRGO basal plane, which presumably induce aggregates domains. However, H 2 TPP-4CP/CRGO forms complex with more hydrogen bonds and should be attached more likely on the edge of graphene favoring nano-sheets structures. This latter is achievable thanks to the presence of oxygen functions on the peripheries of graphene. Similar interactions were also observed with the 5-(4hydroxyphenyl)-10,15,20-triphenylporphyrin with graphene oxide [56] . Thus, no effect on the electron transfer of redox marker p-benzoquinone/hydroquinone was observed and however, the negatively charges on the surface of graphene provided by porphyrins prevent electron transfer for negatively charged redox marker. Two mechanisms of the H 2 TPP/CRGO association could be then proposed,

where the H 2 TPP-1CP and H 2 TPP-4CP were associ g π-π f p g p w the graphene sheet especially in the basal plane-like and the carboxyl groups interact through planeedge structure of graphene (Schema 2).

Schema 2: General presentation of interactions

In the case of electronics properties, it appears clearly from spectroscopic analysis and electrochemical measurements with outer-sphere redox marker that the density of fermi level of graphene are modified. This effect is higher in the case of the composite associated with H 2 TPP-4CP.

This can be explained by the presence of the large number of alkoxy chain attached to the tetraphenylporphyrin with donating effect favoring the increase of DOS in CRGO. The two nanocomposites are integrated into DNA sensors for electrochemical DNA detection and are compared.

Application to DNA biosensors

Comparison of Detection of on ssDNA/H 2 TPP-nCP/CRGO

The DNA biosensors, ssDNA/H 2 TPP-1CP/CRGO and ssDNA/H 2 TPP-4CP/CRGO were prepared.

Single stranded DNA probes (ssDNA) were covalently attached to the porphyrin through an amide link between an 5' p b g ps of the mono or tetrakis-(carboxylphenyl)porphyrin (H 2 TPP-1CP/CRGO or H 2 TPP-4CP/CRGO complex) as described previously [49] .

Hybridization reaction was performed by successive addition of complementary targets (cDNA) onto ssDNA/H 2 TPP-nCP/CRGO sensor. The evaluated concentrations of targets were ranging from 1 aM to 1 nM (10 -18 ~10 -9 M). Electrochemical detection of DNA targets was monitored by SWV in 1 mM benzoquinone (Figure 6 A, B). The choice of the redox marker is related to its insensitive properties with the various modified surface as demonstrated in (2.3) and could inform on the hybridation reaction on the surface. After each addition of concentration of cDNA targets, the redox current of benzoquinone species into hydroquinone decreases whatever the nature of the biosensor.

This electrochemical response is merely due to the formation of double-strand DNA structure on the electrode surface, which further blocks the diffusion of p-benzoquinone species onto the electrode. A plot of average current peak versus the concentration of complementary targets obtained for the two biosensors shows a significant variation with similar behavior (Figure 6C). Calibration curves show dynamic ranges from the concentration 10 -18 to 10 -12 M for biosensor ssDNA/H 2 TPP-1CP/CRGO (Figure 6C, curve a) and from 10 -18 to 10 -11 M for biosensors and ssDNA/H 2 TPP-4CP/CRGO (Figure 6C, curve b). The detection limit was calculated from linear part of the calibration curves and based to S/N 3, the value is 2×10 -18 M for biosensor ssDNA/H 2 TPP-1CP/CRGO and 7×10 -19 M for biosensor ssDNA/H 2 TPP-4CP/CRGO. Thus, the two biosensors demonstrate high sensitivity to DNA hybridization thanks to electronic and structural properties provided by the composite, high electron transfer ability, large surface area and high loading ability for covalent attached bioreceptor provided by carboxyl function. The small variation in sensing properties obtained between the two biosensors could due probably to the number of carboxylic functions on the surface that rise the density of immobilized ssDNA. The reproducibility of the DNA hybridization platform was also tested with measurements with four independent biosensors. Each sensing layer was freshly prepared. A relative standard deviation (RSD) of 3% value was estimated, which showed the high reproducibility of the biosensor layer.

The selectivity was evaluated by the detection of non-complementary target (nc-DNA) in all range of concentration and compared to results obtained in presence of complementary target (c-DNA) (Figure 7 D,E). A relative change of peak current in the case of the biosensor formed with H 2 TPP-1CP/CRGO is obtained with nc DNA (Figure 7D, curve b) compared to the result obtained with c-DNA (Figure 7D, curve a). The average variation of 30% is obtained for larger concentration of nc-DNA and since 10 atm notable variation in biosensor response was obtained with c-DNA compared to nc-DNA. This could be due to the non-specific adsorption of DNA to basal plane of graphene which remains non-modified by porphyrins. However, the biosensor formed with H 2 TPP-4CP/CRGO shows smaller variation when hybridization occurs with nc-DNA target (Figure 7E), with less than 15% for higher concentration of nc-DNA target and distinguish variation since 1atm c-DNA target. This result can be explained by the large amount of carboxylate functions on phenyl groups of H 2 TPP-4CP porphyrin attached to CRGO that leads to a negatively charged surface preventing the non-specific interaction. Thus, the H 2 TPP-4CP/CRGO nanomaterial appears as the best platform for limiting the non-specific interaction and with high sensitivity of detection where the atomolar concentration could be detected.

Detection of PCR samples of M.tuberculosis and SNP mutation

The polymerase chain reaction (PCR) samples of M.tuberculosis were composed of DNA fragment of 411 bases length from ropB gene with the wild-type sequence and mutated sequence with single nucleotide polymorphism (SNP) of TTG mutation. The hybridization reaction with the PCR sample with wild-type sequence (w-PCR) and the sample with TCG/TTG mutation (mt-PCR) were performed with the biosensor ssDNA/H 2 TPP-4CP/CRGO bearing wild-type ssDNA probe. The response of the biosensors after hybridization with the w-PCR sample and mt-PCR at 1fM concentration was performed by SWV (Figure 7A) and show that high variation is obtained with the complementary wt-PCR sequence compared to mt-PCR which present single mutation. Thus, hybridization with w-PCR sample leads to a decrease of current at 23 ± 2.5%, where hybridization with mt-PCR sample gives a small variation in current of 4 ± 1.9% (Figure 7B).

These results demonstrate that biosensor based on H 2 TPP-4CP/CRGO nanocomposite is able to measure DNA from PCR samples extracted from M.tuberculosis strains. The homogeneous distribution of ssDNA target on the surface allows the preparation of a high dense biosensor leading to high sensitivity at atomolar range. The residual negative charge on graphene surface provided by porphyrins prevent non-specific interactions without any other coating.

Conclusion

Carboxyalkoxyphenylporphyrins functionalized with one or four carboxylic groups were successfully synthesized and associated to CRGO by a simple and effective way. Stable association of CRGO with acidic alkoxyphenylporphyrins could be obtained, where the dispersion of CRGO as well as electronic properties are efficiently enhanced. We demonstrated that the association between H 2 TPP-1CP and CRGO is performed through π-π interactions between phenyl groups and graphene nano-sheet with formation of some aggregates. However, with the porphyrin bearing four carboxylic groups H 2 TPP-4CP, the anchoring of the macrocycle is favored at the peripheries of the graphene, through hydrogen bonds with hydroxyl or carboxylic groups of RGO. This association is in favor of macrocycle attachment on the edge structure and improves the electronic properties of graphene. In this case, the ks value of outer-sphere redox marker is arisen. Thus, we demonstrated that the non-covalent interactions between terta-phenylporphyrin bearing carboxylic groups and CRGO, lead not only to improve the dispersion and the electronic properties of CRGO but also allow the introduction of the functional groups on CRGO surface for further functionality. DNA biosensors was demonstrated and show that the nanocomposite formed with H 2 TPP-4CP.CRGO leads to LOD of 1atm and high selectivity is obtained thanks to carboxylic group on the surface and efficient interaction of porphyrins which improved electronic properties. Detection of PCR sample of M.tuberculosis rpob gene and mutated with SNP was successfully demonstrated. Thus, this study highlights the interest of the CRGO modification by modified porphyrins and the role of the chemical function such as alkoxy group and carboxylate and their number to obtain nanomaterials with new properties.

Experimental Section

Reagent

Graphene oxide was purchased from Aldrich. All others reagents are purchased from Sigma-Aldrich France. DNA probe and target are shipped from Eurogentec France. DNA probe are modified with six carbonyl chain bearing amine group at terminal position.

The DNA sequence for probe, complementary and non-complementary are probe NH 2 -C6 -5'-TCA-ATC-TCG-GGA-ATC-TCA-ATG-TTA-G-3', complementary target 5'-CTA-ACA-TTG-AGA-TTC-CCG-AGA-TTG-A-3' -p 5'-CTA-ACA-TTG-AGA-TTC-CGA-GAT-TGA-GAT-CTT-C-3'.

DNA probe specific for PCR products contained 18 bases with sequence is NH2-C6-5'CCG-ACT-GTT-GGC-GCT-GGG3'.

A thee-electrode cell was used and consisted of a glassy carbon electrode (GCE, 0.071 cm 2 ) as working electrode, a platinum as counter-electrode and Ag/AgCl as reference electrode. The redox marker solutions were prepared in 0.1 M of KCl with 5 mM [Fe(CN) 6 ] 3-/4 , 1 mM benzoquinone and 10 mM [Ru(NH 3 ) 6 ] 2+/3+ .

Synthesis of 5-[4-(carboxylbutyloxy)phenyl]-10,15,20-triphenylporphyrin (H 2 TPP-1CP)

This compound was synthesized in one step from the diphenylporphyrin according to the literature as described in a previous paper [49] .

Synthesis of 5,10,15,20-[4-(carboxylbutyloxy)phenyl]tetraphenylporphyrin (H 2 TPP-4CP)

The symmetrical tetraphenylporphyrin bearing four carboxylic groups (H 2 TPP-4CP, 9) is prepared from the 5, 10, 15, 20-tetrakis (p-methoxyphenyl)triphenylporphyrin ( 6) in three steps (Scheme 3) following the same procedure as H 2 TPP-1CP. Due to the symmetry of the porphyrin (6), the strategy applied for the synthesis is Adler and Longo method, in one step via a condensation reaction from four equivalents of pyrrole units and four equivalents of p-methoxybenzaldehyde in propionic acid.

Preparation of H 2 TPP/CRGO hybrid nanomaterial suspensions

The CRGO investigated in this work was obtained by chemical reduction of graphene oxide in DMF according the method described by Ai and coll [18] . The characterization of the efficiency of reduction process was performed through XPS and Raman spectroscopy and was discussed in supplementary materials (See SI 1, Figure S1, Table S1 and Figure S2).

The designated CRGO solution was prepared by dispersing 1 mg of CRGO (see synthesis in SI 1) in 1 mL DMF, and ultrasonicated for 1h. The H 2 TPP-1CP and H 2 TPP-4CP solutions were prepared by dispersing 0.1 mg of porphyrins in 1 mL DMF, separately, and ultrasonicated for 30 min. The hybrid nanomaterial was prepared by mixing a 0.1 mg.mL -1 solution of porphyrin in a 1 mg.mL -1 suspension of CRGO in DMF to obtain a ratio of 10/1 (w/w) of CRGO/H 2 TPP. This ratio graphene-porphyrin was optimized to give the best sensitive surface as we demonstrated in our previous work [48] . The two kinds of porphyrin/CRGO nanomaterial suspensions, named H 2 TPP-1CP/CRGO and H 2 TPP-4CP/CRGO, were prepared by mixing 10 µL of CRGO suspension (1 mg•mL -1 ) and 10 µL volume of H 2 TPP-1CP and H 2 TPP-4CP solutions (0.1 mg•mL -1 ) separately to obtain 10:1 weight ratio of CRGO to porphyrin, followed by sonication for 30 min.

Construction of DNA biosensor

A drop of 2 µL of the prepared 0.

DNA hybridization

The DNA hybridization of complementary 25-bases targets was performed by immersing each DNA sensor in solution of various concentration of DNA target of sequence model from hepatitis C gene for 40 min at 47 ºC. The target concentrations were added successively from 1 aM, 10 aM, 100 aM, 1 fM, 10 fM, 100 fM, 1 pM, 10 pM to 1 nM. After each incubation, the modified electrode was washed with distilled water for three times to eliminate no hybridized single strand DNA and then analyzed by SWV method in benzoquinone. The detection of non-specific interaction was conducted by immersing the electrode in solution of different concentration of non-complementary DNA target in PBS buffer from 1 aM, 10 aM, 100 aM, 1 fM, 10 fM, 100 fM, 1 pM, 10 pM to 1 nM and incubated in the same conditions as for complementary target. To check the reproducibility, the experiment was repeated four times with freshly prepared biosensors individually.

PCR samples detection

The DNA from PCR samples was denaturized by heating at 95 ℃ for 5 min, and then immersed in an ice bath for 1 min. The hybridization of wt-PCR samples and mut-PCR sample was performed by immersing the electrode previously modified with wt-DNA probe in solution of 1 fM wt-PCR sample for 40 min at 47 ºC. The same conditions were applied for the detection of the same concentration of mut-PCR.

Methods

FT-IR was performed by spectrometer Bruker FS66 equipped with ATR platinum Pike and MCT detector. UV-Visible spectra were realized with Spectrometer Carry and Raman spectroscopy was performed at room temperature with a Renishaw spectrometer, using a 532 nm laser physics argon laser focused on the sample with a DMLM Leica microscope with a 100x (NA=0.75) objective. XPS measurements were realised with a K Alpha spectrometer from Thermo-Fisher, using a monochromated X-A Kα 1486.6 V .

SEM images were recorded using a JEOL-2100F Scanning electron microscope and an internal charge coupled device (CCD) camera and AFM withTapping mode topography and phase imaging were accomplished using an Innova AFM Bruker with NanoDrive v8.02 software.

Electrochemical measurements were performed using an AUTOLAB PGSTAT 100

Figure 1 .

 1 Figure 1. SEM (A) and AFM (B) images of (a) CRGO , (b) H 2 TPP-1CP/CRGO and (c) H 2 TPP-4CP/CRGO.

Figure 2 .

 2 Figure 2. UV-visible spectra recorded after successive addition of 1 mg/mL DMF solution of CRGO ((0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 μL) to porphyrin solution [(A) H 2 TPP-1CP and (B) H 2 TPP-4CP] in EtOH. (C, D) The affinity constant calculation.

Figure 4 .

 4 Figure 4. CV measurements of GCE modified electrodes with (solid curve) CRGO, (dot) H 2 TPP-1CP/CRGO, and (dash dot) H 2 TPP-4CP/CRGO (A) in 5 mM [Fe(CN) 6 ] 3-/4-(B) in 1 mM benzoquinone and (C) in 10 mM [Ru(NH 3 ) 6 ] 2+/3+ , scan rate 50mV s -1

  3.3 for details). The ESI data shows a lower impedance after H 2 TPP-1CP and H 2 TPP-4CP association which directly correlated to a decrease of the charge transfer (R ct ). The value of R ct w 2 TPP-1CP/CRGO and H 2 TPP-4C/CRGO (Table

Figure 5 .

 5 Figure 5. Nyquist plots measured in PBS solution at applied potential -0.3V with Dc potential 10mV at frequency range [0.01Hz -100 KHz] (a) CRGO, (b) H 2 TPP-1CP/CRGO and (c) H 2 TPP-4CP/CRGO, Inset: equivalent circuit. R s electrolyte solution resistance.

  electrochemical analysis system controlled by NOVA software (Methrom). The three-electrode cell was purchased from BASI and consisted of glassy carbon electrode (GCE) (surface 0.071 cm2) as working electrode, a platinum as counter-electrode and Ag/AgCl as reference electrode. The analysis was performed by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). from I2BC University Paris-Saclay for providing PCR sample and Dr Ali Madouri from C2N CNRS for RAMAM analysis.List of Figures and Schemes(B) Figure. 1. SEM (A) AFM (B) images of (a) CRGO, (b) H 2 TPP-1CP/CRGO and (c) H 2 TPP-4CP/CRGO.

Figure. 2 .

 2 Figure. 2. FT-IR spectra, (A,B) Raman spectra, (C) of CRGO (a), H 2 TPP-1CP (b), and H 2 TPP-

Figure. 3 .

 3 Figure. 3. UV-visible spectra recorded after successive addition (0μL, 2μL, 4μL, 6μL, 8μL, 10μL, 12μL, 14μL, 16μL, 18μL, and 20μL) of 1 mg/mL CRGO in DMF to porphyrin solution in ethanol [(A) H 2 TPP-1CP and (B) H 2 TPP-4CP]. (C, D) The curve of variation of optical density versus concentration of RGO for the complex with H 2 TPP-1CP (C) and H 2 TPP-4CP (D).

Figure 4 . 1 0Figure 5 .

 415 Figure 4. CV measurements of GCE modified electrodes with (solid curve) CRGO, (dot) H 2 TPP-1CP/CRGO, and (dash dot) H 2 TPP-4CP/CRGO (A) in 0.1M KCl solution with 5 mM [Fe(CN) 6 ] 3-/4- (B) in 1 mM benzoquinone and (C) in solution of 10 mM [Ru(NH 3 ) 6 ] 2+/3+ , scan rate 50mV s -1

Figure 6 .

 6 Figure 6. SWV experiments during detection of biosensors based on (A) H 2 TPP-1CP/CRGO and (B) H 2 TPP-4CP/CRGO at various concentration of targets (1 aM to 1 nM); (C) Plot of the relative changes of the current peaks versus concentration of DNA targets on the (a) H 2 TPP-1CP/CRGO and(b) H 2 TPP-4CP/CRGO; Plot of the relative changes of the current peaks versus concentration of (a)

Figure 7 .

 7 Figure 7. Detection of wt PCR and mut PCR sequence in benzoquinone (A) SWVs measurements scan rate 12.5 mV.s -1 , (a) ssDNA/H 2 TPP-4CP/CRGO; (b) mut PCR; and (c) wt PCR; (B) Histogram comparing hybridization signal intensities for detection of 1 fM of wt PCR and mut PCR sample. Data points are the mean values ± RSD obtained with three independent experiments.

Fe

  CO 3(1.46 mmol, 20 eq) under argon for stirring 2h at room temperature. And then 0.17 ml of 5bromopentanenitrile was added (1.46 mmol, 20 eq) into the solution and the mixture was heated at 60 °C during 2 days. The solution was concentrated under vacuum and extracted with CH 2 Cl 2 and washed with H 2 O. The organic phase was then dried over Na 2 SO 4 and concentrated to give 25 mg (34%) of the crude product (8) and was directly used for the next step.1 H NMR (300MHz, CDCl 3 ): δ 8.87 [8H, d,

	Schema 3. Synthesis of H 2 TPP-4CP (9) (i) BBr 3 , DCM, rt, 18h, 86%; (ii) CsCO 3 , 5-bromovaleronitrile DMF, 60°C, 24h, 34%; (iii) dioxane, HCl 12M, 80°C, 12h, 32% 5,10,15,20-(4-hydroxyphenyl) tetraphenylporphyrin (7) Boron tribromide (1.16mL, 22 eq.) was added at 0°C onto 5,10,15,20-(4-methoxyphenyl) tetraphenylporphyrin (6) (400 mg, 54 mmol) dissolved in 80 mL CH 2 Cl 2 under argon. The crude solution was mixed during 18 hours at room temperature. The solution was then basified with a saturated Na 2 CO 3 solution and washed with water until the neutrality. The organic phase was then dried over Na 2 SO 4 and concentrated to give 319 mg (86%) of the crude product (7). Compound 7 was directly used in the next step without any purification. 1 H NMR (250MHz, CDCl 3 ): δ 8.88 (m, 8 H, pyrrol-H meso ), 8.24 (m, 6 H, Ar-H), 8.07 (d, 3 J H,H = 8.5 Hz, 2H, Ar-H), 7.80 (m, 9H, Ar-H), 7.12 (d, 3 J H,H = 8.25 Hz, 2H, Ar-H), -2.06 (ls, 2H, NH). 5,10,15,20-[4-(valeronitrile)phenyl] tetraphenyl-porphyrin (8) On a solution of porphyrin (7) (50 mg, 0.073 mmol, 1 eq) in DMF (25 mL) was added 0.48g Hz], 4.3 [2H, t, J=5.55Hz], 2.6 [2H, t, J=6.75Hz], 2.19 [4H, m],-2.74 [2H, s]; HRMS (MALDI-MS) m/z calcd. for C 64 H 58 N 8 O 4 , M= 1002,45, found 1002.47. FT-IR (ATR) 2093 cm -1 (ν C≡N ). 5,10,15,20-[4-(carboxylbutyloxy)phenyl] tetraphenyl-porphyrin (9) Nitrile was hydrolyzed by HCl 12M and afforded carboxylic porphyrin 9. On a solution of porphyrin (8) (100 mg, 0.1 mmol) in 4 mL dioxanne, was added 1.8 mL of a concentrated HCl solution. The reaction was running vigorously at 80 °C during 12 hours. Dichloromethane was added and the crude mixture was washed with water, dried over Na 2 SO 4 . Crude product was obtained with 32% of yield (35 mg) as a pure powder. 1 H NMR (300MHz, CDCl 3 ): δ 8.9 [8H, m]; 8.3 [6H, m]; 8.2 [2H, d, J= 8.1 Hz]; 7.8 [9H, m]; 7.2 [2H, d, J= 7.5Hz]; 4.2 [2H, t, J=4.8 Hz]; 2.6 [2H, t, J= 6.9Hz]; 2.0 [4H, m]; -2.7 [2H, s, NH]. HRMS (APCI MS) m/z calcd. for C 64 H 62 N 4 O 12 , (M+H) + 1079.22, found Cs 2 J=7.8 Hz], 8.24 [6H, d, J=6.9 Hz], 8.14 [2H, d, J=8.4 Hz], 7.77 [9H, t, J=6.6 Hz, Ha], 7.28 [2H, d, J=6 1079.22. UV-vis spectrum in DMF: λ max , 419 nm; 515 nm; 551 nm; 592 nm; 647 nm.

  1 mg mL -1 H 2 TPP-1CP/CRGO or H 2 TPP-4CP/CRGO suspension was deposited onto the glassy carbon electrode surface followed by drying step under the lamp, and rinsing with distilled water. Finally, the H 2 TPP-1CP/CRGO or H 2 TPP-4CP/CRGO was activated in a freshly prepared 10 mM solution (pH=7.4) PBS of 20 mM EDC, 32 mM NHS. The activation step was running for 1h at room temperature. Then the electrode was immersed in 1µM solution of ssDNA probe prepared in PBS buffer for 1h at room temperature. Subsequently, the obtained DNA sensor based on H 2 TPP-1CP/CRGO or H 2 TPP-4CP/CRGO was thoroughly rinsed with distilled water and stored in PBS at 4 °C before use. After each step of construction of the DNA sensor, the surface modifications were controlled by SWV method in benzoquinone.

Table 2 :

 2 Electrochemical parameters obtained for redox marker analyzed with electrodes modified with various nanomaterials.

		Do (10 -6 cm 2 s -1 )	k s (cm.s -1 )	Do (10 -6 cm 2 s -1 )	k s (cm.s -1 )	Do (10 -6 cm 2 s -1 )	k s (cm.s -1 )
	CRGO	5.43	0.22	1.17	0.01	8.96	0.3±0.03
			±0.02		±0.001
	H 2 TPP-	6.11	0.002	0.99	0.02	9.72	0.15±0.03
	1CP/CRGO		±0.0002		±0.001
	H 2 TPP-	2.47	0.0003	0.56	0.01	3.92	0.95±0.02
	4CP/CRGO				±0.001

Schema 1. Schematic representation of modified material

Acknowledgements

The authors are grateful to the Chinese government and the university Paris-Saclay for financial support (Chinese Scolarship Council -No. 201408420128). The authors are grateful to Prof C. Sola


Mariona Ferrandiz-Rovira

Thierry Lengagne
email: thierry.lengagne@univ-lyon1.fr

Clara Superbie

Irene Figueroa

Coraline Bichet

Bernat Claramunt-Lopez

Aurélie Cohas

James F Hare
email: james.hare@umanitoba.ca

Dan Blumstein

Roberto A Delgado
email: roberto.delgado@hunter.cuny.edu

Irene Figueroa3

Mariona Ferrandiz
email: mariona.ferrandiz@uab.cat

Mariona Orcid

Ferrandiz

Rovira Id

Geographic variations in marmots' alarm calls cause different responses

Keywords: acoustic communication, dialect, genetic differentiation, geographic variation, alarm call

Population differences in acoustic signals, have been investigated for five decades to better understand the evolution of communication. When receivers are able to discriminate among signals and to react accordingly, geographic differences can have major impacts on the ability of conspecifics to communicate. Surprisingly, population differences in alarm calls and their consequences on the communication process have been so far neglected despite their crucial role on individuals' survival. Working with four wild populations of Alpine marmots (Marmota marmota), we found differences in the acoustic structure of their alarm calls. These differences can neither be explained by geographic, nor genetic distances but rather by other mechanisms including random processes. Moreover, playback experiments provided evidence that receivers discriminate among alarm calls from their own versus other populations, with responses being lower in intensity when the call bout played back originated from their own population. Research on the mechanistic causes of geographical markers and on the relationship between geographical variation, reliability of the signal and behavioural responses are now required to better understand how predation pressure and natural selection could drive the evolution of communication.

Introduction

Considerable geographic differences among populations in acoustic signals, usually termed dialects, geographic markers or geographic variations [START_REF] Conner | Dialects versus geographic variation in mammalian vocalizations[END_REF], have been documented across the whole animal kingdom (in mammals [START_REF] Lameira | Review of geographic variation in terrestrial mammalian acoustic signals: Human speech variation in a comparative perspective[END_REF]; in birds [START_REF] Krebs | Repertoires and geographical variation in bird song[END_REF]; in anurans [START_REF] Velásquez | Geographic variation in acoustic communication in anurans and its neuroethological implications[END_REF]; in fish [START_REF] Parmentier | Geographical variation in sound production in the anemonefish Amphiprion akallopisos[END_REF]; in invertebrates [START_REF] Zuk | Geographical variation in calling song of the field cricket Teleogryllus oceanicus: the importance of spatial scale[END_REF]; for review, see (Wilczynski and Ryan 2001). This signal variation could arise from various processes, such as genetic and/or cultural drifts between isolated populations [START_REF] Baker | Genetic population structure and vocal dialects in Zonotrichia (Emberizidae)[END_REF]Davidson and Wilkinson 2001;[START_REF] Mundinger | Microgeographic and macrogeographic variation in the acquired vocalizations of birds[END_REF][START_REF] Janik | Traditions in mammalian and avian vocal communication[END_REF], and are particularly relevant to our understanding of the divergent evolution in communication systems [START_REF] Campbell | Geographic variation in the songs of neotropical singing mice: testing the relative importance of drift and local adaptation[END_REF]Wilczynski and Ryan 2001). Signals have been shown to be influenced by many factors, including geographic barriers [START_REF] Thomas | Variations in underwater vocalizations of Weddell seals (Leptonychotes weddelli) at the Vestfold Hills as a measure of breeding population discreteness[END_REF][START_REF] Cleator | Underwater vocalizations of the bearded seal (Erignathus barbatus)[END_REF][START_REF] Thomas | Geographic variation in leopard seal (Hydrurga leptonyx) underwater vocalizations[END_REF][START_REF] Perry | Variation of harp seal (Pagophilus groenlandicus) underwater vocalizations among three breeding locations[END_REF], environment (, habitat structure, background noise [START_REF] Hunter | Geographical variation in the song of the great tit (Parus major) in relation to ecological factors[END_REF][START_REF] Wiley | Adaptations for acoustic communication in birds : sound transmission and signal detection[END_REF][START_REF] Van Parijs | Vocalizations and movements suggest alternative mating tactics in male bearded seals[END_REF]Nichols and Goldizen 2006)) and sexual selection (e.g., female preferences [START_REF] Slater | The cultural transmission of bird song[END_REF])).

Signal geographic variation implies a structural variation from the emitter point of view but must also involve detection and discrimination processes by the receiver to have a major impact on the ability of conspecifics to communicate. For instance, an extensive literature on songbirds shows that dialects can strongly affect breeding behaviour, especially mate attraction and intra-sexual competition [START_REF] Searcy | Geographic song discrimination in relation to dispersal distances in song sparrows[END_REF].

Many studies suggest that individuals discriminate among songs from conspecific populations, preferring the signal of their local population in a context of mate choice and intra-sexual competition [START_REF] Baker | Genetic population structure and vocal dialects in Zonotrichia (Emberizidae)[END_REF][START_REF] Searcy | Geographic song discrimination in relation to dispersal distances in song sparrows[END_REF][START_REF] Gray | Does courtship behavior contribute to species-level reproductive isolation in field crickets?[END_REF][START_REF] Boul | Sexual selection drives speciation in an Amazonian frog[END_REF][START_REF] Podos | Discrimination of geographical song variants by Darwin's finches[END_REF]Nichols 2008;[START_REF] Uy | Plumage and song differences mediate species recognition between incipient flycatcher species of the Solomon Islands[END_REF]Bradely et al. 2013;[START_REF] Mortega | Behavioural response of a migratory songbird to geographic variation in song and morphology[END_REF][START_REF] Lin | Behavioural response of the greater horseshoe bat to geographical variation in echolocation calls[END_REF]; even in close populations [START_REF] Leader | Dialect discrimination by male Orange-Tufted sunbirds (Nectarinia osea): reactions to own vs. neighbor dialects[END_REF]; but see [START_REF] Colbeck | Asymmetric discrimination of geographical variation in song in a migratory passerine[END_REF][START_REF] Danner | Female, but not male, tropical sparrows respond more strongly to the local song dialect: implications for population divergence[END_REF]. Dialects can then act as pre-zygotic barriers and play an important role in speciation [START_REF] Baker | The biology of bird-song dialects[END_REF][START_REF] Slabbekoorn | Bird song, ecology and speciation[END_REF][START_REF] Price | Speciation in birds[END_REF][START_REF] Wilkins | Evolutionary divergence in acoustic signals: causes and consequences[END_REF]. Similar patterns and processes have been suggested in mammals, although evidence is much more limited [START_REF] Maeda | Locale-specific vocal behaviour of the tamarin (Saguinus I. labiatus)[END_REF].

Studies investigating geographic variation in acoustic signals and its perception in behavioural contexts other than sexual selection in avian species remain scarce [START_REF] Charrier | Bearded seal males perceive geographic variation in their trills[END_REF]. Alarm calls, shaped by natural selection, could be as relevant to speciation as acoustic sexual signals because of their direct consequences on individuals' survival. Despite this crucial role, dialects in alarm calls have been so far poorly investigated because they are not as complex as bird nuptial and territorial songs.

Although some of them are sophisticated (e.g., syntax in mobbing calls [START_REF] Suzuki | Experimental evidence for compositional syntax in bird calls[END_REF]; complex signal structure in monkey screams [START_REF] Zuberbühler | Survivor signals: the biology and psychology of animal alarm calling[END_REF])), alarm calls are often a repetition of one same call unit [START_REF] Randler | Heterospecifics do not respond to subtle differences in chaffinch mobbing calls: message is encoded in number of elements[END_REF]. Both the structure of the note and its repetition rate can be used to encode information [START_REF] Manser | The acoustic structure of suricates' alarm calls varies with predator type and the level of response urgency[END_REF] see Rendall et al. 2009). Nevertheless, it has been shown that alarm calls with highly stereotyped structures can convey information about the age or the sex of the emitter (Blumstein and Muñoz 2005). Geographic differences in alarm calls have been documented in several mammalian species [START_REF] Slobodchikoff | Geographic variation in alarm calls of Gunnison's prairie dogs[END_REF][START_REF] Matrosova | The between-population variation of the alarm call in the speckled ground squirrel (Spermophilus suslicus, Rodentia, Sciuridae): effects of sex, age and body mass[END_REF]Schlenker et al. 2014[START_REF] Francescoli | Geographic variation in vocal signals of Ctenomys pearsoni[END_REF]Eiler and Banack 2004) but were found sometimes absent in yellow-bellied marmots, Marmota flaviventris [START_REF] Blumstein | Alarm calling in yellow-bellied marmots: I. The meaning of situationally variable alarm calls[END_REF]. When present, their perceptual salience remains unknown [START_REF] Zuberbühler | Survivor signals: the biology and psychology of animal alarm calling[END_REF]. Hence the importance of such variation for the communication process is currently unknown and further playback experiments are necessary to determine their biological relevance.

We investigated the existence of structural differences in alarm calls produced by Alpine marmots (Marmota marmota) from two native (Alps) and two reintroduced (Pyrenees) populations, and whether receivers discriminate among them. Alpine marmots are cooperatively breeding ground-dwelling territorial squirrels living in family groups. Territory surveillance is insured by all individuals from a family and, once a predator is detected, marmots can produce alarm calls warning other individuals before hiding in their burrows [START_REF] Perrin | Socio-spatial organization and activity distribution of the Alpine marmot Marmota marmota -preliminary results[END_REF]. Marmot alarm calls are usually composed of one stereotyped and frequency modulated single note [START_REF] Perrin | Socio-spatial organization and activity distribution of the Alpine marmot Marmota marmota -preliminary results[END_REF].

The comparison between the native populations in the Alps and the reintroduced populations in the Pyrenees is of particular interest to investigate the relationship between signal divergence and genetic differentiation, as well as their consequences in terms of between-population recognition. We first investigated whether geographic variations are encoded in the acoustic structure of alarm calls produced by marmots from the four studied populations. Although dynamic interplay between song learning mechanisms and geographic isolation have been evidenced to be at the origin of dialects in birds songs [START_REF] Podos | The evolution of geographic variation in birdsong[END_REF], the processes underlying the evolution of alarm call dialects remains to be described. Moreover, due to the fact that songs in oscines are usually learned [START_REF] Kroodsma | The diversity and plasticity of birdsong[END_REF]), song dialects have been found to result from short-term, cultural, or ecological processes rather than to have a genetic basis (e.g., [START_REF] Halfwerk | A behavioural mechanism explaining noisedependent frequency use in urban birdsong[END_REF][START_REF] Ruegg | Divergence in mating signals correlates with ecological variation in the migratory songbird, Swainson's thrush (Catharus ustulatus)[END_REF]. Conversely, alarm calls structure have a substantial genetic basis [START_REF] Blumstein | The evolution, function, and meaning of marmot alarm communication[END_REF][START_REF] Blumstein | Ontogenetic variation of heritability and maternal effects in yellow-bellied marmot alarm calls[END_REF]) and consequently, cultural drift is unlikely to occur. Therefore, we expect to observe a strong relationship between alarm call divergence and genetic differentiation between the two reintroduced populations, which are geographically and genetically isolated from the two native populations [START_REF] Bichet | Multiple geographic origins and high genetic differentiation of the Alpine marmots reintroduced in the Pyrenees[END_REF]. We also conducted playback experiments to determine whether receivers perceived the differences in alarm calls produced by different populations and changed their anti-predatory response.

Methods

Ethical Note

Fieldwork was conducted under permit number AP n82010/121 by the Préfecture de la Savoie. All handling and sampling were done by three co-authors who are authorized for experimentation with animals by the French Ministry of Agriculture and Fisheries or Catalan Government (diplomas 0ETRY20090520, R45GRETAF110 and 53707-UAB-FELASA). The protocol was approved by the ethical committee of the University of Claude Bernard Lyon 1 (n8BH2012-92 V1).

Study populations

We studied Alpine marmots from four populations (Supplementary Material S1):

Sassière and Tignes are native populations of the Western Alps located 10 km apart, while Cerdanya (La Bastida) and Ripollès (Pardines) are reintroduced (from 1948(from until 1988(from , Couturier 1955;;[START_REF] Ramousse | Twenty years of re-introduction policy of alpine marmots from the national park of La Vanoise (French Alps)[END_REF]) populations situated 500 km away from the first ones in the South-eastern Pyrenees and which are 45 km apart. Ripollès is genetically closer to the two native populations than Cerdanya [START_REF] Bichet | Multiple geographic origins and high genetic differentiation of the Alpine marmots reintroduced in the Pyrenees[END_REF].

Within each population, intensive behavioural observations allowed us to precisely identify family groups and to locate main burrows and territory borders. To conduct genetic analyses, 151 unrelated individuals were captured in these four populations (all the details about the sampling and the genetic analyses are given in the Supplementary Material).

Does the acoustic structure of alarm calls differ between populations?

Each year (2011)(2012)(2013)(2014), alarm calls were recorded in the four populations between mid-April and mid-July from 8:00 a.m. to 6:00 p.m. the main activity period of the marmots.

An omnidirectional microphone (Sennheiser ME62-K6P) connected to a Fostex FR2LE recorder (frequency sampling: 44.1 kHz, resolution: 16 bits) was placed approximately 2 m from a main burrow entrance in order to maximize the chances to record alarm calls. Once a recording was performed, we moved to another territory not adjacent to the previous one to record another individual (Sassière: N = 38 alarm calls; Tignes: N =31; Cerdanya: N = 92 and Ripollès; N =35), Marmots typically retreated into their burrows during the setup of the recording material but they re-emerged within few minutes. Then, individuals were exposed to a threatening situation in order to trigger the emission of alarm calls by a focal individual: a human or a human with a domestic dog (Canis lupus familiaris) appeared promptly from a hide situated 60 to 80 m away from burrows and ran in the direction of the marmot. This protocol worked in almost all cases. Alarm callers were aged up to 3 years (when they reach adult size and stop growing), which enabled us to distinguish three age classes: pups, yearlings and adults.

Since body size and age might impact frequencies of vocalizations neither pups' nor yearlings' calls were analysed [START_REF] Tubaro | Acoustic frequencies and body mass in New World doves[END_REF][START_REF] Blumstein | Individual, age and sex-specific information is contained in yellow-bellied marmot alarm calls[END_REF] but see [START_REF] Matrosova | Pups crying bass: vocal adaptation for avoidance of age-dependent predation risk in ground squirrels?[END_REF]). As marmots are monomorphic, it was not possible to sex the recorded emitters. Hence, if sex variation is present in the alarm call of Alpine marmot, there will likely be "noise" in our analysis. A sex difference could bias our analysis at the population level in case of (i) an acoustic difference among male and female which is currently not known in Alpine marmot, (ii) a large difference in the proportion of male and female responding to alarm call playback in our studied populations, and, (iii) a reverse proportion of males and females recorded according to populations. Although an absence of literature does not mean that sex does not play a role in alarm calls. Currently, we do not have evidence for none of the above, but we are confident that sex information did not bias our analysis.

Alarm call recordings were analysed using Avisoft SASlab Pro (version 5.2.09).

They were low-pass filtered to remove all noise corresponding to low frequency (i.e. less than 1 kHz, mainly wind noise, Hamming windows, FFT 1024pts). The number of calling bouts recorded varied between individuals and whenever it was possible we processed a maximum of 6 alarm calls per calling bout. From the 38 calling bouts from Sassière, the 31 from Tignes, the 92 from Cerdanya and the 35 from Ripollès, we selected 137, 143, 459 and 145 alarm calls, and described the modulation of their fundamental frequency. For each call, we performed a spectrogram (FFT 1024pts, overlap 93.75%, Hamming windows, frequency resolution, 47Hz) and we then researched the frequency modulation pattern through a zero crossing analysis. All analysis were performed with the same settings (i.e. same measurements accuracy). The same person (CS) measured on zero crossing the initial, maximum and final frequencies (in kHz) and the durations (in ms) of the ascendant (AD), stationary (SD) and descendant (DD) phases of the fundamental frequency (Fig. 1). By doing so, we avoided to measure parameters which are highly altered during the propagation.

Statistical analyses

To test for differences in the acoustic structure of alarm calls between the four populations, we fitted four linear mixed-effect models with either the ascendant phase (AD), the stationary phase (SD), the descendant phase (DD) duration or the initial frequency (F1) as the response variable, the population as a fixed effect and the calling bout as a random effect on the intercept. The maximum and final frequencies were discarded due to their strong correlation with the other variables (see Table 1).

We then tested for potential differences in the acoustical structure (AD, SD, DD and F1) of alarm calls among the four populations using a Linear Discriminant Analysis (LDA). To further compare each pair of populations, we built six other LDAs. To overcome bias due to uneven sample size between populations, we randomly selected N calls per population without replacement with N equal to the number of alarm calls of the population with the smallest sample size involved in the comparison.

Reclassification rates using a cross validation were then calculated with half the data set the training samplerandomly selected to build the model and the other halfthe test sampleused to calculate the percentage of correct classification. The entire procedure, including the random selection of N calls/population, was repeated 1,000 times. We calculated the mean of all obtained percentages of correct classification and their 95% confidence intervals. We assumed that the acoustic structure differed between the populations if the percentage of correct classification corresponding to a random classification (%R) was not included in the estimated 95% confidence intervals.

Then, we tested whether the acoustic distance (log-transformed) between populations correlated with either the genetic distance (linearized FST=FST/(1-FST)) or the geographic distance (log-transformed) using Mantel tests (all permutations, Mantel 1967) implemented in the package vegan [START_REF] Oksanen | Vegan R package version 2.5-2[END_REF]. Acoustic distances between populations were quantified as the pairwise differences in the linear discriminant functions. The genetic distances were quantified using pairwise FST values [START_REF] Weir | Estimating F-statistics for the analysis of populationstructure[END_REF] and the geographic distances as the linear distance in kilometres (see the Supplementary Material S2).

Does marmots' response to alarm calls vary depending on the population of origin of the emitter?

Between mid-May and mid-July, 2015, a playback experiment was carried out on marmots from La Sassière and Tignes from 8:00 a.m. to 6:00 p.m. Each individual was tested once with a call from Cerdanya, Sassière or Tignes. Forty-three tests were performed in La Sassière (16, 17 and 10 tests with alarm calls from La Sassière, Tignes and La Cerdanya respectively) and 34 tests were done in Tignes (12, 8 and 13 tests with alarm calls from Tignes, La Sassière and La Cerdanya respectively). To limit pseudoreplication, we picked up and broadcasted 7 recordings of calls (See previous section "Does the acoustic structure of alarm calls differ between populations?") for each of the three populations used. We chose signals with a high signal to noise ratio so as the response observed cannot be due to eventual uniqueness of one soundtrack.

Moreover to avoid a neighbour-stranger effect we chose carefully the sound track used for each test to ensure that it was recorded in another part of each population, away from the tested marmot (at least 500m). To avoid habituation of the tested marmots due to repetitions of trials over a short time, the same calling bout was never displayed more than once to a given territory and two neighbouring territories were never tested successively.

Each playback bout was composed of 5 alarm calls within 3 s, mimicking an average natural bout (measurement performed on 30 bouts recorded in our study). The amplitude of alarm calls of Alpine marmots has not been measured accurately before (i.e. the precise distance between the emitter and the sound level meter, taking the exact position of the head of the marmot into account). Hence, we decided to match by ear the amplitude of the calls used during our test to a natural call. Alarm calls were emitted at 100 dB(C) using a speaker (SMC8060 Beyma amplified loudspeaker) connected to a Fostex FR2LE, measurement done with a Lutron SL-4001, C weighting, slow settings). In one case over 77, more than a single animal was present, we thus conducted the statistical analyses with and without this trial but given that the results were qualitatively identical and quantitatively similar, we chose to kept this experiment in the analyses. We collected the occurrence (coded as a binary outcome) of vigilance, flight/running, entrance into a burrow, time spent vigilant and time until the focal individual resumed foraging. Vigilance behaviour was defined as any posture where marmots were standing on their rear feet, or standing on their four feet but suddenly putting their head up and maintaining it above the horizontal plane of their body.

Flight/running was defined as an escape-related behaviour towards a burrow entrance and was considered to be a more extreme response than any vigilance posture, without necessarily entering in the burrow.

Statistical analyses

To test whether the response to alarm calls depends on the origin of the signal, the occurrence of flight and of the entry in a burrow were entered as response variables in two generalized linear models (GLMs) with a logit link and the variance given by a binomial distribution. Since in nearly all playback trials (72 over 77), individuals became vigilant, the frequency of this behaviour could not be considered.

The time spent vigilant and the time before resuming a normal activity were further entered as response variables in two other GLMs with a logarithmic link and the variance given by a Gamma distribution. In the last model, we categorized the intensity of the response of the focal individual in four categories: no response, vigilance only, flight and entry in a burrow; and we entered this ordinal variable in an ordered logistic regression model. In each of these five models, we first tested whether the responses were different when the playback alarm call originated from the population of the focal individual or from another population by entering the origin of the playback as a two-modalities factor (same or other population) in interaction with the population of the focal individual (Tignes or Sassière) as explanatory variables. Second, we tested whether the geographic distance between the focal individual and the signaller could further impact the responses by entering the origin of the playback as a three-modalities factor (same, close, far) in interaction with the population of the focal individual (Tignes or Sassière) as explanatory variables. All analyses were performed using the R software (Version 3.1.1) and packages 'nlme' [START_REF] Pinheiro | Correlated evolution of morphology and vocal signal structure in Darwin's finches[END_REF], 'multcomp' (Hothom et al. 2008), 'ade4' [START_REF] Dray | The ade4 package: implementing the duality diagram for ecologists[END_REF], 'MASS' [START_REF] Venables | Modern Applied Statistics with S. Fourth Edition[END_REF].

Results

Does the acoustic structure of alarm calls differ among populations?

Marmot alarm calls from the four populations differed significantly in their initial frequencies (F1, 190 = 4.75, P < 0.01), ascendant phases' durations (F3, 190 = 11.58, P < 0.001), stationary phases' durations (F3, 190 = 9.57, P < 0.001) but not in their descendant phases' durations (F3, 190 = 2.45, P = 0.07).

Despite a strong overlap among the four populations when compared altogether (Supplementary Material S3), alarm calls were always assigned more often to the population their emitter originated from than to any other population. Effectively, calls were correctly classified in 41. 96 [35.71; 48.35] % of the cases (while the percentage of random classification would have been 25%). When populations were compared two by two, alarm calls were once again always better attributed to the population their emitters originated from than to the other one (percentage of random classification: 50%).

Percentages of correct classification ranged from 62.49 [54.17; 69.79] % to 71.64 [63.70; 79.41] % when comparing Tignes with Ripollès and Tignes with Cerdanya respectively (Table 2). All the acoustical variables (initial frequencies, ascendant, stationary and descendant phases' durations) contributed to the discrimination among the populations, but they seemed contribute differently to the differentiation between each pair of populations (Fig. 2).

The acoustic distances between two populations was neither explained by their genetic distance (Spearman = -0.77, N = 6, p = 0.92) nor by their geographic distance (Spearman = -0.71, N = 6, p = 1.00). Moreover, there is no evidence of genetic isolation by distance in our four populations (i.e. the genetic distances and the geographic distances were not correlated, spearman's Rho = 0.43, N = 6, p = 0.17).

Does marmots' response to alarm calls vary depending on the population of origin of the emitter?

Marmots exhibited lower intensity responses to an alarm call from their own population than to an alarm call from another population (β = -1.23 ± 0.52, z = -2.40, N = 76, p = 0.02). The odds of a marmot showing a higher response decreased by a factor of 0.29 [0.10; 0.78] when the alarm call originated from its own population compared to another one. More specifically, marmots showed a significantly lower propensity to flee (β = -1.45 ± 0.58, z = -2.50, N = 76, p = 0.01, fig. 3-a) when the alarm call originated from their own population than from nearby versus geographically remote population.

Although the rest of our results were not significant, marmots tended to be less likely to enter their burrow (7.14 ± 1.86% vs. 16.67 + 6.67%, β = -0.96 ± 0.83, z = -1.15, N = 76, p = 0.25, fig. 3-b), to remain vigilant for a shorter time (median = 23.37s vs. 34.58s, β = 0.002 ± 0.006, z = 0.40, N = 74, p = 0.68, fig. 3-c), and to resume a normal activity more rapidly (median = 24.88s vs. 39.35s, β = 0.003 ± 0.005, z = 0.49, N = 68, p = 0.62, fig. 3-d) when the alarm call originated from their own population. The intensity of the response was similar for alarm calls produced in a nearby or in a geographically distant population (odds ratio = 0.73 [0.19, 2.64], β = -0.31 ± 0.66, z = -0.47, N = 76, p = 0.64).

The propensity to flee (β = -0.20 ± 0.83, z = -0.24, N = 76, p = 0.81, fig. 3-a), to enter in a burrow (near: 88.00 + 2.64% vs. far: 78.26 + 3.91%, β = -0.71 ± 0.80, z = -0.89, N = 76, p = 0.37, fig. 3-b), the amount of time spent vigilant (near = 34.58s vs. far = 31.9s, β = -0.02 ± 0.33, z = -0.06, N =74, p = 0.95, fig. 3-c) and elapsed time before resuming a normal activity (near = 38.16s vs. far =39.35s, β = -0.001 ± 0.006, z = -0.29, N =68, p = 0.77, fig. 3-d) did not vary with the geographic distance between the focal and the other population.

Discussion

In the present study we described for the first time the acoustic structure of alarm calls produced by adult Alpine marmots in two native and two reintroduced wild populations.

We found that the acoustic structure of Alpine marmots alarm calls differed among the four populations, which enabled us to assign calls described by four acoustic parameters to their population of origin greater than by chance. Neither the genetic distance nor the geographic distance explained the acoustic differences between populations. Finally, the playback experiments provided evidence that receivers discriminate among alarm calls from their own versus other populations. Surprisingly, intensity of marmots' responses was lower when the playback calling bout originated from their own population than when it came from another population. Again, these responses did not differ depending on the genetic nor on the geographic distances.

Although alarms calls are a stereotyped signal, we found that the acoustic structure of Alpine marmots alarm calls differ among the four studied populations. Indeed, using four acoustic parameters to describe each alarm call it was possible to assign the signal to the population their emitters originated from with accuracy exceeding that expected by chance. The presence of geographic markers in acoustic signals have been shown repeatedly in bird songs [START_REF] Nottebohm | The song of the chingolo, Zonotrichia capensis, in Argentina: description and evaluation of a system of dialects[END_REF][START_REF] Mundinger | Microgeographic and macrogeographic variation in the acquired vocalizations of birds[END_REF]Zimmermann et al. 2016). The richness of avian vocal repertories offer a wide range of possibilities for differentiation of geographic markers: changes in note combinations, presence or absence of some notes (particularly during new population finding) and/or rhythm of emission of different notes [START_REF] Baker | Founder effect and cultural evolution of songs in an isolated population of chaffinches, Fringilla coelebs, in the Chatham Islands[END_REF][START_REF] Handford | Trill rate dialects in the Rufous-collared Sparrow, Zonotrichia capensis, in northwestern Argentina[END_REF][START_REF] Shieh | Experimental evidence that distinct song phrases in the Grey-cheeked Fulvetta Alcippe morrisonia permit species and local dialect recognition[END_REF]. Nevertheless, such markers have also been found within single stereotyped acoustic elements, e.g. those produced by bottlenose dolphins, Tursiops spp. [START_REF] Campbell | Quantitative comparison of bottlenose dolphin (Tursiops spp.) whistles from three geographic regions[END_REF]. Similarly to [START_REF] Campbell | Quantitative comparison of bottlenose dolphin (Tursiops spp.) whistles from three geographic regions[END_REF] study, we found that the general shape of acoustic elements, as short as 0.2s, differed among marmot populations.

We further found a clear difference in the shape of alarm calls used by two populations originating from two mountain ranges but also between populations separated by only 5 km (i.e., Sassière and Tignes). Acoustic divergence is well documented at a large spatial scale [START_REF] Lougheed | Vocal dialects and the structure of geographic variation in morphological and allozymic characters in the rufous-collared sparrow, Zonotrichia capensis[END_REF]Wilczynski and Ryan 1999). For instance, [START_REF] Shizuka | Range-wide patterns of geographic variation in songs of Golden-crowned Sparrows (Zonotrichia atricapilla)[END_REF] reported the existence of 13 discrete song types in golden-crowned sparrows (Zonotrichia atricapilla) over 3,100 km in Alaska. But, geographic variations in acoustic signals can also be observed at a microgeographic scale. [START_REF] Leader | Song dialects do not restrict gene flow in an urban population of the orange-tufted sunbird, Nectarinia osea[END_REF] documented dialects in orange-tufted sunbird (Nectarinia osea) within two sub-populations separated by a sharp boundary but only 100 m apart from each other. Studies conducted at these different geographic scales still remain scarce in mammals. At least two studies working on a phylogenetically close species to the Alpine marmot, the Gunnison's prairie dog (Cynomys gunnisoni), have documented large or microgeographic differences in acoustic signals [START_REF] Slobodchikoff | Geographic variation in alarm calls of Gunnison's prairie dogs[END_REF][START_REF] Perla | Habitat structure and alarm call dialects in Gunnison's prairie dog (Cynomys gunnisoni)[END_REF].

Genetic distance has been repeatedly hypothesized to explain acoustic differences among populations (Wilczynski and Ryan 1999). However, in the present study, we did not find any correlation between the acoustic and the geographic distances or between the genetic and the acoustic distances. The peculiar status of our studied populations is unlikely to explain such a lack of relationship: the two reintroduced Pyrenean populations originated from Alpine populations that were farther away both geographically and genetically than the two Alpine populations considered here [START_REF] Bichet | Multiple geographic origins and high genetic differentiation of the Alpine marmots reintroduced in the Pyrenees[END_REF]. While [START_REF] Balaban | Bird song syntax: learned intraspecific variation is meaningful[END_REF] found a correlation between signal characteristics and genetic distance in two populations of swamp sparrow (Melopsiza georgiana), a lack of congruence in divergence of acoustic signal and genetic characteristics has been repeatedly observed in birds [START_REF] Hafner | Song dialects and gene flow in the white-crowned sparrow, Zonotrichia leucophrys nuttalli[END_REF][START_REF] Payne | A genetic and behavioral analysis of mate choice and song neighborhoods in indigo buntings[END_REF][START_REF] Wright | Population genetic structure and vocal dialects in an amazon parrot[END_REF].

Four main hypotheses (i.e., learning, morphology and body mass differences, antipredator strategies and local adaptation) have been proposed to explain the occurrence of dialects. The learning hypothesis attributes acoustic differences among populations to the colonization of a new area by young individuals before they have learned the song structure from their parents [START_REF] Thielcke | On the origin of divergence of learned signals (songs) in isolated populations[END_REF]. [START_REF] Baker | Founder effect and cultural evolution of songs in an isolated population of chaffinches, Fringilla coelebs, in the Chatham Islands[END_REF] invoked this cultural bottleneck to explain the presence of dialects in isolated populations of chaffinches. However, an interspecific cross-fostering experiment with ground squirrels failed to provide evidence for the learning hypothesis [START_REF] Matocha | Vocal communication in ground squirrels, genus Spermophilus[END_REF].

Although the failure to learn allospecific calls does not preclude learning the nuances of conspecific calls, the learning process is probably not very important in marmots.

Moreover, the founders caught for the reintroduction events in Pyrenees were all adults.

For all these reasons, the learning hypothesis is unlikely to explain geographic variation in the acoustic structure of alarm calls found in our study.

Among other factors that could lead to geographic variation, indirect selective pressures on acoustic signals may alter vocalization characteristics (Podos 2001). In Alpine marmot, mechanistic processes may cause the alarm call divergence. For instance, significant differences in both morphology and body mass between two (Sassière and Cerdanya) of the four studied populations have been found (Ferrandiz-Rovira et al, in prep). Mechanical links between morphology and acoustic signals exist in many groups (e.g. in anurans [START_REF] Hoskin | Ecology and taxonomy-driven deviations in the frog call-body size relationship across the diverse Australian frog fauna[END_REF]Lengagne 2017), in birds [START_REF] Nowicki | Vocal tract resonances in oscine bird sound production: evidence from birdsongs in a helium atmosphere[END_REF]Fletcher and Tarnopolski 1990)). For example, a study conducted on Darwin's finches showed that the adaptive evolution of beaks for feeding also influences the acoustic structure of their songs (Podos 2001). Although less investigated, such link has also been found for alarm calls in the speckled ground squirrel (Spermophilus suslicus, [START_REF] Matrosova | The between-population variation of the alarm call in the speckled ground squirrel (Spermophilus suslicus, Rodentia, Sciuridae): effects of sex, age and body mass[END_REF]. Nevertheless, further studies are needed to firmly establish a direct link between morphology and alarm call structure.

Antipredator strategies such as the use of alarm call is also known to strongly interact with predator dangerousness and predation risk [START_REF] Dutour | Mobbing behaviour varies according to predator dangerousness and occurrence[END_REF][START_REF] Dutour | Mobbing behaviour in a passerine community increases with prevalence in predator diet[END_REF]. Indeed, signal characteristics will differ according to predator characteristics (Zuberbülher, 2009). For instance, mustached tamarins (Saguinus mystax) produce different alarm calls for aerial versus terrestrial predators [START_REF] Kirchhof | Functionally referential alarm calls in tamarins (Saguinus fuscicollis and Saguinus mystax) evidence from playback experiments[END_REF] or for the level of perceived threat [START_REF] Coss | Threat-related acoustical differences in alarm calls by wild bonnet macaques (Macaca radiata) elicited by python and leopard models[END_REF]). In the present study, this is unlikely to explain the observed marmots' geographic variation because alarm calls have been elicited by a human approach at the same distance from burrows (60-80m) and hunting was forbidden in the different marmot populations.

In agreement with the local adaptation hypothesis, dialects could also result from the optimization of sound transmission within local environment along various transmission channel [START_REF] Morton | Ecological sources of selection on avian sounds[END_REF]. Signal alteration depends on vegetation cover density, atmospheric turbulence, or height above the ground at which a signal is transmitted [START_REF] Wiley | Physical constraints on acoustic communication in the atmosphere: Implications for the evolution of animal vocalizations[END_REF][START_REF] Lengagne | Acoustic communication in a king penguin colony: importance of bird location within the colony and of the body position of the listener[END_REF]. Moreover, information transfer efficiency is linked to the intensity and the quality of the background noise [START_REF] Ryan | The role of body size phylogeny and ambient noise in the evolution of bird song[END_REF][START_REF] Lengagne | The effects of rain on acoustic communication: tawny owls have good reason for calling less in wet weather[END_REF]. In birds, a study on dialect suggested that call structure have been shaped by local propagation conditions [START_REF] Doutrelant | Two distinct song populations of blue tit Parus caeruleus in the French mediterranean[END_REF]) and another study with Gunnison's prairie dog emphasized a link between alarm call structure and habitat characteristics: geographic variant calls resulted in significant differences in transmission performance through different environments [START_REF] Perla | Habitat structure and alarm call dialects in Gunnison's prairie dog (Cynomys gunnisoni)[END_REF]. Nevertheless, in our case, although we did not measure vegetation coverage, the predominant vegetation formation consisted in high altitude alpine meadows which should be quite similar from a population to another. Vegetation is thus unlikely to affect alarm call propagation here. Hence, a minimal effect of the propagation conditions on the evolution of alarm call structure in the four studied populations is expected in agreement with previous studies on marmots conducted by [START_REF] Daniel | A test of the acoustic adaptation hypothesis in four species of marmots[END_REF]. Background noise level, one major component of the transmission channel also influence signal evolution. One way to improve signal efficiency in such case is to avoid overlapping with low frequency corresponding to background noise and thus to modify signal spectrum towards high pitched frequencies. Anthropogenic effects on alarm calls had already been hypothesized to explain differences in alarm calls' dialects of speckled ground squirrels [START_REF] Matrosova | Genetic and alarm call diversity across scattered populations of speckled ground squirrels (Spermophilus suslicus)[END_REF]. We observed important noise differences among the studied populations. The "Tignes" population is located in a mountain resort and could be considered as disturbed by anthropogenic noises (traffic, helicopter…). However, we did not observe higher, minimum or maximum frequencies in the alarm calls recorded in Tignes compared to the other studied populations for which there was no evidence of environmental noise.

In addition to direct or indirect selection processes we cannot preclude the fact that acoustic differences observed among populations were due to a random process.

Consistent with this explanation, we found that multiple sets of acoustical variables stoodd out to discriminate between each pair of populations. This lack of consistency suggests that evolution of alarm call characteristics may not be explained by a unique selective process.

As fleeing in response to alarm calls should provide a selective advantage by increasing survival, one predicts that marmots should react to all alarm calls despite discrimination abilities (but see trade-off between vigilance and foraging, [START_REF] Lima | Behavioral decisions made under the risk of predation: a review and prospectus[END_REF]. Surprisingly, our tests revealed that receivers perceived acoustic differences and categorized alarm calls as local or non-local calls. Such behavioural consequences imply that these acoustic differences are meaningful for them [START_REF] Soha | Non-salient geographic variation in birdsong in a species that learns by improvisation[END_REF]. Most studies focused on male territorial defence or female attraction in birds and their results are contrasted with stronger response to local songs in some cases, absence of preferences or mixed responses in others (see [START_REF] Becker | The coding of species-specific characteristics in bird sounds[END_REF][START_REF] Catchpole | Bird song[END_REF].

In an alarm context, experimental approaches with playbacks emphasized that intensity of animal response varies according to the past reliability of the signaller [START_REF] Cheney | Assessment of meaning and the detection of unreliable signals by vervet monkeys[END_REF])) or to the familiarity of the members of the colony [START_REF] Hare | The song remains the same: Juvenile Richardson's ground squirrels do not respond differentially to mother's or colony member's alarm calls[END_REF]. Nevertheless, no study has been conducted so far to determine whether animals discriminate and react differently to conspecific alarm calls according to its population (i.e. geographical origin). Our results thus show for the first time that animals discriminate among calls recorded in different populations and react accordingly. We expected that a divergence in the alarm signal induce an alteration of alarm information and, according to [START_REF] Hanson | Age differences in the response of California ground squirrels (Spermophilus beecheyi) to conspecific alarm calls[END_REF], we expected a reduced behavioural response to alarm calls of non-local congeners in tested animals. Surprisingly, behavioural responses obtained after playback of alarm calls from different populations showed the reverse. Indeed, animals reacted more strongly to playback of alarm calls originating from foreign populations. Moreover, the intensity of the response was similar for alarm calls produced in a nearby or in a geographically distant population suggesting that these signals represent an equally threatening situation for animals. Our results may be explained by a difference in the predator pressure or predator strategy [START_REF] Dutour | Mobbing behaviour varies according to predator dangerousness and occurrence[END_REF]. Even though we did not measure predator pressure precisely, the two main predators of marmots, the golden eagle (Aquila chrysaetos) and the red fox (Vulpes vulpes), were present in the four studied population. Hence, predator pressure is unlikely to explain differences observed in marmot responses to playback. Previous experiments on rodents showed that they discriminate alarm calls from neighbours (i.e. familiar) versus unfamiliar individuals. In the present study, marmots displayed a stronger response to alarm calls originating from foreign populations compared to their native one. Nevertheless, this familiar versus unfamiliar discrimination process cannot explain our results because all animals were tested with unknown signals even when they were tested with calls of their own population. Our results may be explained by the fact that marmots estimate the information reliability received and reacted accordingly. [START_REF] Blumstein | Reliability and the adaptive utility of iscrimination among alarm callers[END_REF] observed a stronger response to unreliable than reliable alarm calls in yellow-bellied-marmot (Marmota flaviventris) and suggests that such difference was due to the fact that a marmot hearing an unreliable alarm call makes its own independent assessment of relative risk and thus invests more in antipredator behaviour (but see opposite results in Richardson's ground squirrels Spermophilus richardsonii:

Hare and Atkins 2001).

The presence of geographic markers in acoustic signals involved in a context of mate choice and intra-sexual competition have been repeatedly found in avian species [START_REF] Nottebohm | The song of the chingolo, Zonotrichia capensis, in Argentina: description and evaluation of a system of dialects[END_REF][START_REF] Mundinger | Microgeographic and macrogeographic variation in the acquired vocalizations of birds[END_REF]Zimmrmann et al. 2016). Both random and selective processes linked to sexual selection that may act via genetic or cultural transmission pathways could explain their origin. In the context of alarm, further research on the proximate causes of geographical markers as well as on the relationship between geographical markers, reliability of the signal and behavioural responses are now required to better understand how predation pressure and natural selection could drive the evolution of communication.

Electronic supplementary material

The online version of this article contains supplementary material, which is available to authorized users.

TABLES AND TABLE LEGENDS

  This amplitude is closed to the value measured on yellow-bellied marmot Marmota flaviventris (95-100 dB[START_REF] Lea | Age and sex influence marmot antipredator behavior during periods of heightened risk[END_REF].The speaker was placed on the ground in the upper part of the focal territory directed towards the main burrow entrance. Marmots typically retreated into their burrows during the installation but they re-emerged within minutes. Once a marmot was between 5 m and 10 m away from any burrow entrance and displayed a normal activity such as foraging, an observer placed outside the focal family group triggered the alarm call playback (beginning of the trial). At the same time, observers filmed the focal adult individual of the focal family group with a digital video camera (Sony® Handycam model DCR-DVD650 or JVC® digital video model GZ-E 209). The trial was considered completed when the focal individual entered into a burrow, resumed a normal activity (i.e., foraging) or 5 minutes after the beginning of the trial. All video recordings were displayed in AVS Video Editor (version 7.1) in slow motion (x0.25) by a unique observer, blind to playback treatment, to ensure an accurate identification of behaviours as well as to record their duration with an accuracy of 0.01s.

FIGURES

  FIGURES

Figure 1

 1 Figure 1

Figure 2

 2 Figure 2

832 833 Table 1

 8331 Pearson's correlation coefficients between acoustic features (N = 894 alarm 834 calls). *: 0.10 < P > 0.05; **: 0.05 < P > 0.001; ***: P < 0.001.

	835
	836
		Maximum	Final	Ascendant	Stationary phase	Descendant
		frequency	frequency	phase		phase
	Initial frequency	0.55***	0.67***	-0.17***	-0.02 (P=0.58)	0.09**
	Maximum frequency		0.74***	0.36***	0.08**	0.30***
	Final frequency			0.09**	0.07*	0.03 (P=0.36)
	Ascendant phase				0.05 (P=0.11)	0.30***
	Stationary phase					0.01 (P=0.84)

Table 2

 2 Percentage of correct classification and acoustic structure (AD, SD, DD and F1) correlations with the Linear Discriminant Analysis (LDA) canonical axis of the LDAs comparing each pair of studied populations. N: number of calls; AD: ascendant phases' durations; SD: stationary phases' durations; DD: descendant phases' durations; F1: initial frequency.

							Pairwise
		Mean correct	Mean correlation [95% CI] of acoustical structures with the first canonical axis of each	differences
	Compared populations	classification [95%			LDA		in LD
		CI] (%)					functions
			AD	SD	DD	F1
	Sassière vs. Tignes (N=137)	65.03 [56.04; 73.63] 0.35 [ 0.30; 0.40] -0.51 [ -0.56; -0.47]	0.77 [ 0.73; 0.80]	0.19 [ 0.13; 0.25]	44.45
	Cerdanya vs. Ripollès (N=145)	65.13 [56.25; 73.96] 0.69 [ 0.58; 0.78] 0.61 [ 0.48; 0.72]	0.13 [-0.04; 0.31]	-0.24 [-0.41; -0.07]	42.21
	Sassière vs. Cerdanya (N=137)	64.41 [56.04; 72.53] 0.63 [ 0.51; 0.75] 0.58 [ 0.47; 0.68]	-0.18 [-0.35; -0.01] -0.65 [-0.75; -0.54]	41.10
	Sassière vs. Ripollès (N=137)	62.95 [54.95; 71.43]	0.04 [-0.02; 0.09]	0.06 [ 0.01; 0.12]	-0.36 [-0.41; -0.30] -0.52 [-0.56; -0.47]	43.08
	Tignes vs. Cerdanya (N=153)	71.64 [63.70; 79.41] 0.90 [ 0.84; 0.95] 0.13 [-0.02; 0.28]	0.50 [ 0.38; 0.61]	-0.47 [-0.58; -0.35]	41.85
	Tignes vs. Ripollès (N=145)	62.49 [54.17; 69.79] 0.47 [ 0.43; 0.51] -0.57 [ -0.61; -0.53]	0.55 [ 0.51; 0.59]	-0.38 [-0.43; -0.33]	44.56

Acknowledgements

We thank all the students involved in marmot recording. We are grateful to the golf of Tignes for allowing us to work on its green. We are deeply grateful to the commune of Tignes for the use of the Santel chalet. In the Pyrenees, we warmly thank all the people involved in field campaigns and special thanks to A. Planella and M. Unzeta (supported by a COLAB grant from the Catalan Government). MFR thanks the Generalitat de Catalunya (2017 SGR 1006). We further acknowledge James F. Hare, Dan Blumstein and two anonymous reviewers for helpful and constructive comments and suggestions that helped us to improve a previous version of this paper.

Agència de Gestió d'Ajuts Universitaris i de Recerca (2017 SGR 1006)

FIGURE LEGENDS