Maxime Couture

Thibault Brulé

Stacey Laing

Wenli Cui

Mitradeep Sarkar

Benjamin Charron

Karen Faulds

Wei Peng

Michael Canva

Jean-Francois Masson
email: jf.masson@umontreal.ca

High figure of merit (FOM) of Bragg modes in Au-coated nanodisk arrays for plasmonic sensing

Keywords: Gold-coated nanodisk array, high FOM, multi-well plate reader, SERS, plasmonic sensing, Bragg mode

We report that gold-coated nanodisk arrays of nearly micron periodicity have high figure of merit (FOM) and sensitivity necessary for plasmonic refractometric sensing, with the added benefit of suitability for surface-enhanced Raman scattering (SERS), large scale microfabrication using standard photolithographic techniques and a simple instrumental setup. Gold nanodisk arrays were covered with a gold layer to excite the Bragg modes (BM) which are the propagative surface plasmons localized by the diffraction from the disk array. This generated surface-guided modes, localized as standing waves, leading to highly confined fields confirmed by a mapping of the SERS intensity and numerical simulations with 3D finite element method (3D FEM). The optimal gold-coated nanodisk arrays were applied for refractometric sensing in transmission spectroscopy with better performance than nanohole arrays and they were integrated to a 96-well plate reader for detection of IgY proteins in the nM range in PBS.

The potential for sensing in biofluids was assessed with IgG detection in 1:1 diluted urine. The structure exhibits a high FOM of up to 46, exceeding the FOM of structures supporting surface plasmon polaritons (SPPs) and comparable to more complex nanostructures, demonstrating that sub-wavelength features are not necessary for high performance plasmonic sensing.

Introduction

The field of plasmonics has generated great interest in the past years due to a broad applicability for a series of important contemporary issues. 1 Specifically, the usefulness of plasmonics has been shown for biomedical, environmental and food safety applications. [2][3][4] While the potential of plasmonic sensing has been shown for clinical sensing 5 , several issues remain to be addressed. For example, most sensors reported early on relied on a single measurement channel or low throughput. A significant number of applications require the measurement of multiple markers or reaching higher analytical throughput. Therefore, the field is rapidly moving towards higher multiplexing of plasmonic sensors [6][7][8][9] . To reach this goal, a high reproducibility must be achieved for a series of parameters, which include reproducible sensing performance of the device (plasmonic materials and fluidics), robust surface chemistry, repeatable immobilization of bioreceptors, optimization of the analytical conditions, and exquisite control on the external factors such as bulk properties of the sample and temperature among other factors. As one of the essential parameters to reaching the potential of plasmonic devices, the fabrication and characterization of reproducible nanostructures with high sensitivity are subject to intensive research activities [10][11][12] . Plasmonic substrates support surface plasmons (SP), which are coherent oscillations of free electrons at the interface of a metal and a dielectric. 13 SPs are sensitive to refractive index variation, which allows a suite of plasmonic techniques to be used to detect binding events at the vicinity of the sensor's surface, hence the source of the general applicability and popularity of the technique to detect (bio)molecules. The coupling of light with SP in the classical technique of surface plasmon resonance (SPR) generates SPs that propagates on a thin gold film. 1,3,14 Other coupling methods of light with SPs involve the use of nanoplasmonic structures such as 2D grating structures (in reflection or in transmission) or through resonances with colloidal nanoparticles leading to localized surface plasmon resonance (LSPR). 1 2D grating structures can be fabricated using several lithographic techniques [15][16][17] . While highly regular and reproducible, these fabrication techniques often necessitate infrastructure that can be costly to operate. However, they offer high sensitivity and currently constitutes a vibrant research field.

Colloidal nanoplasmonics benefit from simple synthetic methods of the nanomaterials, which can then be self-assembled into more complex and functional architecture using self-assembly 10,18 . Colloidal nanostructures are also suited for the naked eye detection 19 , leading to simple design of sensors. The integration in lab-on-a-chip devices thus constitutes a significant advantage of 2D and colloidal nanoplasmonic materials [20][21][22] .

Thereby, nanoplasmonic structures can solve current limitations of classical SPR, such as low-throughput capabilities for analyzing large numbers of samples and difficulty in miniaturization of equipment, 23 and are highly sensitive for use in surface-enhanced spectroscopy such as SERS and metal enhanced fluorescence (MEF). [START_REF] Stiles | Annual Review of Analytical Chemistry[END_REF] The identification of nanoplasmonic structures exhibiting advantageous analytical properties is central to the development of the next generation of nanoplasmonic devices.

Different nanostructures have been proposed to achieve highly sensitive and high figure of merit (FOM) plasmonic sensing. Colloidal nanostructures supporting LSPR have been widely popular for sensing due to recent advances in synthesis of metal colloids. [START_REF] Grzelczak | [END_REF][26][27] These nanostructures are characterized with strong extinction in the visible to nearinfrared (NIR) spectral region, moderate sensitivity to bulk refractive index, but high sensitivity to molecular adsorbates in plasmonic sensing and in SERS. These structures are of different shapes and sizes such as nanorods, 28 nanospheres, 29 nanocubes, 30 or nanostars, 31 among others. Arrays of individual nanostructures have also been fabricated on solid substrates with properties similar to colloids in suspension, including arrays of nanotriangles, 32 nanorings 33 , nanocrescents 34 , or nanodisks 35 among others. Since the first report on nanodisks 36 , they have been increasingly used for various applications in plasmonics [37][38][39][40][41] . Alternatively, there has also been a lot of interest in developing nanostructures that support propagating SPs. These nanostructures typically have higher bulk refractive index sensitivity than LSPR, higher FOM (FOM = bulk RI sensitivity / full width at half-maximum (FWHM)) and achieve better refractive index resolution. 42 For example, nanoslits 43 , nanovoids 44 , nanohole arrays 45 , and nanogratings 46 have been reported for plasmonic sensing. Despite all these significant advances in nanostructure design, it remains one of the contemporary challenges to develop a reproducible nanostructure with high sensitivity, high FOM and broad applicability in a series of plasmonic techniques.

To address these challenges, we studied gold-coated nanodisk arrays and report the generation of sharp resonances in transmission spectroscopy measurements in the near-infrared (NIR) region of the spectrum ideal for biosensing applications. In periodic structures with an underlying metallic film, the Bragg Modes (BMs) are excited due to the change of the in-plane wavevector of the PSP (kPSP) owing to the periodicity of the array. For this work, we selected a nanodisk array structure with a large periodicity (1200 nm) and disk diameter (720 nm) where resonant coupling of the localized and propagative plasmons are not expected to occur for the wavelengths used in this work.

Advantages of working with larger structural parameters include tuning of the resonance in the so-called biological window in the NIR and the possibility to fabricate the arrays by conventional photolithography. Microfabrication on a large scale with high surface uniformity is highly desirable for integration of plasmonic structures in devices. While most plasmonic structures reported excel in few of the analytical parameters of importance for sensing using refractometric (such as sensitivity, FWHM, FOM, RI resolution, S/N, and reproducibility among others) and surface-enhanced spectroscopies, we report excellent analytical properties of the gold-coated nanodisk arrays in this series of important parameters.

Results and Discussion

Gold nanodisk arrays are usually fabricated by electron beam lithography (EBL) and exhibit sub-wavelength features. 47,48 The fabrication of gold-coated nanodisk arrays was based on the same approach we used in a recent paper to manufacture nanohole arrays of the same periodicity (Figure 1). 49 In brief (detailed experimental procedures are provided below), the microfabrication of nanodisk arrays was achieved by exposing UV light through a chromium mask on a positive photoresist, which was spin-coated on a 4" glass wafer. The mask consisted of an array of chromium disks with a periodicity of 1200 nm and a diameter of 720 nm. After the exposition step, the wafer was developed and a nanohole arrays pattern was produced on the glass wafer. Thereafter, the wafer was metalized with a layer of chromium (1 nm) and a layer of gold (10 to 75 nm) was deposited to yield a nanodisk array on the wafer. Then, the photoresist was lifted off the wafer to display the array of gold nanodisks. The last step of the process consisted of coating another layer of gold (10 to 75 nm) onto the nanodisk arrays. The gold-coated nanodisk arrays were named for their nanodisk height (NDXX, where XX is the height in nm) and their gold film thickness (filmYY, where YY is the thickness in nm), thus a substrate with disks of 75 nm and a film of 75 nm is termed ND75film75. This fabrication technique generated gold-coated nanodisk arrays on a large area (SEM image provided in Figure 1), with great speed and high surface uniformity (<10% variation).

We first studied the optical properties of the nanostructures. The localized plasmon resonance of the isolated disks was measured at 1.6 m for free nanodisks.

When the array was coated with a gold layer, the plasmon resonance of the disk was expected to shift, but not significantly. Thus, we did not expect any localized surface plasmon resonance of the gold-coated nanodisk arrays in the working wavelengths (Visible-NIR) used for this article. Thus, we did not consider any further coupling possibilities of the PSP and the localized plasmon resonances of the disks. 50,51 The 75 nm layer of gold below the nanodisk arrays (disk height of 75 nm) led to a series of plasmonic bands associated to BM in the visible-NIR region (Figure 2). The plasmon resonances were excited when the wavevector of the PSP matched the wavevector of the diffracted orders from the array. The equation for the condition of exciting the diffracted orders (i,j) is given in terms of the excitation wavelength (), dielectric constants () of the metal (m) and solution (d), the refractive index of the glass support (D) and incidence angle () as

λ = P √ 4 3 (i 2 + ij + j 2 ) ( √ ε m ε d ε m +ε d -η D sinθ) (1)
λ = P √ 4 3 (i 2 + ij + j 2 ) (√ ε m ε d ε m +ε d -η D sinθ (i cos(ϕ) + j cos(ϕ + 60) + i sin(ϕ) + 𝑗 sin(ϕ + 60))) (2)
These plasmonic modes highly depend on the periodicity (P) of the array rather than the nanodisk shape. 52 Equation 1 is valid only for hexagonal arrays of nanostructures such as used in this article and considered a rotation angle of 0 o (Figure 1) 49 , while equation 2 considers all rotation angles (ϕ), also for hexagonal arrays. Analytical calculations using equation 1 were then carried out to estimate the BM coupling conditions and to determine the position of the different orders of BM (Figure 2). The predicted resonances calculated with equation 1 were in good agreement with the experimental positions of the BM for different orders (Figure 2A). For example, the experimental resonance wavelength of 641 nm was also in good agreement with the 3D FEM prediction at 644 nm. Based on equation 1, the plasmonic resonances observed in air for the incidence angles of 0° and 5° were assigned to the (1,1) and (2,-1) modes

(shifting to shorter wavelengths) and the (-1,-1) and (-2,1) modes (shifting to longer wavelengths) (Figure 1). We must mention here that the current experiments were not performed with polarized light, which is common for sensing applications of plasmonic nanostructures. However, polarization has been previously shown to influence the spectra of disordered nanohole arrays 53 . In that case, p-polarization showed angular dependence, which was not observed for s-polarization due to the electric field orientation in the plane of the nanostructure for the latter case. These results showed that a large number of BM can be excited with the gold-coated nanodisk arrays and the impact of the excitation conditions on these resonances.

The sample was then rotated 90 o to demonstrate the effect of rotation angle on the excitation of the BM and the difference in the plasmon dispersion curves (equation 2 and We then measured the plasmonic properties of 16 gold-coated nanodisk arrays with various disk heights and film thicknesses to evaluate the optimal excitation conditions of the BM. These different plasmonic structures were studied in transmission for a range of incident angles of 0° to 30° and all plasmonic dispersion curves are reported in figure S1. Equation 1 accurately predicted ( 12 nm) the resonances observed at an incidence angle of 0 o for the gold-coated nanodisk arrays of different heights and film thicknesses. This observation further supported the excitation of BM for the goldcoated nanodisk arrays of the different nanodisk heights and film thicknesses. In addition, the resonance of the gold-coated nanodisk arrays was invariant ( 5.5 nm) with the height of the nanodisk (10 to 75 nm). If a localized plasmon was excited in the NIR spectral range, the plasmon resonance would have strongly depended on the aspect ratio of the disk, as previously reported 54 , which was not the case for the gold-coated nanodisk arrays. However, the plasmon resonance slightly increased from 631 to 644 nm for thicker Au films (from 25 to 75 nm), which is also in agreement with a redshift observed for thicker Au films in classical propagative SPR measurement in the Kretschmann configuration. This observation is valid for symmetric modes, but it is important to mention that other modes can exist in finite films. We also observed that nanodisks of heights of 10 nm were insufficient to efficiently excite the BM, excepted with a film thickness of 75 nm for which a very weak resonance was observed at 640 nm, a resonance wavelength in agreement with other disk heights. Nanodisks of small heights failed to effectively diffract the surface plasmon and thus, light cannot couple to the propagating plasmon. Film heights on the order of 10 to 20 nm do not typically support propagating surface plasmons in classical SPR experiments using smooth films and the Kretschmann configuration of SPR. In agreement, we generally did not observe the excitation of the propagating surface plasmon of gold-coated nanodisk arrays with 10 nm Au films, with the exception of nanodisks of 50 nm, for which, a weak resonance was observed. Overall, these results demonstrated that sufficient thicknesses (50 and 75 nm)

of the gold film and nanodisks are the necessary conditions to strongly supports a propagative surface plasmon and excite the BMs for gold-coated nanodisk arrays.

The sensitivity of the gold-coated nanodisk arrays was then evaluated for sensing purposes. The excitation of these nanostructures at normal incidence in aqueous solutions shifted the BM, which was at nearly 640 nm (normal incidence) in air, towards wavelengths of nearly 840 nm. The bulk refractive index sensitivity (~600 nm/RIU), and RI resolution (1.8 x 10 -5 RIU) were relatively similar for every structure (Table 1), which was expected since the refractive index sensitivity (RIS) of a plasmonic grating nanostructure varies mainly from the variation of the periodicity. 55 The refractive index resolution was measured from the noise level measured from a temporal series of spectra.

The RI resolution obtained with the gold-coated nanodisk arrays was identical to a cavity-coupled plasmonic device reported recently 42 and of nanodisk arrays (estimated from data in ref. 56 ), and was better than RI resolution of nearly 10 -4 RIU typically reported for the classical spherical Au nanoparticle 57 . If optical detection methods other than transmission are used (phase, microscopy, optical fibers), refractive index resolution of 10 -6 RIU or better can be achieved [START_REF] Špačková | Proceedings of the IEEE[END_REF] but oftentimes with added complexity of the instrumental setup. Furthermore, the plasmonic signal of the BM in water was very sharp with a full width at half maximum (FWHM) of 14 nm for the gold-coated nanodisk arrays. In comparison, nanodisk arrays had a smaller RI sensitivity than nanohole arrays of the same periodicity (900 nm/RIU) but their FWHM was 5-fold smaller in comparison to the same nanohole arrays. 49 The sensitivity and FWHM of gold-coated nanodisk arrays were close to the best values reported for other plasmonic nanostructures. In brief, the sensitivity is typically less than 400 nm/ RIU for LSPR nanostructures, and while it varies with the periodicity for arrays of nanostructures, it is generally comprised between 200 and 700 nm/RIU for arrayed nanostructures with resonances below 1000 nm. 42 In addition, the FWHM is typically on the order of a few tens to hundreds of nanometers for LSPR and it was reported within the range of 4 to 36 nm for SPP. 42 Our reported value for the FWHM of 14 nm for gold-coated nanodisk arrays was also close to the best nanostructures. The narrow linewidth was due to the excitation of the Bragg mode of the nanostructure array, which are typically narrower than propagating plasmons (PSP) on smooth films. The area of perfectly ordered nanostructure also contribute to narrowing the linewidth and achieving small FWHM.

The FOM (FOM = RIS/FWHM; calculated in using values in nm) in plasmonic structure is a quantitative parameter providing an indication of the resolution in refractometric sensing. High FOM structures generally have better resolution and thus, lower noise and ultimately leading to sensors with better limits of detection. It is thus customary to compare the performance of plasmonic structures using FOM. From this data, we obtained a FOM that varied from 42 to 46 for gold-coated nanodisk arrays, which once again fared as one of the best overall plasmonic nanostructures reported to date. For example, structures supporting a LSPR resonance such as nanoparticle arrays (FOM = 25) [START_REF] Offermans | [END_REF] , nanobar arrays (FOM = 4.5) 60 , nanodisk (FOM = 29) 61 , Au octamers (FOM = 5.7) 62 , or a "XI" nanostructure (FOM = 4.7) generally had FOM from 0.6 to 29, while if a Wood's anomaly was excited, the FOM increased to 38 63 . Structures supporting a SPP are generally on the order of FOM = 23 to 39, which includes the FOM of a thin Au film classically used in SPR sensing (FOM = 25 to 39) 64 . Significantly higher FOM can be achieved with SPP with Fano resonances, where the FOM ranges from 39 65 and 252 43 , and often exploits subwavelength nanostructures such as the nanoslits structures 43,66 , grating structures 67 , suspended nanohole arrays 68 or nanomushrooms 69 to reach higher FOM. A more extensive list of FOM for a series of nanostructures was provided elsewhere for comparison purposes. 42 However, one must keep in mind that the FOM of a plasmonic structure is not an absolute measurement of the sensing performance and low FOM plasmonic structures have been shown to perform well in sensing. 70 Hence, the gold-coated nanodisk arrays are well positioned due to their combination of high FOM, sensitivity and RI resolution to achieve highly sensitive refractometric measurements. The relatively simple photolithographic fabrication is also an advantage of the gold-coated nanodisk arrays.

While some nanostructures may have greater performance for one of the parameters mentioned in the previous paragraph, it is rare to find a nanostructure excelling in all these analytical parameters, especially with micron size features. For this purpose, we compared the performance of gold-coated nanodisks arrays with nanohole arrays, nanodisk arrays and crossed nanogratings, which are nanostructures bearing structural similarities with gold-coated nanodisk arrays (Table 2). While a list provided here cannot be exhaustive, readers are referred to Homola and coworkers [START_REF] Špačková | Proceedings of the IEEE[END_REF] and Martin and coworkers 42 , which provided recently comprehensive overviews of the analytical parameters for nanoplasmonic structures. Crossed gratings had similar performances to gold-coated nanodisk arrays with the exception of a lower FOM of 14, nanodisk arrays and nanohole arrays consistently fared worse or equally to gold-coated nanodisk arrays depending on the parameter compared. We then compared our results to two plasmonic substrate with exceptional FOM. Nanomushrooms had superior sensitivity, FWHM and FOM, but had a RI resolution 10x worse than the gold-coated nanodisk arrays. SERS performance for this nanostructure has not been reported. Suspended nanohole arrays have a FOM of 162 and excellent sensitivity and FWHM, but are expected to have lower SERS EF (close to conventional nanohole arrays). They are relatively complex to fabricate and are suspected to be more fragile due to the suspended thin film. One clear distinction between these nanostructures and the gold-coated nanodisk arrays is that all nanostructures had features smaller than 300 nm, with a periodicity smaller than 600 nm.

The gold-coated nanodisk arrays are significantly larger with features of 720 nm and 1200 nm periodicity, which shows that sub-wavelength features are not necessary for achieving high analytical performance.

One of the main parameters dictating the performance of a plasmonic sensor for biosensing purposes is the distribution of the EM field. 4 The surface sensitivity of a sensor characterizes the interaction of the plasmonic field at the metal/air or metal/solution interface for the detection of biological recognition events. Surface sensitivity of the gold-coated nanodisk arrays was assessed with a biosensor for IgY in PBS. For this test, a custom multi-well plate reader was used in order to achieve a broadly multiplexed platform. The multi-well plate reader allowed monitoring of up to 96 wells (volume of 100 L/well) in a single experiment and was used without modification from a previous report. 49 The biosensor was constructed to include a peptide monolayer 71, 72 for minimizing nonspecific adsorption of biofluids, on which a polyclonal anti-IgY was immobilized with standard EDC-NHS chemistry as previously reported. 73,74 The sensor was then subjected to direct detection of 100 nM of IgY in triplicate measurements (in different wells of the 96-well plate reader), followed by a secondary detection using the same polyclonal anti-IgY antibody. A control in absence of IgY was performed with a triplicate of wells to validate the specificity of the secondary detection step. We obtained a plasmonic shift of 0.1 ± 0.1 nm in absence of IgY, confirming the specificity of the secondary detection step. From these data, we estimated the surface sensitivity at the limit of detection to be approximately 5 pg/mm 2 . Differences in gold film and nanodisk thicknesses did not significantly affect the surface sensitivity of the sensors, but the goldcoated nanodisk array with ND75film75 exhibited the best overall performance (Table 1)

and was thus used for the following experiments. We then measured the surface sensitivity of the gold-coated nanodisk arrays with a calibration curve of IgY (Figure 3).

The noise level of gold-coated nanodisk arrays was lower than for nanohole arrays. Both calibration curves were nearly identical, but the plasmonic response was slightly better for nanohole arrays at higher concentration (> 100 nM).

Biosensing in complex matrices is one of the main challenges of plasmonic sensing because of the nonspecific adsorption of background proteins on refractometric sensors. 1,71 To demonstrate the potential of this sensing platform for more realistic sample measurements, biosensing of human IgG was performed in human urine. IgG detection in urine can serve for detection of urinary tract infections among other pathologies. The sensor was modified with anti-human IgG to capture IgG in the sample.

Since human urine contains IgG at concentrations on the order of a few µg/mL (about a few tens of nanomolar) 75 , we performed a standard addition assay where human urine was spiked with an additional concentration of 100 and 1000 nM of human IgG. Once again, a control consisting of injecting human urine without further spiking with human IgG led to a plasmonic shift 3-fold smaller (Δλ = 0.15 ± 0.02 nm) than for the detection of a human urine sample supplemented with 100 nM human IgG (Δλ = 0.39 ± 0.02 nm).

The shift of the sensor further increased with the addition of 1000 nM IgG in human urine (Δλ = 0.52 ± 0.03 nm). This indicated that the sensor was able to differentiate human IgG from background noises from analytes in a complex matrix.

To further characterize the gold-coated nanodisk arrays and to evaluate the potential of this platform in surface-enhanced spectroscopy, we have studied the properties of this plasmonic substrate in SERS and numerically simulated the field distribution on the nanostructure. Typical nanodisk arrays (without the gold film) have been extensively reported in SERS applications with enhancement factors (EF) of 10 6 -10 7 . [76][77][78] The high localization of the plasmonic field near nanodisks allowed a strong enhancement of the EM field intensity. The optimal conditions will depend on the geometrical parameter of the nanodisk, as the EM enhancement is wavelength-dependent and requires coupling of the wavelength of the laser with the plasmonic resonance of the nanostructure. [START_REF] Stiles | Annual Review of Analytical Chemistry[END_REF]79 For our gold-coated nanodisk arrays, the plasmon resonance was excited at wavelengths in air (λ = 641 nm) and water (λ = 840 nm) close to the range of wavelengths from common Raman lasers of 633 and 785 nm.

A false color Raman map of gold nanodisk arrays (ND75film75) was generated to assess the localization of the plasmonic field (Figure 4) using the intensity of the peak at 1335 cm -1 from the spectrum of nitrobenzenethiol (4-NBT). The Raman map revealed that the highest intensity of Raman scattering was located near the edge of the nanodisks.

These results correlated with the previously reported theory that the plasmonic field of the BM should be highly confined at the edges of the nanodisk. 51 Slight differences were observed in the experimental and 3D FEM data (Figure 4). The Raman maps showed a relatively even field around the circumference of the nanodisks, while the 3D FEM simulations reported a dipolar mode of the field. This difference is likely due to the conical irradiation of the substrate due to the numerical aperture of the objective.

The Raman map could not distinguish if the highest EM field was located at the bottom or top of the nanodisk. Therefore, we simulated the field distribution using 3D FEM, where in these simulations the field impinged at the metal-air interface, as for the Raman experiments. The Raman map was in good agreement with the 3D FEM data of the electromagnetic field distribution in the X-Y plane for the same gold-coated nanodisk arrays (Figure 4). Indeed, the plasmonic field at a wavelength of 644 nm described a donut shape on the edges of the nanodisks for Z values of 75 nm (gold film/nanodisk interface) and 150 nm (top of nanodisk). These results demonstrated that the field intensity at the edges of the disk was higher at the interface of the nanodisk and air (Z = 150 nm). In addition, the simulations showed the plasmon field leaking in the gold film and creating interference patterns around the nanodisk (Z = 75 nm). The 3D field simulations demonstrated the high confinement of the field distribution near the top of the nanodisk, a location in agreement with the Raman images. The field distribution was also similar to 3D FEM simulations for transmission measurements, such as the ones reported for the plasmonic sensing experiments above.

Enhancement factors (EF) are often compared to evaluate the potential of plasmonic structures in SERS. Gold-coated nanodisk arrays led to an estimated EF of 10 7 , which is slightly higher than that reported by Gillibert et al. for a similar structure 52 and slightly lower than the SERS enhancement of 10 8 recently reported on a nanotriangle arrays on a gold film. 80 SERS EF on the order of 10 7 to 10 8 were reported to be sufficient for single molecule studies 81 and typical SERS EF reported in the literature 82 ranges from 10 5 to 10 7 , such as for cavity nanohole arrays 83 , but can exceed this value in aggregated nanoparticles. Higher SERS EF could be achieved using nanodisk arrays with smaller features, as reported elsewhere using nanoimprint lithography 84 . The SERS response from different locations was highly reproducible with about 7% of coefficient of variation measured on 576 spots on the gold-coated nanodisk arrays, which was also evident from the relative homogeneity of the SERS intensity on the Raman map (Figure 4). The periodic structure and the lower EF can explain the better SERS reproducibility of this plasmonic substrate. Thus, gold-coated nanodisk arrays can also serve as effective SERS substrates.

Conclusions

In conclusion, we developed gold-coated nanodisk arrays with great potential for biosensing applications. A screening of the plasmonic properties of 16 structures demonstrated that the plasmonic properties were superior for thicker gold film and nanodisk heights (≥ 50 nm). The refractive index sensitivity (about 600 nm/RIU), resolution (1.8 x 10 -5 RIU), and FOM (up to 46) were superior to most plasmonic substrates reported to date. While higher refractive index, higher FOM or SERS enhancement can be achieved in a few plasmonic nanostructures, the previously reported nanostructures required features with significantly smaller dimensions and often excelled in one of these parameters. The gold-coated nanodisk arrays performed well in all these analytical parameters, were fabricated using simpler techniques and also showed good surface sensitivity towards protein detection. We also demonstrated that gold-coated nanodisk arrays can be easily integrated with a 96-well plate reader and we confirmed their great potential for high throughput label-free biosensing. Finally, a mapping of the SERS intensity and 3D FEM simulations revealed that the plasmonic field of the BM was highly confined at the edges of the nanodisk. This enhanced EM field generated a high SERS EF of approximately 10 7 . Thus, gold-coated nanodisk arrays showed great potential for plasmonic applications.

Methods

Microfabrication

Gold-coated nanodisk arrays and nanohole arrays were manufactured by photolithography similarly to our previous publications. 49,85 The fabrication of goldcoated nanodisk arrays was achieved with a positive photoresist (OIR 674-11) and a sacrificial layer (LOR1A) using the same patterned mask as for nanohole arrays. A layer of Cr (1 nm) and different thicknesses of Au (10, 25, 50 or 75 nm) were evaporated on the patterned wafer using electron beam deposition. Resins were lifted off from the wafer by sonication in remover PG. This resulted in a wafer covered with gold nanodisk arrays where the deposited thickness of gold corresponded to the height of the disk. The periodicity and diameter of the nanodisk arrays were respectively 1200 ± 20 and 650 ± 20 nm. Then, another layer of gold was evaporated on the gold nanodisk arrays for different gold thicknesses (10, 25, 50 or 75 nm). A total of 16 gold-coated nanodisk arrays were manufactured using this methodology. The structures were identified from the height of the nanodisk (ND75 for a 75 nm high nanodisk) and gold film (film75 for a 75 nm thick gold film) evaporation steps. Hence, a structure labeled ND75film75 had a nanodisk arrays of 75 nm thick followed by a deposition of 75 nm of gold covering the nanodisk arrays. The structures of the nanohole arrays and nanodisk arrays were confirmed by scanning electron microscopy (SEM, Jeol). The periodicity and diameter of the nanodisk were measured by atomic force microscopy (AFM, Witec).

Optical measurements and plasmonic sensing

Gold-coated nanodisk arrays were excited in transmission spectroscopy using a homemade fluidic cell and a halogen lamp. The plasmon dispersion curves were obtained by measuring the plasmonic signal in air for each gold-coated nanodisks arrays at various incident angles from 0° to 30° using a manual goniometer. Spectra were collected with a PI-Acton spectrophotometer equipped with a grating of 150 g mm -1 (set from 415 to 975 nm) and a CCD camera (Andor model DU401A-BR-DD-152). The transmission signal of the corresponding gold film thickness was used as a reference for the normalization of the plasmonic signal for each gold-coated nanodisk arrays. The bulk refractive index sensitivity of the sensors was determined with aqueous sucrose solutions (1.33-1.36 RIU) using a fluidic cell. A screening of the surface sensitivity of several nanodisks arrays was achieved with a custom multi-well plate reader and 96-well plate as described elsewhere 49 . For these measurements, a grating of 600 g mm -1 was used and set from 785 to 914 nm. Direct and secondary detection of IgY proteins (Cedarlane) using anti-IgY antibodies (Cedarlane) was achieved with the plasmonic reader for several structures based on previously reported methods. 73,74 Each sensor was measured in triplicate and a control was achieved to assess nonspecific adsorption. A calibration curve for detection of IgY in PBS (10 to 1000 nM) was performed for gold-coated nanodisk arrays and compared to nanohole arrays using the fluidic cell setup. Detection of human IgG protein (100 nM, 1000 nM and control) was also done in PBS and diluted urine (1:1 in PBS:urine) with the fluidic cell for the nanodisk arrays. Furthermore, the LSPR signal of the nanodisk arrays and gold-coated nanodisk arrays (ND75film75) was measured with a Cary 6000i UV-vis-NIR spectrophotometer set from 500 to 1800 nm.

Raman spectroscopy measurements

SERS enhancement factors (EF) of gold-coated nanodisk arrays (ND75film75)

was estimated using a confocal Raman microscope (Renishaw inVia) with a grating of 600 g mm -1 , a 633 nm laser excitation with 4.4 mW at the sample and a 50x/0.75 objective. Samples were functionalized overnight in a 1 µM solution of 4nitrobenzenethiol (4-NBT). Raman spectra were acquired at a frequency of 1 Hz for a total of 20 spectra average per acquisition. A Raman image of the nanodisk array (ND75film75) was collected on a WITec alpha 300 with a 633 nm laser excitation wavelength using the piezo-driven XYZ scan stage and a grating of 600 g mm -1 coupled to a CCD camera. The spectra were collected with an integration time of 0.1 seconds using a 100x/0.9 objective with a laser power of around 1 mW at the sample. Image scans of 10 µm x 10 µm were obtained using a 100 nm step size. WITec Project software was used to background subtract the spectra using a first order polynomial fit and false color images were generated based on the intensity of the 4-NBT peak at 1335 cm -1 .

The SERS EF of the ND75film75 structure was determined using equation 2

where ISERS and IRaman correspond respectively to the intensity of the 4-NBT band at 1335 cm -1 for SERS and normal Raman. [START_REF] Stiles | Annual Review of Analytical Chemistry[END_REF]

𝐸𝐹 = (

Simulation

The theoretical dispersion curves of the BM were calculated using a Matlab algorithm based on equation 1. The values were determined for an array having a periodicity of 1200 nm and for incident angles of 0° to 30°. 3D finite element method (3D FEM) simulations were also carried for the gold-coated nanodisk arrays (ND75film75) in air using COMSOL multiphysics software package for the wavelength region of 600 to 1000 nm. All spectra were normalized with the incident light field. The normalized electromagnetic field distribution in the X-Y plane was plotted at the peak wavelength under polarized normal incidence. The region from Z = 0, 75, and 150 nm corresponded to the BK7 glass substrate (Z = 0 nm), interface of the gold film (Z = 75 nm) and top of the nanodisks (Z = 150 nm).

Figure

  Figure 2B). At a rotation angle of 90 o , several bands such as the (1,0) and (-1,0) bands are orthogonal to the plane of incidence, which has a consequence of invariability of the plasmon resonance wavelength (excited at nearly 1.1 m) in relation to the excitation angle, an effect that was also seen for the (-1,2) and (1,-2) bands at a rotation angle of 0 o . Consequently, the plasmon dispersion curves at a rotation angle of 90 o had fewer bands than at a rotation angle of 0 o .

  NSERS and NRaman are respectively the number of excited molecules generating the SERS and Raman signal. The value of NSERS was based on the surface coverage of 4-NBT around a nanodisk. This value was determined based on the coverage ratio given in Saverot et al. (0.54 nm 2 ) 86 and the estimation of the active area of high intensity EM field was approximately 12,000 nm 2 based on figure 5. NSERS was thus approximately 6,400 molecules. The value of NRaman was determined based on the Raman scattering from a solution of 4-NBT where the number of 4-NBT molecules present in the solution within the laser interrogation volume was estimated to be approximately 4x10 7 molecules. ISERS and IRaman were respectively 3,984 counts/s and 3.3 counts/s.

Figure 1 .

 1 Figure 1. Top) Representation of manufacture process for gold-coated nanodisks; A positive photoresist layer was spin-coated on a 4" glass wafer (A), then UV-light cured through a mask and developed to create an array of holes in the exposed resist (B), followed by the deposition of a gold layer (C), and liftoff of the resist (D) to create an array of nanodisks (E), and finally, the deposition of a gold overlayer to yield the goldcoated nanodisk arrays (F). Bottom left) SEM image of a gold-coated nanodisk arrays with a periodicity of 1200 nm and a disk diameter of 720 nm. Bottom center) Plasmonic response of nanodisk arrays (ND75film75 -definition in the text) excited in transmission in air for incident angles of 0° (blue curve) and 5° (red curve). Bottom right) Representation of the incidence angle (θ) and rotation angle (φ).

Figure 2 .

 2 Figure 2. Plasmonic dispersion curves for different orders of BM for a gold nanodisk arrays (ND75film75) with a periodicity of 1200 nm. The calculated modes (white lines) were for the metal/air interface. The color map represents the relative transmission intensity of the gold-coated nanodisk arrays using a smooth Au film of an equivalent thickness as a reference. The gold-coated nanodisk arrays were aligned at A) 0 o rotation angle (incident light oriented with the (1,0) mode) and B) 90 o rotation angle (perpendicular to the (1,0) mode). Other modes can also be seen on the plasmon dispersion curve, which can be associated to the BM of the plasmon at the glass-metal interface.

Figure 3 .

 3 Figure 3. Left) Sensorgrams for the detection of IgY with the gold-coated nanodisk arrays. Concentrations of 10, 50, 100, 500 and 1000 nM were successively injected in a fluidic cell. Right) IgY calibration curves for structures of gold-coated nanodisk arrays excited in transmission using a fluidic cell.

Figure 4 .

 4 Figure 4. A) Raman imaging of a gold-coated nanodisk arrays (ND75film75) with 4nitrobenzenethiol (4-NBT). The image was acquired with a 633 nm laser and 100 nm steps between pixels and 100 ms integration time per pixel. The image represents a false color Raman map of the intensity of 4-NBT peak at 1335 cm -1 for a scanning area of 10 µm x 10 µm. The inset shows a cross-section of the Raman intensity corresponding to the white line in the SERS image. B-D) The field distribution at resonance wavelength (644 nm) was calculated in 3D under polarized light for the gold-coated nanodisk arrays. The light was impinging from the top of the image B, directly on the nanodisk array-air interface, similar as to the Raman imaging experiment. The field was normalized to the incident 1 V/m. The arrow represents the direction of the polarization. The region of highest field intensity was located at the top rim of the nanodisk. The field distribution at resonance wavelength (644 nm) was calculated in the X-Y plane under polarized light for the glass-gold interface for the gold film-nanodisk interface (Z = 75 nm, C) and for the nanodisk-air interface (Z = 150 nm, D). The images also represent normalized false color maps of the field distribution and the arrows represent the orientation of the polarization of the incident light.

Table 1 .

 1 Plasmonic sensing properties of gold-coated nanodisk arrays.

		ND50film50 ND50film75 ND75film50 ND75film75
	Sensitivity (nm/RIU)	583 ± 1	601 ± 2	599 ± 2	599 ± 3
	FWHM (nm)	14 ± 2	14 ± 4	13 ± 1	14 ± 1
	FOM	42	43	46	43
	RI resolution (RIU)	1.9 x 10 -5	1.8 x 10 -5	1.8 x 10 -5	1.9 x 10 -5
	∆λSPR (nm) IgY	0.5 ± 0.1	0.4 ± 0.1	0.2 ± 0.1	0.5 ± 0.1
	∆λSPR (nm) anti-IgY	2.3 ± 0.2	2.6 ± 0.2	1.9 ± 0.2	2.8 ± 0.2

Table 2 .

 2 Comparison of analytical parameters of 2D plasmonic structures

	Structure	Sensitivity	FWHM	FOM	Resolution	SERS	Features	Periodicity	Reference
		(nm/RIU)	(nm)	(RIU -1 )	(RIU)	EF	(nm)	(nm)
	Crossed gratings	647	50	14	10 -5	NA	NA	550	87
	Nanodisk arrays	161 a	10.5 a	15 a	2 x 10 -5 b	2.6 x	100	500	a 37; b 56 ; c 76
						10 7 c
	Nanohole arrays	313 d	14.5 d	23 d	2 x 10 -5 e	10 5 f	100	400	d 88; e 89; f 90
	Nanomushrooms	1015	9.5	108	2 x 10 -4	NA	285	610	69
	Suspended	717	4	162	NA	NA	230	580	91
	nanohole arrays
	(Fano)
	Gold-coated	599	14	43	1.9 x 10 -5	10 7	720	1200	this work
	nanodisk arrays

Acknowledgements

The authors thank financial support from the National Science and Engineering Research Council (NSERC) of Canada (MC and JFM), of the Canada Foundation for Innovation (JFM), the Leverhulme Trust through Research Project Grant RPG-2012-758 (KF and SL), from the National Natural Science Foundation of China Grant Nos. 61137005 and 61520106013 (WC and WP), the project support (ANR-11-IDEX-003-02) from the ANR French National Research Agency as well as MS IDI 2012 thesis funding by the IDEX Paris-Saclay, LN2 is an international joint research laboratory, Unité Mixte Internationale UMI 3463, managed by CNRS, Université de Sherbrooke, UdeS, as well as Université de Lyon, UdL, especially including Ecole Centrale de Lyon, INSA Lyon et CPE Lyon, and Université Grenoble-Alpes, UGA.

Associated content

Supporting Information

The Supporting Information is available on the web at DOI: 10.1021/XXX

Figures on the dispersion curves in transmission measurements


Sylvie Guillerme
email: sylvie.guillerme@univ-tlse2.fr

Hugues Barcet

Nicolas De Munnik

Eric Maire

Claire Marais-Sicre

Guillerme Sylvie

Barcet Hugues

Maire Eric

Marais-Sicre Claire

Evolution of traditional agroforestry landscapes and development of invasive species: lessons

Keywords: Agroforestry, Land abandonment, Landscape dynamics, Invasive Alien Species (IAS), Perceptions, Place attachment, UAV Technology

published or not. The documents may come   L'archive ouverte pluridisciplinaire

Introduction

Agroforestry systems, based on tree components, are usually viewed as sustainable land use options [START_REF] Rigueiro-Rodríguez | Agroforestry systems in Europe: productive, ecological and social perspectives[END_REF][START_REF] Mcadam | Classifications and functions of agroforestry systems in Europe[END_REF][START_REF] Jose | Agroforestry for conserving and enhancing biodiversity[END_REF], offering the potential to solve important ecological and biodiversity problems while enabling the production of food, wood products and/or fodder for animals at the same time [START_REF] Nerlich | Agroforestry in Europe: a review of the disappearance of traditional systems and development of modern agroforestry practices, with emphasis on experiences in Germany[END_REF]. The term "High Nature Value Farming" [START_REF] Baldock | Farming at the margins: Abandonment or Redeployment of Agricultural Land in Europe[END_REF] reflects the advantages offered by traditional agroforestry systems as well as the valued semi-natural habitats that are associated with them, the majority of which are found in remote areas under extensive land use.

While the pressure on agricultural land is increasing on a global scale in terms of availability, the dynamics of European landscapes is characterized by contrasting trends, which have been particularly marked since the end of World War II: i/ the disappearance of tree elements in areas most suitable for large-scale cultivation and ii/ the developing encroachment of natural forest where farming practices (particularly those linked to livestock) have been abandoned or are in decline.

Thus, although trees represent a structuring element of many rural landscapes associated with traditional forms of agroforestry, in the first case, the pursuit of increased productivity has led to a specialization of modern agricultural or livestock systems, while the second has led to the non-use of trees. Both trends have resulted in a decline in the use and value of trees outside forests [START_REF] Guillerme | L'arbre hors forêt en France. Diversité, usages et perspectives[END_REF].

Agricultural land abandonment is one of the main processes of change in land use in Europe [START_REF] Keenleyside | Farmland Abandonment in the EU: an Assessment of Trends and Prospects[END_REF][START_REF] Van Der Zanden | Trade-offs of European agricultural abandonment[END_REF][START_REF] Vinogradovs | Assessment of sitespecific drivers of farmland abandonment in mosaic-type landscapes: A case study in Vidzeme, Latvia. Agriculture[END_REF]. It leads to a homogenization of landscapes and has been increasing since the 1950s [START_REF] Cramer | What's new about old fields? Land abandonment and ecosystem assembly[END_REF]). Land abandonment is a complex and multidimensional process [START_REF] Munroe | Alternative trajectories of land abandonment: causes, consequences and research challenges[END_REF]) that causes changes in rural livelihoods and agricultural practices. It has become a major concern due to its environmental, socio-economic and landscape dimensions [START_REF] Lasanta | Space-time process and drivers of land abandonment in Europe[END_REF]. It also constitutes an important challenge in terms of sustainability and land use planning, especially in marginal agricultural areas and remote and/or mountainous zones.

Agricultural land abandonment and rural depopulation provide a special opportunity for cultivated alien plants (Pandi et al. 2014). Species that are nonnative and non-invasive can become invasive as habitats and dispersers change. This combination of changes can cause rapid increase of a non-native species, allowing it to become invasive (Lenda et al. 2012).

Invasive species have become an issue all over the world, on all types of land (agricultural or natural). Over the past few decades, humans have greatly accelerated the process of biological invasions, to the point where they are now recognized as the second cause of accelerated decline in biodiversity (Simberloff and Rejmánek 2011;[START_REF] Lévêque | Les espèces exotiques envahissantes, pour une remise en cause des paradigmes écologiques[END_REF]. The impact of invasive plants on natural vegetation is currently being widely studied (Powel et al. 2011; [START_REF] Barney | Invasive plant management must be driven by a holistic understanding of invader impacts[END_REF][START_REF] Mccary | Invasive plants have different effects on trophic structure of green and brown food webs in terrestrial ecosystems: a meta-analysis[END_REF]. It has been shown that, when non-native plants invade an environment, they are capable of outcompeting native plant species through either direct [START_REF] Daehler | Performance's comparisons of co-occurring native and alien invasive plants: Implications for conservation and restoration[END_REF] or indirect competition [START_REF] Tanner | The impact of two non-native plant species on native flora performance: potential implications for habitat restoration[END_REF]. Invasive weeds are an increasing problem because they are difficult and costly to control.

One of the difficulties is that, even when an invasion is territorially assessed, it remains very complex to quantify, and its spatial extension is hard to assess. Although IAS are widely explored by research in the ecological sciences, knowledge of their spatial dynamics and characterization remains complicated [START_REF] Michez | Mapping of riparian invasive species with supervised classification of Unmanned Aerial System (UAS) imagery[END_REF], which is a barrier to their management. Since the 1990s, remote sensing has been used to monitor and map the first outbreaks [START_REF] Huang | Applications of Remote Sensing to Alien Invasive Plant Studies[END_REF][START_REF] Große-Stoltenberg | Evaluation of Continuous VNIR-SWIRSpectra versus Narrowband Hyperspectral Indices to Discriminate the Invasive Acacia longifolia withina Mediterranean Dune Ecosystem[END_REF][START_REF] Alvarez-Taboada | Mapping of the Invasive Species Hakea sericea Using Unmanned Aerial Vehicle (UAV) and WorldView-2 Imagery and an Object-Oriented Approach[END_REF], to identify optimal periods for applying mitigation treatments [START_REF] Wallace | Mapping Presence and Predicting Phenological Status of Invasive Buffelgrass in Southern Arizona Using MODIS, Climate and Citizen Science Observation Data[END_REF], to model invasion risks [START_REF] Huang | Applications of Remote Sensing to Alien Invasive Plant Studies[END_REF][START_REF] Blanchet | Remote Detection of Invasive Plants: A Review of Spectral, Textural and Phenological Approaches[END_REF] or to monitor the effectiveness of management measures [START_REF] West | Using multi-date satellite imagery to monitor invasive grass species distribution in post-wildfire landscapes: An iterative, adaptable approach that employs open-source data and software[END_REF].

High spatial resolution imagery (about 10 m resolution) has been widely used to detect and monitor invasive plants but it is only effective when plots and/or stands occupy a large area [START_REF] Wallace | Mapping Presence and Predicting Phenological Status of Invasive Buffelgrass in Southern Arizona Using MODIS, Climate and Citizen Science Observation Data[END_REF]) and remains unsatisfactory for detecting alien plants in the ecotone or for mapping a small area in detail [START_REF] Alvarez-Taboada | Mapping of the Invasive Species Hakea sericea Using Unmanned Aerial Vehicle (UAV) and WorldView-2 Imagery and an Object-Oriented Approach[END_REF]. However, the evolution of technologies, and of drones in particular, is removing a number of scientific barriers by allowing very high spatial resolution (~10 cm) acquisition.

During the last decade, in which the advent of these technologies has been particularly marked, several studies have found effective approaches for identifying invasive plants [START_REF] Blanchet | Remote Detection of Invasive Plants: A Review of Spectral, Textural and Phenological Approaches[END_REF][START_REF] Alvarez-Taboada | Mapping of the Invasive Species Hakea sericea Using Unmanned Aerial Vehicle (UAV) and WorldView-2 Imagery and an Object-Oriented Approach[END_REF]. However, the difficulty of measuring the spatial expansion of these species accurately, especially in a treecovered environment, has also been highlighted [START_REF] Dash | Early Detection of Invasive Exotic Trees Using UAV and Manned Aircraft Multispectral and LiDAR Data[END_REF].

Various studies [START_REF] Arim | Spread dynamics of invasive species[END_REF][START_REF] Davidson | Using a Novel Spatial Tool to Inform Invasive Species Early Detection and Rapid Response Efforts[END_REF][START_REF] Akodéwou | Problématique des plantes envahissantes au sud du Togo (Afrique de l'Ouest): apport de l'analyse systémique paysagère et de la télédétection[END_REF]) have been conducted with the aim of understanding the spatial dynamics of invasive species but recent decades have seen a marked shift in focus from topics addressing the mechanisms of invasions and the underlying process towards more a complex perception of invasions, where the socioeconomic and social perspective on invasive species is recognized as increasingly important [START_REF] Pysek | Current trends in plant invasion research[END_REF][START_REF] Shackleton | The role of invasive alien species in shaping local livelihoods and human well-being: A review[END_REF].

Many non-native trees (NNT) have been planted worldwide in agroforestry systems for timber, fodder, erosion control, shade and aesthetics, and some of them are now qualified as invasive [START_REF] Richardson | Trees and shrubs as invasive alien species -a global review[END_REF]. Some research projects conducted in Brazil [START_REF] Ramos | Environmental filtering of agroforestry systems reduces the risk of biological invasion[END_REF][START_REF] Cordeiro | Agroforestry systems reduce invasive species richness and diversity in the surroundings of protected areas[END_REF]) have shown that agroforestry systems provide less favourable habitats for exotic species, filtering them from the understory, but the mechanisms that may select against exotic species in agroforestry remain unknown [START_REF] Ramos | Environmental filtering of agroforestry systems reduces the risk of biological invasion[END_REF] and data are lacking in other places, including temperate environments.

The relationship between agroforestry and invasive plant species is rarely addressed. Yet the spread of invasive species is an important issue, not only ecologically but also economically and socially. The objective of the present work was to study the evolution of the traditional agroforestry system in the Oussouet valley (foothills of the Pyrenees mountains in France) where invasive plant species are abundant. Here (as in other parts in Europe), the abandonment of agricultural activities is a major problem, having environmental, socio-economic and landscape implications. The paper considers the landscape dynamics as well as the diverse perceptions expressed by the stakeholders we interviewed concerning the invasive species that have spread. The issue of mapping the invasive species is then addressed. Finally the link with sustainable development is discussed.

Materials and methods

The approach used to address these issues integrates social geography and spatial sciences, and provides an analytical framework for understanding how territorial and agrosystem changes interact with the dynamics of social systems.

Study area

The area surveyed in France is located in the Oussouet valley, which is representative of local traditional agroforestry landscapes based on a combination of grazing lands, trees, and livestock, located in the Pyrenees mountains in the south of France (Fig. 1A). This sector of the Hautes-Pyrénées department is on the fringe of the Pyrenees National Park and near the town of Bagnères-de-Bigorre. It is a mid-mountain area with a climate having oceanic influences. Rainfall is high due to the location in the foothills of the mountain range. The altitude and climatic conditions in this area result in two types of landscapes characterized by specific vegetation: -i/ in the hillside level (<800-1000m), where precipitation is normally high, habitats are composed of meadows and stream banks in the valley bottoms. On the slopes, deciduous forest and thickets dominated by beech [Fagus sylvatica L.) cover most areas, but there are also some grasslands and some bracken fields (extensive, often closed, communities of the large fern Pteridium aquilinum L. [Devillers et al. 1991]).

-ii/ in the mountainous level (>800-1000m), which is cool and very humid with strong cloud cover, the vegetation consists of deciduous and coniferous forests, bracken fields and recolonizing thickets (mainly of holly [Ilex aquifolium L ] and willow trees [Salix spp.]).

This area -where the population is gradually decreasing -is characterized by three main types of human activities, based upon the exploitation of the natural resources of this rural mid-mountain region (data provided by the municipal authorities): i/ pastoralism (associated with bovine, sheep, goat and horse breeding); ii/ forestry, which is quite an important activity in numerous areas of coniferous and beech forest, mainly in the mountain area; and iii/ slate mining, which used to be the particularity of the Oussouet valley. The municipality of Labassère (Fig. 1A) is situated on an outcrop of high-quality slate schist and three slate quarries are present in the area but all have ceased mining operations. The region is also characterized by a strong neo-rural population and numerous second homes.

As far as vegetation is concerned, this very rural and pastoral valley ultimately presents the paradox of benefiting from high specific biodiversity with a diversified flora [START_REF] Corriol | Massif du Monné, vallée de l'Oussouet[END_REF]) while, at the same time, being a place where many invasive alien species have spread [START_REF] Fontaine | Plan régional d'actions : Plantes Exotiques Envahissantes en Midi-Pyrénées[END_REF].

Survey method

Practices and perceptions were placed in the spatial and temporal context of the territory, which was considered as a set of elements in dynamic interaction. It also corresponds to a functional spatial entity, with landscapes reflecting different environmental and anthropogenic processes. People's perceptions and practices concerning the evolution of the agroforestry landscape and invasive species were studied through qualitative interviews, and assessed by a botanical survey.

A qualitative approach to agroforestry and invasive (or qualified as such) species present in the study area was sought. We wished to adopt an "intermediate" posture between the "theoretical saturation" and "qualitative tradition" described by [START_REF] Savoie-Zajc | L'entrevue semi-dirigée[END_REF], in order to define the corpus of semi-directed interviews. In qualitative research based on an ethno-phenomenological and discursive approach, several authors refer to a maximum number of about ten interviews. In the aim of remaining as close as possible to the experience of those acting "in the field" (Retaillé, 2010) and capturing a broader diversity of perceptions, we finally conducted 18 open or semi-directive interviews. The local stakeholders were randomly selected, in order to triangulate data. We interviewed people of the valley having diverse activities, including livestock farmers, local elected officials, teachers, actors in the biodiversity field, the slate mining activity, and residents (formerly settled and neo-rural residents (Table 1).

Table 1. Description of the sample of interviewees in terms of their main activity (Note: in case of multiple activities the main one considered during the interview is mentioned)

The interviews were conducted in the field and/or in the homes of the respondents, in a conversational mode rather than following a strict pre-defined list of questions. All interviews started with broad questions on the interviewee's professional and associative activities, and life history in connection with the valley. It was followed by questions about the experience of the respondents regarding the valley, the evolution of landscapes and activities, biodiversity and their perception of invasive species. An interview guide with the main themes and questions addressed during the interviews was used as a guideline but enriched by additional questions in accordance with each person's profile and responses. The agroforestry concept was never explicitly used in the interviews, but was a conceptual lens to analyse the transcripts. In contrast, the term invasive species was deliberately used in order to understand the way respondents understood this expression, and what they personally considered as "invasive".

The interviews were recorded (with the oral consent of the respondents), transcribed in full, and manually coded using a thematic coding. A regressive approach was used to understand the processes of evolution of this landscape, and the associated agrarian practices and perceptions, in a spatial and temporal dimension. The objective was to start from the stakeholders' experience in order to reconstruct "the structural aspect of the situation or the intervention area considered in terms of stakeholders, challenges, interests […] that give meaning and coherence to this life experience" [START_REF] Friedberg | Invasive Species as Ecological Threat: Is Restoration an Alternative to Fear-based Resource Management?[END_REF]. In identifying these representation systems and local practices, we sought to seize the perceptions of the landscape's evolution and the issues related to it, and the meaning that the stakeholders assigned to their practices and to the events they may have witnessed [START_REF] Blanchet | Remote Detection of Invasive Plants: A Review of Spectral, Textural and Phenological Approaches[END_REF].

Hence, the results of the qualitative analysis of the interviews focus on i/ the evolution of the landscape (in order to describe the structure and history of the agroforestry landscapes of the valley from the beginning of the century to the present day and to understand the context in which the invasive species have spread), ii/ the factors perceived as favouring invasiveness, and iii/ a typology of perceptions about invasive species To achieve more detailed insight into the spread of IAS, we also carried out a mapping process that was complementary to this social survey.

Mapping method

In order to characterize the spatial dimension of invasive species, a fine (metre scale) mapping of a pastoral area of the valley (upstream sector of the Oussouet valley -Labassere, Fig. 1C) was carried out. The species targeted was Spiraea japonica (Fig. 1D), which was easy to identify for the purpose of testing the method.

The areas occupied by these invasive plants were assessed by means of very high spatial resolution drone-based technologies (UAV's -Unmanned Aerial Vehicles) and aerial photography. However, this method could not be implemented everywhere (places with heavy tree cover were not suitable for UAV investigation, except with LIDAR sensors). Aerial photography helped to understand the evolution of a landscape over the last two decades, while UAV images showed the spatial extent and recent progression of Spiraea japonica over the last 2 years and attested to the evolution of this pastoral area of the valley. For the classification generated from the drone images, we used the spectral bands provided by the sensor (G-R-NIR-RE), to which we added various indices highlighting vegetation, soil or the chlorophyll content of plants (NDVI [START_REF] Trucker | Red and photographic infrared linear combinations for monitoring vegetation[END_REF], RVI [START_REF] Jordan | Derivation of leaf-area index from quality of light on the forest floor[END_REF], DVI [START_REF] Richardson | Distinguishing vegetation from soil background information[END_REF], TSAVI [START_REF] Baret | TSAVI: A vegetation index which minimizes soil brightness effects on LAI and APAR estimation[END_REF]], MTVI 2 [START_REF] Haboudane | Hyperspectral vegetation indices and novel algorithms for predicting green LAI of crop canopies: Modeling and validation in the context of precision agriculture[END_REF]) that enhanced the detection of relevant objects. The classification was also based on data collected during field investigations and/or photo-interpreted on the RGB drone image. These data were divided into two independent sets, 60% of the data being used for training and 40% for validation. The findings from this method are presented in the last part of the results section.

Results

An age-old agroforestry landscape: a consensual description of the evolution of landscapes and practices

A key initial result emerging from the interviews was the existence of a consensual description of the evolution of the landscapes and activities in the valley by the respondents who had always lived there, or had heard about it (from neighbours or relatives) if they were too young to have had their own experience or if they were recently settled. Only two persons (neo-rural residents) were unable to speak much about the past of the valley. Not surprisingly, the livestock farmers provided a multitude of details and narratives. The main features evoked concerning the practices and the evolution of the landscapes are given below.

Hedges were a key element of the landscapes, with fenced fields, hay meadows and grazing meadows. Trees were fundamental in the lives of the families, not only for the timber, firewood and fruit they provided, but also for the protection they furnished for animals and the soils. Livestock farmers qualified trees as essential in the lives of the families. However, they also pointed out that open, extensive meadows found at higher altitudes were crucial for the animals as well. Livestock rearing and traditional cultivation methods described by the interviewees created a landscape mosaic with various species-rich grasslands. Such landscapes were the result of labour-intensive agricultural land-use.

The farming system of the valley was based on a strong predominance of smallscale livestock farms (cattle, sheep and goats, linked with cattle breeding) when rearing animals was a major activity. This was a prosperous activity for the valley's livestock farmers grew corn for the family, the poultry and other animals. No-one in this valley lived from crop farming alone as yields were low. Peasants used to go onto communal grazing lands to collect bracken to use as bedding for animals. Most of the older stakeholders and farmers insisted on the presence of a large rural population involved in agricultural activities in the valley. This age-old agroforestry that shaped the landscapes of the area was also linked to the economic activity of slate quarries. The availability of what the respondents used to call "high-quality slate schist" was responsible for the presence of three slate quarries in the valley. Their activity was linked with the agricultural decline that started 70 years ago. The population began to abandon unprofitable agriculture to work in the slate quarries, which were hiring manpower. All the respondents who knew this period were unanimous in stating that at least one member of each family was employed there in order to improve the family income, and managed to combine this job with the farming activities.

It was underlined that the valley has later underwent significant depopulation and a decline in traditional farming and agroforestry practices. Many farmers have left the valley and their activity to look for work in the cities, so the agricultural land of the valley has gradually became abandoned. The depopulation of the valley was reported by various stakeholders (and could also be assessed from data provided by the village), some of them specifying that they had witnessed the disappearance of one third of its farmers in the last 20 years. Some farmers remain, but have not been able to maintain the agroforestry structure. Now they only grow fodder crops. Livestock breeding is also declining, even though most of the remaining farmers still continue some pastoral activities. However it was mentioned that the herds that can be seen in the valley no longer belong to the valley's breeders only. Hence the farmers are finding the pastoral activity overly constraining and are withdrawing from it.

Interviews revealed that nowadays, half the inhabitants of the valley are said not to be farmers. While many farming families have gradually left in the past two decades, neo-rural residents have recently been settling in the valley and buying some of the old farms. These new settlers are attracted by the proximity of the town where they work, the lower price of land, and the landscapes. They are mostly people who have come to live in the area but have other professional activities in the nearby town of Bagnères-de-Bigorre or its surroundings.

A decline of the agroforestry complex: factors perceived as favouring invasiveness

The narrative data collected through the interviews about the evolution of landscapes and practices highlighted what species were considered as invasive by the respondents and the factors behind their spreading in the valley. Two types of drivers were identified: the first is linked with the farming activity, and the second with the slate mining.

i/ The set of explanations given first and foremost concerned the gradual abandonment of agricultural activity in connection with the general depopulation of the valley. Hence only the few farmers remaining in the valley still help to maintain the landscape through pastoralism, and the presence of trees in the landscapes bears witness to their past use in agroforestry. As the hedges are no longer being exploited or managed, many trees (such as ash [Fraxinus excelsior L.] or birch [Betula L.], the tree species that were most easily named by the respondents) were pointed out as having grown spontaneously from hedgerows.

Here, the feeling of invasiveness is expressed to qualify the development of this vegetation which has gradually colonized the abandoned fields and meadows, contributing to a "landscape closure" by encroachment of natural afforestation. A young livestock breeder said "I like a valley when it's clean and tidy. You say to yourself, "Here, they worked". Arriving in a valley like ours, which is wooded everywhere, you say to yourself, "But there are no farmers here, what's going on? ».

Questions about the family history and activities of the interviewees showed that many farming families have left the valley to settle and seek work in cities. In the majority of the farming families who have decided to stay in the valley, at least one member has been compelled to develop a second activity in order to supplement their income because neither the farm nor the quarries can ensure a livelihood for their families, as mentioned earlier. And this reduces the time they can invest in maintaining the landscapes, a situation again favouring the spread of unwanted vegetation. On the one hand, the number of people able (and willing) to maintain the landscape as it was in the past is decreasing and, on the other hand, potential young farmers are discouraged from settling here (by many obstacles, such as high investment costs). In consequence new establishments are rare. The land left vacant following the cessation of agricultural activity has mainly been bought or rented by farmers from outside the valley. The local farmers who mentioned that point were complaining that these non-resident farmers do not have the same practices (the practices of the newcomers being considered more extensive and failing to maintain the landscape of the valley as finely as they themselves do). The last correlated argument expressed to explain the spread of invasive vegetation is the lack of mechanization possibilities in this mountainous area: pasture lands cannot be maintained and are gradually being abandoned, part of the reason being because the steep slopes make mechanization impractical. To sum up this first set of factors, "Landscape changes have occurred because man was no longer there to control nature", as a mayor put it.

ii/ The second type of explanation for the feeling of invasiveness is linked with the presence of the slate mining quarries. It was mentioned that, in addition to the weakening of agriculture, international competition has put an end to the slate mining activity. The three slate quarries, which had become economically unprofitable in their turn, have gradually closed (two of them 20 years ago and the other 5 years ago). These sites, left abandoned, have been pointed out as the first places to be invaded by Spiraea japonica. Other IAS, such as Reynoutria japonica, Impatiens glandulifera (Fig. 2) and Buddleja davidii [note: most interviewees could recognize and show them in the valley, but few were able to name them, either by a scientific or a local name] were also mentioned on the slate quarry sites, but mostly for having invaded the banks of rivers and roads. The consequence of this double dynamics is that woods, brambles (Rubus fruticosus L.), bracken (Pteridium aquilinum L.) and the various invasive species have also gradually spread into the abandoned agricultural plots, contributing to a profound change in the landscapes (Figs. 3 and4). Most interviewees, especially the elderly, identified the slate quarries as the cradle for the development of the invasive alien species currently present in the valley, through the extensive movement and transport of soil and rock. The subsequent cessation of slate quarry activity left large areas of "disturbed" schist that would then have favoured the installation and dispersal of these species. The spread of IAS to the forest understorey as well as to the agroforestry plots and meadows was facilitated because the vegetation was no longer managed through the agricultural activity. The abandonment of agroforestry practices gradually facilitated IAS invasion in the landscape. However, none of the respondents were able to assess the process and rapidity of the invasion by IAS more precisely.

A contrasting perception of invasive alien species (IAS)

The emergence and development of several IAS in the valley (mainly Reynoutria japonica, Impatiens glandulifera, Buddleja davidii and Spiraea japonica) have contributed to the landscape changes and are seen as an environmental impact of the decline of agriculture. Inductive analysis of qualitative data compiled from interview transcriptions revealed three types of perceptions concerning the IAS in the valley: two took an environmental standpoint (but oriented towards different views), and one was heritage oriented. i/ Protecting habitats: a conservationist view For environmental technicians and managers, and also for ecologists involved in different associations, IAS are considered as species to be eradicated. In their opinion, these species, because they are exotic, should not be there. These people are convinced that the presence of such plants is evidence of a loss of biodiversity. They do not consider IAS as a possible additional component of landscape diversity but as a sign of habitat degradation because, in some spots, IAS dominate the landscapes of the valley. They also fear that their expansion will become uncontrollable. Only a minority is of the opinion that biodiversity also includes these species and that it is sufficient to avoid their excessive spread.

ii/ A so-called "wilderness": aesthetic attractiveness for a neo-rural population Most of the neo-rural people interviewed were not deeply aware of the local history and activities of the valley. One of the motivations of these interviewees, who mostly worked in the nearby town, to settle in this valley was what they termed its "wilderness" quality. For them, the valley looks "wild" due to its forest landscapes. They do not realize that what they call "wilderness" is, in fact, not natural but the result of abandonment of traditional agroforestry practices. It is now the "wild" character conferred by the natural afforestation and the aesthetic value of these IAS (qualified as "colourful and flowery" because Spiraea japonica and Impatiens glandulifera in particular colour the valley pink during summer) that make the valley attractive for some neo-rural people who have decided to settle here, as well as for tourists, who do not know these plants and think that they have always been present in the valley (according to reports).

iii/ What "invasive species" means: the point of view of farmers and the elderly Farmers, on the other hand, do not care much about IAS. However, all the farmers we met stressed that, at present, mostly brambles, but also bracken and various trees, are invading the land very quickly. This factor was stressed as the main argument explaining the spread of a dense vegetal cover: "The vegetation swallows up everything, it's moving at breakneck speed" (an old, long settled resident). Farmers no longer use bracken for animal bedding and, to have good, clean grazing lands, brambles and bracken have to be eradicated, which is difficult and costly. It is essentially brambles and brackens that they consider as invasive species to be eradicated, because they overcome the vegetation of the grazing lands: "It is the brambles that cause the invasion..." (a livestock farmer). They are, in fact, nostalgic for the very well maintained landscapes of the past that testified to agricultural work and a rural way of life that no longer exists "The hedges, they were trimmed, it was neat" (an old farmer). For some of those concerned, brambles, ferns and IAS are simply reminders of the past landscapes to which they were attached and which have now disappeared. For others, they evoke fantasized, wild landscapes. Finally, only beekeepers see either brambles or IAS as a resource, since they increase the honey potential of their bees.

The difficulty of mapping invasiveness

In this section we will consider the results of the mapping method tested. To assess the rapid spread of IAS, mentioned by most of the stakeholders interviewed, and to alleviate the problem of a lack of available data for mapping these species, highlighted by ecologists, we attempted to carry out a fine mapping of an invaded pastoral area of the valley (Fig. 1C).

Mapping of IAS has mostly been achieved on a limited scale so far and based on occasional botanical records. The few existing IAS maps have been drawn up on the basis of spot surveys (SI FLORE database) or established by botanical inventory directly in the field but with very limited geographical extents (at best several hundred linear metres) due to the complexity of the work. Yet mapping patterns of invasive plants requires metre or sub-metre spatial resolution to capture the complex nature of the invasion process in order to understand its spatial dynamics. The use of a UAV was therefore preferred. Airborne technology with a drone (UAV) equipped with a multispectral sensor enables a sub-metre land-use map to be produced by remote sensing and, in particular, allows targeted invasive plants to be extracted. It was first used on an open space of grazing land colonized by Spiraea japonica, near one of the old slate quarries.

Detection of invasive species such as Spiraea japonica was especially used to identify the changes in habitats and agroforestry landscapes. The first observations of Spiraea japonica in the selected area (Fig. 1C) were made by the interviewees in the 1950s but, as mentioned above, these plants have been expanding spatially in this area since the early 2000s (Table 2), as also recorded in the SI FLORE data base (http://siflore.fcbn.fr).

Table 2. % of the total area surveyed

The mapping provides milestones for characterizing "landscape closure" by natural afforestation. The image in Fig. 5B shows the results obtained from an orthorectified aerial image (Fig. 5A) by classifying the bracken and brambles that have accompanied the natural afforestation since 2013. For 2013, we did not have accurate information about the presence of Spiraea japonica. Consequently, due to the absence of previous field data, we were unable to distinguish Spiraea japonica from brambles or bracken and therefore grouped them together in a single class because our botanical surveys show that, in its initial stage, Spirea develops preferentially under the cover of these plants.

The image in Fig. 5D shows the result obtained with a UAV (Fig. 5C) for the classification of Spiraea japonica in 2018. The decimetre resolution obtained with the UAV provides images of the spatial distribution of the invasive Spiraea japonica plants that are not visible on conventional aerial photos at a resolution of 40 cm. The classification based on the drone image shows 88% of Spiraea japonica, and 56% of bracken/brambles. The understanding of this fine spatial dynamics of the Spiraea invasion is still in its initial stages because the survey of this testing area by UAV only started in 2017. We will continue to conduct drone flights every year to understand its evolution. The advent of UAV technologies will allow us to investigate the dynamics of invasion further via multi-date annual imagery whereas, until recent years, we could rely only on archives of aerial photographs.

Discussion

Reviews of the literature on land abandonment in European mountains assess its environmental impacts and the decline in traditional farming practices (MacDonald et al. 2000;[START_REF] Antrop | Why landscapes of the past are important for the future[END_REF]). The homogenization of biodiversity as a result of the introduction and migration of species is a global change that profoundly alters the functioning of terrestrial ecosystems [START_REF] Macdougall | Are invasive species the drivers or passengers of change in degraded ecosystems?[END_REF][START_REF] Moser | Impacts of nonnative invasive species on US forests and recommendations for policy and management[END_REF]. Some authors underline its negative impacts on economic and agricultural activities [START_REF] Perrings | Biological invasion risks and the public good: an economic perspective[END_REF][START_REF] Paini | Global threat to agriculture from invasive species[END_REF]) and its contribution to the vulnerability of human populations [START_REF] Andersen | Risk assessment for invasive species[END_REF]). However, the results presented here show that, even if invasive alien plant species are part of the processes of natural afforestation in the study area, they are not perceived negatively by all the stakeholders. The presence of invasive species in this valley is a symptom of two interrelated phenomena: land abandonment and anthropogenic disturbance, but mapping the spatial extent of these species and assessing their evolution remains a major challenge for the management of the territories concerned.

The challenge of assessing the evolution of landscapes and ecosystems

In terms of sustainable development, an approach to invasiveness in agroforestry landscapes requires a precise diagnosis of the spatial extension of invasive plants. This could be obtained by drawing up local maps of IAS distribution in order to compare them with vernacular stakeholders' knowledge. The presence/absence of the species is not enough, even though the inventory of targeted taxa in databases is useful in a first assessment of this environmental issue. It would then be necessary to produce maps reflecting the reality at infra-metre resolution, which could be utilized for discussion in order to confront it with the perceptions of invasive plants voiced by local stakeholders and impacted populations. This question could, hopefully, be answered by the use of multi-date UAV imagery for better monitoring of IAS expansion [START_REF] Martin | Using single-and multi-date UAV and satellite imagery to accurately monitor invasive knotweed species[END_REF].

The success of large-scale control programmes for established invasive species is challenging to evaluate because of spatial variability in expansion rates [START_REF] Pepin | Accounting for heterogeneous invasion rates reveals management impacts on the spatial expansion of an invasive species[END_REF]. Mapping the spatial expansion of invasive species is highly strategic for future management but is also a technical challenge in agroforestry conditions since the presence of trees complicates the use of drones for surveys. That is why the challenge for the next few years will be to combine different UAV technologies in order to ensure high-resolution mapping of the invasiveness from the open landscape to the denser tree cover over large areas (> 200 ha). This will be made possible by multi-date remote sensing combining two types of UAVs: long-range fixed wing vehicles and multicopters equipped with LIDAR sensors for areas located in tree-covered areas of agroforestry landscapes [START_REF] Dash | Early Detection of Invasive Exotic Trees Using UAV and Manned Aircraft Multispectral and LiDAR Data[END_REF]).

In the event of natural afforestation and loss of agroforestry structure, such data may provide a better understanding of how invasive species are expanding spatially. This would also be very useful for making appropriate management choices. But this raises the question of the reference state that will be used as a basis for future management, and the time that will elapse between successive recordings, in order to observe the evolution. This will require not only technical means but also human resources to carry out the analyses of the images that will be taken. The budget required will be considerable, especially if the areas to be monitored are large.

From the work described here, we can also affirm that high-resolution mapping will become one of the major challenges in characterizing the processes that allow invasive plants to spread, the aim being to manage them in the best possible way in order to maintain the sustainability of agroforestry landscapes. At present, this approach, which is still in its infancy, has limitations that will have to be overcome if it is to be used over larger areas. The technique is currently very place-specific, whereas a reflection in terms of sustainable landscape management needs to be undertaken on a broader scale. Nevertheless, the results can provide much essential quantitative and qualitative information about IAS that will be very useful not only for ecologists but also for decision makers responsible for land-use management. Measuring invasiveness dynamics in the landscapes is therefore essential. But the difficulty of assessing the expansion of invasive plants lies not only in the spatial aspect; ecological analyses are largely influenced by stakeholders' contrasting perceptions of the phenomenon and the species.

Social perceptions of agroforestry and invasive plants: whose sustainability?

Manners of perceiving the landscape can be many and varied. The interviews provided an opportunity to understand the main constraints and potentialities perceived by the participants, and thus to explore the landscapes perceived by individual stakeholders and their close connection with the environment. Research in anthropology as well as in geography [START_REF] Buijs | From Hiking Through Farmland to Farming in a Leisure Landscape: Changing Social Perceptions of the European Landscape[END_REF][START_REF] Tilley | Anthropology of landscape. The extraordinary in the ordinary[END_REF][START_REF] Soudière (de La) M | Arpenter le paysage. Poètes, géographes et montagnards[END_REF] shows how the landscape is constructed by the different perceptions that individuals have of it. Mountain agroforestry systems can play a major role by providing high nature value and maintaining the countryside. But the diversity of stakeholders, who do not necessarily share the same conservation values regarding a territory, is a major issue. Hence approaching this question through IAS is a source of learning in terms of sustainable landscape management.

The interest of any study of invasive alien species obviously concerns the consequences of their expansion, both on the environment and on human activities. However, this subject remains highly controversial in the scientific community itself. Some authors describe these species as "ecological threats" or "harmful" (Gobster 2005;[START_REF] Marbuah | Economics of Harmful Invasive Species: A Review[END_REF][START_REF] Doherty | Multiple threats, or multiplying the threats? Interactions between invasive predators and other ecological disturbances[END_REF], while others see actions against these invasive alien species as a form of racism or xenophobia [START_REF] Simberloff | Confronting introduced species: a form of xenophobia?[END_REF][START_REF] Lefeuvre | Plantes envahissantes, attention aux belles étrangères[END_REF][START_REF] Tassin | Espèces invasives/envahissantes[END_REF].

The notion of invasiveness is assessed in a subjective way and we have seen that the value attributed to a species depends on the social perception of the phenomenon. For example no stakeholders interviewed referred to black locust (Robinia pseudoacacia), which is also widespread in the valley. Yet this tree, which was one of the first North American trees introduced into Europe, is ranked among the top plants in Europe having the highest negative impact. In many places it is considered as a part of the rural landscape, economy and culture, having positive aspects such as nitrogen fixation, wide habitat tolerance, fast growth and vigorous vegetative propagation. So the same properties that make Robinia attractive for cultivation are also the source of problems in nature conservation and environmental management (Vitkova et al. 2017).

Agroforestry and alien species management: towards which sustainability?

The difficulty also lies in the fact that the notion of invasiveness is related to what we know (about the proven -or supposed -threats of these plants, for example, and according to the context) but also to how each stakeholder wants to use the environment. For example, managers of natural areas often encounter public misunderstanding of this problem and it can even become an obstacle to action [START_REF] Lefeuvre | Plantes envahissantes, attention aux belles étrangères[END_REF]). The time frame for a manager is very narrow because it is easier to act early to be more efficient and minimize costs (Rejmanek and Pitcairn 2002). If it is already difficult for them to act on public spaces, it is even more problematic on private lands, where they have little authority or influence. Thus, abandoned plots of land can quickly present a high potential for invasions. This is what we observe in the dynamics of the Oussouet valley. The choice of species introduced into an agroforestry system, the management of the plot, and the species present in the surroundings can constitute sources of invasion when the system is no longer maintained. Many authors point out that IAS can cause significant damage to human activities, not only in ecological terms but also in economic ones [START_REF] Simberloff | Confronting introduced species: a form of xenophobia?[END_REF] but scientists themselves are not unanimous on this point [START_REF] Atramentowicz | Les invasions biologiques, une question de natures et de sociétés[END_REF][START_REF] Simberloff | Impacts of biological invasions: what's what and the way forwards[END_REF].

For Udawatta et al. (2019:14) "Agroforestry can promote undesirable species including invasive species if proper measures were not implemented". Based on our results we can also argue that, although agroforestry is seen as a key agrosystem, valuable in terms of sustainability, this is only the case if these landscapes are appropriately maintained, mainly through farming practices. The evolution observed in the Oussouet valley is rich in lessons for many other territories. We have seen that agroforestry landscapes are not immutable, and abandonment of farming practices can be detrimental to them. However, the invasion of these landscapes by exotic species following their abandonment is not necessarily a bad experience for all stakeholders. It is therefore necessary to ask ourselves what sustainable development means: what reference system should we use as a basis? Sustainability as seen by an ecologist will be different from the perception of a farmer or a manager. This consideration is important for decisionmakers and a compromise has to be found between economy, ecology and society, while taking the spatial and temporal dimensions into account [START_REF] Antrop | Why landscapes of the past are important for the future[END_REF][START_REF] Maire | Télédétection de la trame verte arborée en haute résolution par morphologie mathématique comme aide à l'acceptabilité locale des politiques publiques environnementales[END_REF][START_REF] Guillerme | Landscapes of Non-woodland Trees; landscapes which reveal the challenges of sustainable development[END_REF].

Conclusion

In a context of unprecedented environmental crisis, sustainability science has emerged as a new field of research. It aims to create and apply knowledge to support decision-making for more sustainable development [START_REF] Clark | Sustainability science: The emerging research program[END_REF][START_REF] Komiyama | Sustainability Science: building a new discipline[END_REF][START_REF] Lang | Transdisciplinary research in sustainability science: practice, principles, and challenges[END_REF].

Invasive species represent temporal, spatial and human issues for agroforestry, which is an emblematic response promoting a sustainable approach to landscape management. In the study area of the Oussouet valley, the decline of agroforestry by land-use abandonment has favoured the spread of invasive species, as the landscapes are no longer being maintained. New management strategies are difficult to undertake due to the contrasting perceptions of the different stakeholders as to what is considered as invasive and what has to be protected or eradicated.

Our intention was to carry out an assessment of the sensitive dimension of interviewees' perception of the landscapes and the changes induced by the presence of invasive species by initiating a fine mapping process in order to enrich stakeholders' interviews with precise data. The spatial expansion of IAS observed by UAVs gives very precise results, but does not allow us to understand which territorial dynamics it belongs to (field validations not allowing such global understanding). However, this understanding is possible thanks to detailed surveys conducted among local stakeholders. Conversely, social surveys alone cannot accurately spatialize the extent of IASs, their spatial dispersion or the speed at which they spread. This is possible with high-resolution mapping conducted over time. The interdisciplinary approach proposed, combining these two different and complementary methods, represents an asset in understanding what becomes of traditional agroforestry systems when they are left abandoned.

The spread of invasive species is a symptom of such abandonment of practices, and poses challenges in terms of sustainable development. In order to respond to these challenges, it is necessary to consider the diversity of stakeholders with a view to revitalizing agroforestry systems and the multifunctional landscapes associated with them, to get them out of abandonment. This implies assessing the spatial expansion of IAS but also the usefulness -or otherwise -of these invasive plants without qualifying them as threats from the outset.

Fig. 1 .

 1 Fig. 1. Location of the study area in France (A), the presence of Spiraea japonica in the Occitania region (B) and the survey site for UAV capture (C) of Spiraea japonica (D) [The presence of the plants was mapped from the Siflore database (National Botanical Conservatory -France)]

  These supervised classifications, based on the random forest algorithm developed by Breiman in 2001, enabled the elements of a dataset to be quickly labelled from a set of previously known examples. To generate the classification mode used with ortho-photographs, we took the same data set to provide examples for all years. The data were collected in the field in 2018, photo-interpreted on images from 2001 and visually verified for all years.

Fig. 2 .

 2 Fig. 2. Abandoned meadow, totally invaded by Impatiens glandulifera and subject to spontaneous reforestation

Fig. 3 .

 3 Fig. 3. Traditional agroforestry landscape in a process of abandonment materialized by the expansion of tree components in the fields, and the presence of brambles, bracken and invasive species like Spiraea japonica (Upstream of the Oussouet valley, Labassère, Hautes Pyrénées).

Fig. 4 .

 4 Fig. 4. Evolution of traditional agroforestry landscapes of the Pyrenees (Oussouetvalley, Trébons village, France, 1948-2016). The "landscape closure" process by natural afforestation (B, 2016) is a result of the previous agricultural and pastoral activity(A, 1948) that maintained them and avoided their being abandoned, which also favoured overgrowth by various invasive species (IGN aerial images).

Fig. 5 .

 5 Fig. 5. Images obtained by classification of brambles, bracken and Spiraea japonica in the field area (described in Figure 1): A) Aerial ortho image taken in 2013; B) Detection of bracken/brambles in 2013 superimposed on image A; C) UAV ortho image taken in 2018; D) Detection of bracken, brambles and Spiraea japonica in 2018 superimposed on image C.

Acknowledgments

This research was funded by CNRS through the projects INVASCAPE (GEODE), EI2P (Région Occitanie) and INVAVISION (PEPS TOHMIS CNRS). We are grateful to the Botanical Conservatory for its help, especially to J. Dao, and we warmly thank the villagers of Oussouet.