Dr Olivier Henrotte

Alice Boudet

Dr Ndrina Limani

Dr Philippe Bergonzo

Dr Bacem Zribi

Dr Emmanuel Scorsone

Dr Bruno Jousselme

Renaud Cornut

Steady state electrocatalytic activity evaluation with the redox competition mode of SECM: the interests of a gold probe and a boron doped diamond substrate

Keywords: Boron doped diamond, Electrocatalysis, ORR, Redox competition mode, SECM

In the current context of energetic transition, investigations of alternative complex systems require tools as scanning electrochemical microscopy (SECM) offering interesting opportunities as an electroanalytical technique to evaluate innovative catalysts. Herein, we demonstrate how a judicious choice of probe and substrate materials opens up improved performances to achieve steady state measurements for oxygen reduction reaction (ORR) catalytic activity detection through the redox competition scanning electrochemical microscopy (RC-SECM).

On the probe side, we show that using gold enhances the stability of the local oxygen concentration detection in comparison to the regularly used platinum one. On the substrate side, we evaluate boron doped diamond as an appealing alternative to classical support substrate, that shows a low ORR activity, high stability and very good reusability. This work introduces an alternative approach for quantitative measurements with SECM, improving measurement easiness, comfort and reproducibility, thus paving the way towards standardized benchmarking and numerical simulation-based parameter extraction.

Introduction

The global energetic transition requires innovative tools enabling the evaluation of alternative materials and systems as candidates to perform energy. Fuel cells, and proton exchange membranes, for instance, are envisioned to play a key role as an efficient alternative technology. They are considered worldwide with high interests and could be easily adopted. However, the variety of catalysts presently under consideration by the community generates a high demand to enable their comparative characterization. Improved tools would help in their discrimination [1,2] . Presently characterization methods are the limiting part of innovation and improvements, particularly when active carbon based nanomaterials are concerned [3] . During the past decades, local probe techniques, such as scanning electrochemical microscopy (SECM) have made great progress. SECM already provides impressive results in the investigation of local electrochemical properties [4] . SECM is an electroanalytical tool that consists of a four-electrodes setup: two working electrodes (WE), namely a micrometric probe as an electrochemical sensor and a substrate to polarize the investigated material, a counter electrode (CE) and a reference electrode (RE). A potential can be applied on both the probe and the substrate while the probe is moved in the close vicinity of the substrate. The versatility of the technique allows it to be used in many different fields such as electrocatalysis [5,6] , corrosion [7] , photoelectrochemistry [8] or even DNA detection [9] or archaeological remains investigations [10] .

In the global research context of energetic transition, SECM has been used for the investigation of materials involved in energy related systems (batteries [11] , solar cells [12] , supercapacitors [13] , etc…), in particular for fuel cells (e.g. direct methanol fuel cell [14] , polymer electrolyte fuel cell [15] , proton exchange membrane fuel cell (PEMFC) [16][17][18] , etc…). The investigations here mainly focus on hydrogen evolution reaction (HER) [19] and more particularly on oxygen reduction reaction (ORR) [6,16,20] due to its slow kinetics, which constitutes a key point regarding the adoption of fuel cells at a large scale.

Regarding electrocatalytic activity assessment, the redox competition (RC) mode of SECM, introduced by W. Schuhmann's group in 2006, is a recognised reference protocol [21] . It consists in polarizing the probe and the substrate in such a way that the same reaction occurs on both sides: the probe then evaluates the local reactant depletion due to its consumption by the electroactive sample (as shown in Figure 1). A scan over the surface thus reveals the local activity variations. RC mode has already been used to visualize the cell breathing [22] , to understand corrosion mechanism on CrN film [23] , to study the consumption of oxygen from zinc oxide formation [24] or even to determine more accurately enzymatic kinetics [25] thanks to previous modelling studies of such a system [26] . For ORR catalysts, it operates in such a way that the scanning electrode is used as a probe of the local O2 concentration [18,21,27] . However, the use of the technique still remains mostly limited to the SECM community, where studies are usually proof of concepts. We foresee that the expansion of the technique to quantitative studies could provide better knowledge of many catalysts that are currently being investigated within the context of the energetic transition. In fact SECM, and RC-SECM in particular, are presently underused by the community, in comparison to the benefits the technique can provide, namely its high resolution [28,29] .

The lack of a comfortable configuration that would enable better ease and reproducibility, and particularly in complex conditions such as in acidic media to be compatible with PEMFC technology, is a key obstacle to the development of the RC mode approach. Herein, we show how the choice of the probe and substrate materials can help to stabilize the experimental configuration and enable steady state measurements while scanning the surface.

Up to now, Pt microelectrodes are the most frequently used for the RC mode [18,21,27,30] , which is a rational choice considering the established electrocatalytic activity of this material towards ORR. However, Pt presents some drawbacks, for instance measurement instabilities are common at high probe current densities, and further it displays a high sensitivity to impurities that might be present in the solution. Subsequent alternatives such as the use of potential pulses [31] or high scan speeds [27] are required, but that may significantly hinder quantitative studies. On the other hand, gold as a probe metal has been used for other SECM measurements [16] , but -to the best of our knowledge-not for ORR detection. Here, we show that the low catalytic activity of gold is not an issue for ORR detection, which makes it suitable for studying ORR in RC mode, and even in acidic media.

In theory, the high sensitivity of the RC mode enables the evaluation of very low catalytic activities, however in this case, the substrate activity is likely to enter in competition with the catalytic material [32] . Overall ORR studies are usually performed using glassy carbon (GC) substrate [30,33,34] . Boron doped diamond (BDD) catalytic activity toward ORR has been investigated [35] , as other carbon based materials [36] . Furthermore, BDD has been used in SECM as a probe [37] or a studied material [35,38] , but not as substrate for ORR catalytic activity determination. BDD has shown higher overpotential for ORR, higher stability and lower H2O2 production in acidic media compared to other carbon based substrates [39] . Here we demonstrate that BDD is an interesting alternative to standard carbon based substrates. We compared it to a GC substrate, due to its high representation in the literature, and a Siwafer substrate covered with gold, as it is easy to produce, with good reproducibility and permits to achieve highly flat surface. The catalyst used to illustrate the potentiality of the new setup is a noblemetal free material based on carbon nanotubes (CNT) annealed with cobalt and nitrogen precursors [40] .

Experimental Materials

All chemicals and solvents of research grade were purchased in the highest purity from Sigma Aldrich and used as received. All gases (nitrogen, oxygen) were of 99.995% purity. Commercial grade NC3100 (purity >95%) multi-wall carbon nanotubes were obtained from Nanocyl (Belgium).

BDD substrate preparation

BDD was grown onto highly doped 4 inches silicon substrates by microwave plasma enhanced chemical vapor deposition (MPECVD) technique in a Seki Diamond AX6500 diamond growth reactor in a hydrogen plasma containing 1% methane as the source of carbon and trimethylboron as dopant. The resulting boron doping level is approximately 2x10 21 cm -3 as determined by secondary ion mass spectrometry measurements. The polycrystalline diamond film obtained is approximately 1 micron thick.

BDD substrate cleaning process

BDD substrates were immerged into a piranha solution during 30 minutes and then in pure H2SO4 at 300°C for 30 minutes. Afterward, KNO3 was added into the solution until a yellow coloration started to appear and the substrates stayed still 30 minutes more in the 300°C solution. Then, the substrates were rinsed into a 300°C pure H2SO4 solution during 10 minutes and finally rinsed with distilled water.

In the case there was any doubt of catalyst residual traces on the substrate, a micro-wave hydrogen plasma exposure of the BDD surface, at a temperature of 600°C, was used to perform a complete reclaim of the BDD native surface.

Gold substrate preparation

Gold substrates were obtained by vacuum evaporation in a Balzers BAK 600 evaporator: a thin interlayer of chromium (to enhance gold adhesion on glass) and pure gold (99.99 % from Williams Advanced Materials) were evaporated at room temperature on silicium wafers. Prior to evaporation, the silicium wafers were rinsed 10 min under ultrasonication in water, ethanol and acetone successively. The thickness of the deposited layers was 3 nm of chromium and 30 nm of gold monitored in-situ by using a quartz crystal microbalance.

Catalyst preparation

The catalyst was prepared as described previously [40] . Briefly, Co(NO3)2.6H2O, multi-wall carbon nanotubes (MWNTs) and triazolopyridine (TAPy) were mixed in ethanol and sonicated for 30 minutes.

Ethanol was removed under low pressure and the black Co-TAPy/CNTs powder was pyrolyzed at 950°C during 2h under argon. This catalyst will be called Co-N-NTC.

Catalyst deposition

The catalyst ink was prepared by dispersing Co-N-NTC powder (20 mg) with 5% in wt of D-520 Nafion in ethanol (1 mL) under sonication (30 min) with a cup-horn coupled with a Vibra-Cell (VCX 130 PB from Sonics Material). Afterwards, the total volume is increased with ethanol to 2 mL and the process is repeated. The same process continues by increasing the total volume to 5/10/20/50/100/200 mL to obtain a catalyst ink of 0.1 g.L -1 .

The catalyst spot was obtained by two methods. The first one consists of dropping 2 mL of the catalytic ink onto the substrate heated at a temperature of 100°C with a micropipette. The second method uses the ExactaCoat apparatus from Sono-Tek to pulverize the solution onto the substrate. A mask with micropatterns (from Micron Laser Technology) was used to obtain a controlled square spot in size and volume of solution deposited.

Instrumentation AFM and SEM measurements

AFM images were performed on a Veeco Dimension 3100 equipped with a Nanoscope IIIa controller and analysed with the software Gwyddion. SEM images were recorded with a Hitachi S-4500 scanning electron microscope.

Electrochemical measurements

Electrochemical measurements were performed in sulfuric acid (Sigma Aldrich). The gold and platinum microelectrodes from Sensolytics had an active radius of 5 microns and a RG (the ratio between the inactive part radius and the active part radius of the probe) of 30. SECM experiments were performed on a modified M470 SECM Workstation from Bio-Logic Science Instruments. A four-electrode setup was used for the SECM experiments. It involved a platinum or a gold microdisk as first working electrode, a GC, a gold or a BDD substrate as second working electrode, a saturated calomel electrode (SCE) as reference and a net made of platinum wires as counter electrode. Both reference and counter electrodes are placed in sintered guards to avoid direct contact between the analysed solution, the reference and the counter electrodes. The probe-substrate distance was evaluated by approaching the probe in the vicinity of the area of interest, somewhere without catalyst, and by letting the substrate at open circuit potential (OCP). In this case a negative feedback was obtained, and a comparison with theory provides the relationship between the probe position and the probe-substrate distance [41,42] .

Unless mentioned, the analysis of the ORR activity was made in H2SO4 0.1 M solution. The probe was stabilized 300 s before each measurement.

Results & discussion

The working electrodes are central to the SECM technique. Improving both of them to specific demands allows progresses in the quality of measurements. First, in order to investigate the first working electrode, i.e. the probe, a linear sweep voltammetry (LSV) is performed from OCP up to the solvent reduction signal, in Ar saturated solution and O2 saturated solution. At this step, the probe is put in solution without the substrate to avoid any interaction. As shown in Figure 2.a, a well-defined plateau is observed between -0.2 to -0.4 V/SCE for the ORR at the platinum probe, illustrated by the green bar. Several potentials of this plateau are then tested by chronoamperometry (CA). Each potential is applied for 600 seconds, in order to investigate the stability of the measurement. The probe is polished and cleaned between each measurement and the procedure is repeated several times in the O2 saturated solution. The results presented Figure 2.b show the currents measured at -0.2, -0.3 and -0.4 V/SCE. For the three potentials, an exponential loss of current is observed, followed by a stabilization of the slope to a non 0 value. This behaviour is not the one expected from a well-defined diffusion plateau as observed on the CV. Furthermore, the current variation during the first 200 seconds is not exclusively due to the establishment of the diffusion profile in the vicinity of the probe, as this is a process that has time constant of typically seconds [START_REF] Mirkin | Nanoelectrochemistry[END_REF] . It is also related to the evolution of platinum surface states, with possibly some contamination due to side reactions. Furthermore, the current densities are not the same along the probe surface increasing the complexity of such phenomenon. In any case, the observation is in accordance with what is observed in the literature [21] . In any case, the important element for SECM imaging is to have a steady-state current at the probe. The Pt probe current was the most stabilized after 300s with a linear loss of current. After this stabilization step, the sensitivity, the detection threshold and the instability can be then evaluated. These values are shown in Table 1. We evaluated the theoretical sensitivity by using the equation of the current at a diffusion plateau [START_REF] Aoki | [END_REF] . In the equation 1, n is the number of electrons exchanged (4 in the case of a complete O2 reduction), F is the Faraday constant, D0 is the diffusion coefficient of O2 in H2SO4 (1.4x10 -5 cm².s -1 for 0.5M) [45] , C0 is the concentration of O2 in H2SO4 (in the case of an O2 saturated H2SO4 0.1M solution, it is 1.27x10 -6 mol.cm -3 at 298K) [46] and rT is the radius of the active part of the electrode. The theoritical sensitivity is 10.8 nA/mM(O2). The sensitivity measured at t600 is 1.0, 2.2 and 4.3 nA/mM(O2) for -0.2, -0.3 and -0.4 V/SCE respectively, which are significantly lower values compared to the theoritical one. It can be noticed that the sensitivity measured at t0 is 8.3, 5.9 and 6.4 respectively which is closer to the theoritical one.

𝐼 = 4𝑛𝐹𝐷 𝑂 𝐶 𝑂 𝑟 𝑇

(1)

We decided to consider the detection threshold as the O2 concentration for a current equal to the current measured in absence of O2 in solution. This is calculated by the equation 2 where 𝐼 𝑂 2 is the current measured in the O2 saturated solution and 𝐼 𝐴𝑟 , the current measured in the Ar saturated solution. For -0.2, -0.3 and -0.4 V/SCE, the detection threshold is 47, 31 and 46 µM(O2) respectively.

𝐷𝑒𝑡𝑒𝑐𝑡𝑖𝑜𝑛 𝑡ℎ𝑟𝑒𝑠ℎ𝑜𝑙𝑑 = 𝐶 𝑂 𝐼 𝑂 2 × 𝐼 𝐴𝑟 -1 (2)
Besides, the instability of the measurement is evaluated by considering the current difference between 480 and 600 s to have a well-defined slope compared to 300 s where the stabilization occurs (see equation 3, where I600 is the current at t600, I480 is the current at t480 and 𝐼 𝑚 is the averaged current between t480 and t600).

𝐼𝑛𝑠𝑡𝑎𝑏𝑖𝑙𝑖𝑡𝑦 = | 𝐼 600 -𝐼 480 𝑡 600 -𝑡 480 × 1 𝐼 𝑚 | 𝑥100 (3)
The calculated instability is expressed in %/min which corresponds to the current percentage loss every minute of the measurement. In the case of the Pt probe, the instability is 0.44, 0.62 and 0.44 for -0.2, -0.3 and -0.4 V/SCE respectively.

Exactly the same procedure is applied to the gold probe. The LSV and the CA are presented in Figure 2.c and Figure 2.d respectively. In this case, a pseudo-plateau between -0.4 to -0.7 V/SCE is observed (illustrated by the red bar) in the ORR window instead of a well-defined diffusion plateau. Different CA were done in the same condition as previously for the platinum probe, except that the chosen potentials are -0.4, -0.5 and -0.6 V/SCE, in accordance with the observed lower electrocatalytic activity of gold versus platinum. Here also, stabilisation of measurements lasted about 300 s. The shape of the current curves is different in the case of gold. Here again, the probe's surface state impacts positively or negatively the kinetics at the surface. The difference between Pt and Au probes can be explained by their different ORR mechanisms occurring at the surface, as well as the difference in the stability of the surface states. It can be noticed in the Figure 2.d that the stabilized current is close to the starting current. Thus, for the gold probe, the sensitivity calculated is 3.3, 7.4 and 10.0 nA/mM(O2) for -0.4, -0.5 and -0.6 V/SCE respectively. The detection threshold is 63, 49 and 46 µM(O2) and the instability is 0.62, 0.13 and 0.19 %/min respectively. The sensitivity at -0.6 V/SCE is close to the theoretical one. As an additional advantage, the use of a gold probe permits to avoid the possibility to contaminate the substrate with a highly active material such as platinum. Moreover, the stability of the current at the gold probe allows long time acquisition with steady-state measurements. Therefore, based on the comparison of their sensitivities, their detection thresholds and their instabilities between platinum and gold probes, the latter really appears as a very interesting alternative probe for studying ORR. The supporting substrate used to analyse the activity of a catalyst is another important element for electrochemical measurements. Micrometric planarity, roughness and electrochemical activity are key parameters that influence the analysis. Figure 3 shows AFM (a) and SEM (b) images of a BDD substrate, where one can observe its polycrystallinity. The roughness average (Ra) and the root-mean-square roughness (Rq) of BDD is measured by AFM and is 39.9 nm and 50.0 nm respectively. Moreover, the maximum measured height is 372 nm. In the present work, a micrometric probe is used, and the positioning is performed at the micrometric scale (typically 30 microns, as shown Figure 5), so the ~100 nm roughness of the sample is negligible [49] . Besides, the use of a Si-wafer as substrate support guaranties a negligible non-planarity of the whole sample. Thus, the topography of the BDD substrate is highly acceptable for the investigation of micrometric spots of catalysts with the RC-mode and a micrometric probe. As an alternative to BDD, a Si-wafer covered with gold and a GC substrate can be considered. The gold substrate structure is illustrated in Figure 3.c and d for the AFM image. The maximum measured height is 2.3 nm with a Ra of 178 pm and a Rq of 225 pm which allows to scan on a nanoscale surface where inhomogeneities come from the investigated material only. The AFM and SEM image of a GC substrate are presented in Figure 3.e and f respectively. The maximum height measured on the GC substrate is almost 650 nm despite a Ra of 7.2 nm and a Rq of 11.4 nm. The average value is 58.3 nm meaning there is scarce but important inhomogeneities on the GC substrate. The polishing process of the GC substrate is of the outmost importance to allows a high planarity and so nanoscale measurements on it. In contrary, gold and BDD substrates, thanks to the Si-wafer support, keep their planarity on the whole surface.

Electrochemical results are presented Figure 4 comparing BDD to a GC substrate, a gold substrate and a catalyst spot of Co-N-NTC deposited on BDD. The samples are studied by RC-SECM at a constant height with a probe-substrate distance of 50 µm. The process involved is presented Figure 4.A and is done in acidic media saturated with oxygen (H2SO4 0.1 M) due to the acidic condition used in a PEMFC. The probe was held at -0.6 V/SCE in order to reduce O2 during all measurements. Samples were held at different potentials, from one from which the sample is inactive, to one corresponding to a high activity of the sample (almost no current at the probe) with a 100 mV difference between each measurement. Each potential was held at least 100 s for the current to reach a plateau once stabilized.

The results of the experiment are presented in Figure 4.b.

The competition starting point between the probe and the samples is noticed at a potential between 0.5 and 0.4 V/SCE for the Co-N-NTC spot, 0.1 V/SCE for the gold substrate, between -0.1 and -0.2 V/SCE for the GC substrate and -0.3 V/SCE for the BDD substrate. This means that the competition occurs for more negative potentials applied to the substrate than the one of this starting point. A 50 % contribution of samples is observed at 0.3 V/SCE for the Co-N-NTC spot, -0.1 V/SCE for the gold substrate, -0.41 V/SCE for the GC substrate and -0.48 V/SCE for the BDD substrate. Finally, the probe measures a current almost null at -0.2 V/SCE for the Co-N-NTC spot, -0.5 V/SCE for the gold substrate and -0.7 V/SCE for the BDD. However, for the GC substrate, even at the lowest potentials, the probe current was not null. This can be explained by an uncomplete ORR at the GC substrate. These results

showed that the BDD substrate is less active than the other substrates. Moreover, according to previous investigations made for GC through cyclic voltammetry, the same conclusion was established in acidic [39] and alkaline media [50] .

Overall, the large inactive window of BDD -until -0.3 V/SCE no contribution from the substrate is observed-offers a wider range of experimental conditions that can be used, as illustrated with the Co-N-NTC spot with a loading around 200 µg/cm 2 . There, the O2 consumption by the catalyst is total before the O2 consumption starts at the substrate, which is not the case with gold. This proves that much lower electrocatalytic activities or loadings can be investigated with BDD.

In order to further show that gold probe and BDD substrate are suitable to perform SECM imaging at steady state,

with 𝛽(𝑅𝐺), a correction factor due to the enhancement of diffusion limiting current for the small RG values [51] and NiT(L, RG), a correction factor due to diffusion hindering similar than a negative feedback [42] . With the experimental conditions of This way, the quantification of the oxygen consumption rate will become possible. It will need the support of numerical simulation, explicitly taking into account the experimental parameters such as the probe-to-substrate distance, the probe size (active and inactive part), and the spot size and shape. This is fully justified in a context of performing the benchmarking of catalysts, which will be done in future works.

Conclusion

Herein, we showed how steady state evaluation by RC-SECM for ORR detection in acidic conditions can be achieved with gold probes and BDD substrates. The gold probe exhibited a remarkable current stability, with variation values under 0.2 % variation per minute), further to a good sensitivity and a detection threshold similar to that of the platinum probe. Similarly, we also demonstrated that BDD as supporting substrate shows a very large inactivity window, up to -0.3 V vs SCE, with a submicrometric roughness and a small long-distance non-planarity, thanks to the Si-wafer support underneath. The same setup can also be considered for alkaline media investigations. This may be the basis of future works. Furthermore, preparation of flat BDD substrates is also planned in the future.

Figure 1 .

 1 Figure 1. Scheme of the RC-SECM mode.

Figure 2 .

 2 Figure 2. (a) LSV at the platinum probe from 0.55 to -0.43 V/SCE with a 10 mV/s scan rate. (b) CA at the platinum probe at different potentials (-0.2, -0.3 and -0.4 V/SCE). Black curves for Ar saturated solution and green curves for O2 saturated solution in (a & b). (c) LSV at the gold probe from 0.1 to -0.9 V/SCE with a 10 mV/s scan rate. (d) CA at the gold probe at different potentials (-0.4, -0.5 and -0.6 V/SCE). Blue curves for Ar saturated solution and red curves for O2 saturated solution in (c & d).All measurements were made in H2SO4 0.1M, rT = 5 µm and RG = 30 for both probes.

Figure 3 .

 3 Figure 3. (a) AFM image showing a thickness of 372 nm, Ra of 39.9 nm and Rq of 50.0 nm and (b) SEM image showing crystals smaller than 1 µm in diameter of a BDD substrate. (c) Scheme of the gold substrate structure. (d) AFM image showing a 2.3 nm thickness, Ra of 178 pm and Rq of 225 pm of a gold substrate. (e) AFM image showing a GC substrate with a Ra of 7.2 nm and a Rq of 11.4 nm. (f) SEM image of a GC substrate.

Figure 4 .

 4 Figure 4. (a) Scheme presenting the process involved in the RC-SECM measurement to analyse the ORR activity of BDD, GC, Au and the catalyst spot. (b) Results of chronoamperometry (CA) at different substrate potentials with a gold probe (rT = 5 µm, RG = 30, Eprobe = -0.6V/SCE) in H2SO4 0.1 M solution saturated with oxygen and with a distance probe-substrate of 50 µm. Measurements are made on a clean BDD substrate (∎), a clean GC substrate (▲), a clean gold substrate (♦) and a Co-N-NTC spot (•). The area under the curves represent the potential zone were the competition between the probe and the substrate occurs.

Figure 5 .

 5 a shows a 3 mm² optical image of two Co-N-NTC spots of 120 (a) and 230 (b) µg/cm² sprayed with the ExactaCoat system through a micro-patterned mask to obtain this welldefined square spots. The RC-SECM image of these spots is shown in Figure 5.b at Esubstrate = -0.3 V/SCE. The full RC-SECM image (with both forward and backward scans) is recorded within 2 hours.

Figure 5 .

 5 Figure 5. (a) Optical image of (1) 120 µg/cm² and (2) 230 µg/cm² Co-N-NTC spots on a BDD substrate. (b) RC-SECM image of the spots shown in (a). (c) RC-SECM forward (black curve) and backward (red curve) linescans at Y = 450 µm from the RC-SECM image (b) corresponding. SECM experiments done with a gold probe (rT = 12 µm, RG = 11, Eprobe = -0.45V/SCE) in H2SO4 0.1 M solution saturated with oxygen and with Esubstrate = -0.3 V/SCE, a distance probe-substrate of 30 µm and vscan = 20 µm/s.As shown in Figure5.c, forward and backward linescans overlap above the active spot, and this proves the steady state nature of the measurement. Still, non-stationarity can be seen when the probe is at the extremity of the spots, and in this case the current is smaller if the probe is approaching the center of the spot, and larger in the contrary. This fits the expectations, and does not affect the maximal current variation, which is the most important parameter regarding the spot electrocatalytic activity evaluation. At each line of the image, the current obtained far from the spots can be used to calibrate the relationship between the measured current and the oxygen concentration, by evaluating the effective number of electrons transferred:

Figure 5 .

 5 c (RG = 11, L = 2.5; L being the ratio between the distance probe-substrate with the rT), NiT(L, RG) = 0.79 so n= 1.8 with the current measured at the end of the linescan (-11.5 nA) presented Figure5.c. Alternatively, NiT(L, RG) = 0.78 is the ratio between the current measured far from the spot, divided by the current measured in solution. n is smaller than 2 here probably because the diffusion plateau is not reached at the working potential.

Table 1 .

 1 Averaged values from the CA at the probes at t600s and calculated sensitivity, detection threshold and instability for these values.

	Active part	Eprobe	Current at	Current at	Sensitivity	Detection threshold	Instability
	composition	(V/SCE)	t600s (nA) Arsat	t600s (nA) O2sat	(nA/mM(O2)	(µM(O2))	(%/min)
	Platinum probe	-0.2 -0.3 -0.4	-0.04 -0.07 -0.20	-1.22 -2.83 -5.48	1.0 2.2 4.3	46 31 46	0.44 0.62 0.44
		-0.4	-0.21	-4.27	3.3	63	0.62
	Gold probe	-0.5	-0.36	-9.39	7.4	49	0.13
		-0.6	-0.46	-12.72	10.0	46	0.19

Acknowledgement

The authors acknowledge the SENTINEL project, funded by the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement no 812398, and the PEGASUS project, funded by the European Union's Horizon 2020 research and innovation program FCH-01-2-2017, no779550.

Conflicts of interest

The authors have no conflict of interest to declare.

Table of Contents

This paper demonstrates how a judicious choice of probe and substrate materials opens up improved performances to achieve steady state measurements, in this case, for oxygen reduction reaction (ORR) catalytic activity detection through the redox competition scanning electrochemical microscopy (RC-SECM).

We show that the use of gold enhances the stability of the local oxygen concentration detection in comparison to the regularly used platinum one. We evaluate boron doped diamond as an appealing support substrate, that shows a low ORR activity, high stability and very good reusability.


Clément Zanolli
email: clement.zanolli@gmail.com

Fereidoun Biglari
email: fbiglari@gmail.com

Marjan Mashkour

Kamyar Abdi

Hervé Monchot

Karyne Debue

Arnaud Mazurier

Priscilla Bayle

Mona Le Luyer

Hélène Rougier

Erik Trinkaus

Roberto Macchiarelli

A Neanderthal from the Central Western Zagros, Iran. Structural reassessment of the Wezmeh 1 maxillary premolar

Keywords: Zagros Mountains, Wezmeh Cave, Human premolar tooth, Microfocus X-ray tomography, Neanderthals

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

Introduction

Since Coon's pioneering excavation in the late 1940s at the Middle Paleolithic site of Bisitun Cave [START_REF] Coon | Cave Explorations in Iran 1949[END_REF], the Zagros Mountains of Iran and Iraq have produced a number of Late Pleistocene human remains associated with Middle and Upper Paleolithic technocultural contexts that represent the main bioanthropological source of information about the populations inhabiting the region during the oxygen isotope stages (OIS) 3-2 (for a recent chronometric assessment of some Middle and Upper Paleolithic sites of the region, see [START_REF] Becerra-Valdivia | Chronometric investigations of the Middle to Upper Paleolithic transition in the Zagros Mountains using AMS radiocarbon dating and Bayesian age modelling[END_REF]. Among the excavated sites yielding human remains, with its assemblage of at least ten Neanderthal individuals, the Shanidar Cave, Iraqi Kurdistan, has produced one of the largest sample of fossil human remains from Southwestern Asia thus far [START_REF] Solecki | Prehistory in Shanidar valley, Northern Iraq[END_REF][START_REF] Trinkaus | The Shanidar Neandertals[END_REF][START_REF] Cowgill | Shanidar 10: A Middle Paleolithic immature distal lower limb from Shanidar Cave, Iraqi Kurdistan[END_REF][START_REF] Pomeroy | Newly discovered Neanderthal remains from Shanidar Cave, Iraqi Kurdistan, and their attribution to Shanidar 5[END_REF].

A complex of Paleolithic sites in the Kermanshah region of Central Western Zagros, Iran, produced the second fossil human sample (Fig. 1). Among these sites, the Middle Paleolithic sequence of Bisitun Cave, located east-northeast of Kermanshah, was the first that yielded a human fossil [START_REF] Coon | Cave Explorations in Iran 1949[END_REF], a right radius preserved from just distal of the radial tuberosity to the distal diaphysis. Although its dimensions overlap the upper end of the earlier Upper Paleolithic range of variation, comparisons with Neanderthal and Middle Paleolithic early modern human right radii align it predominantly with Neanderthals, but separate from Middle Paleolithic early modern humans of Southwestern Asia [START_REF] Trinkaus | Middle Paleolithic human remains from Bisitun cave, Iran[END_REF].

In the course of a reanalysis at the Field Museum of Natural History of the Upper Paleolithic collections from the Warwasi rockshelter, a site in the northern outskirt of Kermanshah, an isolated M 2 from a fossil modern human was recently identified in the faunal assemblage from the Baradostian levels [START_REF] Tsanova | The beginning of the Upper Paleolithic in the Iranian Zagros. A taphonomic approach and techno-economic comparison of Early Baradostian assemblages from Warwasi and Yafteh (Iran)[END_REF].

Another Paleolithic site in Western Zagros that provided human remains is Gar Arjeneh, near Khoramabad, in Luristan. First excavated by [START_REF] Hole | The prehistory of Southwestern Iran: A preliminary report[END_REF] for a review see [START_REF] Bazgir | Test excavations and initial results at the Middle and Upper Paleolithic sites of Gilvaran, Kaldar, Ghamari caves and Gar Arjene Rockshelter, Khorramabad Valley, western Iran[END_REF], its Baradostian levels have yielded a human premolar and some bone fragments [START_REF] Trinkaus | Pleistocene human remains from Iran[END_REF].

In the Southern Zagros, the Upper Paleolithic and Epipaleolithic layers of Eshkaft-e Gavi, near Shiraz, produced an assemblage of fragmentary dental, cranial and postcranial remains sampling a minimum of four modern human individuals bearing evidence of stone-tool cutmarks and burning [START_REF] Rosenberg | Report on the 1978 sondage at Eshkaft-e Gavi[END_REF][START_REF] Scott | Paleolithic hominin remains from Eshkaft-e Gavi (southern Zagros Mountains, Iran): description, affinities, and evidence for butchery[END_REF].

Finally, the catalog of human remains from Paleolithic sites of Central Western Zagros Mountains includes Wezmeh 1, an isolated maxillary premolar from Wezmeh Cave, southwest of Kermanshah. Its buccolingual crown diameter places it at the upper limits of Late Pleistocene human maxillary third (P 3 ) and fourth premolar (P 4 ) dimensions and above a terminal Pleistocene Natufian sample [START_REF] Trinkaus | Late Pleistocene human remains from Wezmeh Cave, Western Iran[END_REF].

Given uncertainties concerning the original chronostratigraphic context of Wezmeh 1 [START_REF] Abdi | Islamabad Project 2001. Test excavations at Wezmeh cave[END_REF][START_REF] Trinkaus | Late Pleistocene human remains from Wezmeh Cave, Western Iran[END_REF][START_REF] Mashkour | Carnivores and their prey in the Wezmeh Cave (Kermanshah, Iran): A Late Pleistocene refuge in the Zagros[END_REF] and the difficulties of assessing its taxonomic status based on its external features [START_REF] Trinkaus | Late Pleistocene human remains from Wezmeh Cave, Western Iran[END_REF], we used micro-focus X-ray tomography (µCT) to reassess the metameric position and taxonomic attribution of this specimen.

Using techniques of virtual imaging coupled with geometric morphometrics, we evaluated its tissue proportions, enamel thickness topographic distribution, enamel-dentine junction morphology, and pulp cavity shape. We then compared these data to samples of European Neanderthals, Near Eastern Middle Paleolithic modern humans, an Upper Paleolithic human, and Holocene humans.

The Wezmeh Cave

Wezmeh Cave is located 10 km southeast of the town of Islamabad-e Gharb (34°03'17.04" N, 46°38'41.03" E), at an elevation of ca. 1430 m asl (Fig. 1). The cave, discovered in 1999 during archeological survey as a part of the Islamabad Project (director: K.A.), is in a small valley in the western foothills of the Qazivand Mountain (Fig. 2a). Its entrance (Fig. 2b) faces north and is 2 m wide and 1.2 m high. The cave is about 27 m long and has about 45 m 2 of floor area [START_REF] Abdi | Islamabad Project 2001. Test excavations at Wezmeh cave[END_REF] see Fig. 2c). In 2001, following disturbance by clandestine digging, a 3 × 3 m trench was excavated to the bedrock on the terrace immediately outside of the cave entrance, and six small test pits at 2 m intervals were dug to bedrock within the cave. These excavations yielded Holocene (Chalcolithic) archeological materials [START_REF] Abdi | Islamabad Project 2001. Test excavations at Wezmeh cave[END_REF]. The surface collecting on the exterior slope (Fig. 2b) in 1999, 2001 and 2008 yielded an abundant faunal assemblage, particularly rich in carnivoran and ungulate remains [START_REF] Trinkaus | Late Pleistocene human remains from Wezmeh Cave, Western Iran[END_REF][START_REF] Mashkour | Carnivores and their prey in the Wezmeh Cave (Kermanshah, Iran): A Late Pleistocene refuge in the Zagros[END_REF]. The faunal list includes carnivorans present during the Late Pleistocene in Southwest Asia: Hyenidae (Crocuta crocuta), Canidae (Vulpes vulpes, Canis lupus), Felidae (Panthera leo, Panthera pardus, Caracal/Lynx/Felis chaus, Felis silvestris), Mustelidae (Meles meles, Martes martes, Mustela putorius), Viverridae (Herpestes sp.), and Ursidae (Ursus arctos). Artiodactyl remains include Suidae (Sus scrofa), Cervidae (Cervus elaphus), Bovidae (Bos primigenius, Gazella sp., Capra aegagrus, Ovis orientalis). Among smaller taxa, representatives of Lagomorpha, Rodentia, Insectivora, and Reptilia were also found. The skeletal remains were mainly collected in disturbed late Pleistocene deposits and a small number from excavated early Holocene deposits. Three carnivoran coprolites from disturbed deposits were dated by 14 C [START_REF] Djamali | Pollen analysis of coprolites from a late Pleistocene-Holocene cave deposit (Wezmeh Cave, west Iran): Insights into the late Pleistocene and late Holocene vegetation and flora of the central Zagros Mountains[END_REF] to the Last Glacial Period (19,228 ± 352 cal yr BP and 12,744 ± 370 cal yr BP) and to the recent Holocene (405 ± 59 cal yr BP). Several fragmented human bones and teeth were discovered among the faunal remains, including the premolar Wezmeh 1 (Fig. 2d). A few other fragmented human bones and teeth were also found in the cave; one of them was dated to the early Neolithic, at around 9300 cal yr BP [START_REF] Broushaki | Early Neolithic genomes from the eastern Fertile Crescent[END_REF].

Because of the exceptional nature, size and taxonomic diversity of the assemblage from Wezmeh Cave and the joint presence of extinct mammal taxa including human remains (notably the large and fossilized tooth Wezmeh 1), a program of absolute dating of targeted specimens was developed. According to uranium-series analyses of the fauna by alpha spectrometry, the site was occupied by carnivorans within the interval 70-11 ka (OIS 4-2). The human premolar was directly dated by gamma spectrometry to 25-20 ka [START_REF] Trinkaus | Late Pleistocene human remains from Wezmeh Cave, Western Iran[END_REF][START_REF] Mashkour | Carnivores and their prey in the Wezmeh Cave (Kermanshah, Iran): A Late Pleistocene refuge in the Zagros[END_REF]. However, given that these dates are approximate and may be only minimum ages, Wezmeh 1 is currently assigned to either OIS 3 or early OIS 2 [START_REF] Trinkaus | Late Pleistocene human remains from Wezmeh Cave, Western Iran[END_REF].

Materials and methods

Wezmeh 1 is an unerupted right upper premolar from an 8 ± 2 year-old individual [START_REF] Trinkaus | Late Pleistocene human remains from Wezmeh Cave, Western Iran[END_REF]; see Fig. 2c). The specimen consists of a finely preserved crown and of ca. 7 mm of growing root (developmental stage between R 1/4 and R 1/2 following [START_REF] Moorrees | Age variation of formation stages for ten permanent teeth[END_REF], which is largely complete up to its developing edge despite some local chipping.

The specimen was imaged at the University of Poitiers, France, using an EasyTom XL Duo device (RX-Solutions) set at the PLATINA platform of the IC2MP laboratory. A sealed Hamamatsu microfocus X-ray source (small focal spot mode) was used, coupled to a Varian PaxScan 2520DX imager. The acquisition parameters are: 60kV voltage; 166 µA current; a 0.35 mm Cu filter; 1280 projections, 4 frames/second; averaging of 13 frames/projection; anti-ring shift procedure; source-to-detector distance and source-to-object distance of 416 mm and 41 mm, respectively, and reconstructed with a voxel size of 12.5 µm. Reconstruction of the data was done with the XAct software (RX-solutions) with a filtered back projection algorithm (Felkamp method for cone beam geometry with a Tukey filter) and correction of beam hardening artefacts through linearization with a polynomial function. A semiautomatic threshold-based segmentation was conducted using Avizo v.8.0 (FEI Visualization Sciences Group, Hillsboro), following an adaptation of the half-maximum height method [START_REF] Spoor | Linear measurements of cortical bone and dental enamel by computed tomography: Applications and problems[END_REF][START_REF] Fajardo | Assessing the accuracy of high-resolution X-ray computed tomography of primate trabecular bone by comparisons with histological sections[END_REF][START_REF] Coleman | Technical note: CT thresholding protocols for taking measurements on three-dimensional models[END_REF]. The crown was virtually isolated from the roots [START_REF] Olejniczak | Dental tissue proportions and enamel thickness in Neandertal and modern human molars[END_REF] and 3D surface models of the dental tissues were generated using a constrained smoothing algorithm [START_REF] Kupczik | Mandibular molar root morphology in Neanderthals and Late Pleistocene and recent Homo sapiens[END_REF].

For assessing tissue proportions, the following variables were measured on the 2D buccolingual virtual tooth section [START_REF] Feeney | Enamel thickness in Asian human canines and premolars[END_REF] and calculated in 3D (see [START_REF] Kono | Molar enamel thickness and distribution patterns in extant great apes and humans: New insights based on a 3-dimensional whole crown perspective[END_REF][START_REF] Macchiarelli | How Neanderthal molar teeth grew[END_REF][START_REF] Olejniczak | Dental tissue proportions and enamel thickness in Neandertal and modern human molars[END_REF][START_REF] Bayle | Dental maturational pattern and dental tissue proportions in the early Upper Paleolithic child from Abrigo do Lagar Velho, Portugal[END_REF][START_REF] Smith | Variation in enamel thickness within the genus Homo[END_REF][START_REF] Skinner | Enamel thickness trends in Plio-Pleistocene hominin mandibular molars[END_REF]: enamel area (c; mm²); coronal dentine and pulp area (b; mm²); total crown area (a; mm 2 ); percentage of crown area that is dentine and pulp (b/a; %); enamel-dentine junction (EDJ) length (e; mm); bicervical diameter (BCD; mm); enamel volume (Ve; mm 3 ); coronal dentine and pulp volume (Vcdp; mm 3 ); total crown volume (Vc; mm 3 ); percentage of crown volume that is dentine and pulp (Vcdp/Vc; %); EDJ surface area (SEDJ; mm 2 ). Enamel thickness was assessed by calculating the following parameters [START_REF] Martin | Significance of enamel thickness in hominoid evolution[END_REF][START_REF] Olejniczak | Dental tissue proportions and enamel thickness in Neandertal and modern human molars[END_REF]: 2D average enamel thickness (2D AET = c/e; mm) and 2D relative enamel thickness (2D RET = 2D AET*100/(b 1/2 ); scale-free); 3D average enamel thickness (3D AET = Ve/SEDJ; mm) and 3D relative enamel thickness (3D RET = 3D AET*100/(Vcdp 1/3 ); scale-free).

Three-dimensional maps of topographic enamel thickness distribution were created with the segmented enamel and crown dentine components [START_REF] Macchiarelli | Virtual dentitions: Touching the hidden evidence[END_REF][START_REF] Bayle | Three-dimensional imaging and quantitative characterization of human fossil remains. Examples from the NESPOS database[END_REF]Zanolli et al., 2018a, b), where differences in enamel distribution are rendered by a thicknessrelated pseudocolor scale ranging from dark blue (thinner) to red (thicker). This method maps topographic thickness variation at the outer enamel surface and facilitates the site-specific and synthetic comparative assessments of this feature. The topographic variation of standardized enamel thickness was measured using MPSAK v2.9 (available in [START_REF] Dean | A Digital Radiographic Atlas of Great Apes Skull and Dentition[END_REF] in the lingual and buccal aspects of the virtual buccolingual section [START_REF] Macchiarelli | L'apport des nouvelles technologies à l'étude des Néandertaliens[END_REF][START_REF] Luyer | Hominin Biomechanics, Virtual Anatomy and Inner Structural Morphology: From Head to Toe. A Tribute to Laurent Puymerail[END_REF], and the values were calibrated with respect to a bicervical diameter of 6 mm [START_REF] Schwartz | Taxonomic and functional aspects of the patterning of enamel thickness distribution in extant large-bodied hominoids[END_REF][START_REF] Mazurier | Topographic variation of the enamel thickness in the human anterior deciduous dentition[END_REF][START_REF] Luyer | Roman Empire Bosco Pontini (BP 5, BP 6, BP 7) Bosco Pontini (BP 5, BP 6, BP 7, BP 8, BP 9)[END_REF].

Geometric morphometric (GM) analyses of EDJ were performed on the virtual surface by placing two landmarks at the apices of the paracone and protocone dentine horns and 71 semilandmarks along the marginal ridges (33 on the mesial marginal ridge and 38 on the distal one). We performed the generalized Procrustes analyses, followed by between-group principal component analyses (bgPCA) and Jackknife cross-validated canonical variate analyses (CVA) based on the first 9 (for the separate P 3 and P 4 samples) to 12 (for the combined P 3 -P 4 samples) first principal components explaining ~90% of the total variance [START_REF] Mitteroecker | Linear discrimination, ordination, and the visualization of selection gradients in modern morphometrics[END_REF][START_REF] Krenn | Variation of 3D outer and inner crown morphology in modern human mandibular premolars[END_REF]. Wezmeh 1 was included a posteriori in the analyses. Multivariate analyses were performed using the packages ade4 v. 1.7-6 (Dray and Dufour, 2007) and Morpho v.

2.7 [START_REF] Schlager | Morpho: Calculations and visualizations related to geometric morphometrics[END_REF]) for R v.3.6 (R Development Core Team, 2019). Allometry was tested using the coefficient of determination (R 2 ) of a multiple regression [START_REF] Bookstein | Morphometric Tools for Landmark Data: Geometry and Biology[END_REF] in which the explicative variable is the centroid size and the dependent variables are the bgPC scores (see also [START_REF] Mitteroecker | A brief review of shape, form, and allometry in geometric morphometrics, with applications to human facial morphology[END_REF].

Because of the uncertainty of the Wezmeh 1 metameric position, the comparative analyses used the µCT record of selected unworn/slightly-moderately worn crowns (wear stage ≤ 2 following [START_REF] Smith | Patterns of molar wear in hunter-gatherers and agriculturalists[END_REF] Due to the limited µCT-based comparative evidence directly available to us or exploitable from the literature, the fossil modern human sample (MPMH) is restricted to the material from the site of Qafzeh, thus resulting in a temporal and geographic gap between the fossil and recent comparative samples. However, we also exploited the endostructural signal from an unpublished late Upper Paleolithic (Magdalenian) P 3 from the French site of La Piscine (see Discussion and conclusions), suggesting that this heterogeneous sample is still representative of the fossil modern human variation. Information on the three comparative samples (including the variable number of cases used in the different 2-3D analyses) is reported in Table 1, while Table 2 provides the NEA and MPMH samples. The sample of Holocene premolars used in this study, consisting of archaeological and recent specimens from Western Europe, is provided in Supplementary Online Material (SOM) Table S1.

In a few cases, local virtual integrations of the outer enamel surface and/or of the dentine horn tip contour were made preliminary to the quantitative analyses on 2D virtual sections and 3D models, respectively. Within each sample, integration of the removed enamel was based on the preserved morphology of the unworn crowns as revealed by their serial buccolingual cross-sections [START_REF] Smith | Variation in enamel thickness within the genus Homo[END_REF].

Adjusted Z-scores [START_REF] Maureille | Les dents inférieures du néandertalien Regourdou 1 (site de Regourdou, commune de Montignac, Dordogne): analyses métriques et comparatives[END_REF][START_REF] Scolan | Des nouveaux vestiges néanderthaliens à Las Pélénos (Monsempron-Libos, Lot-et-Garonne, France)[END_REF] were computed to compare the 3D dental tissue proportions and enamel thickness values of Wezmeh 1 to the means and standard deviations of the comparative groups. This method allows the comparison of unbalanced samples, which is often the case when dealing with the fossil record, by using the Student's t inverse distribution, where the +1.0 to -1.0 interval comprises 95% of the variation in the reference sample.

Plots of the common logarithm (log) of 2D AET against the log of coronal dentine and pulp area (b) were used to illustrate the relationship between AET and tooth size [START_REF] Skinner | Enamel thickness trends in Plio-Pleistocene hominin mandibular molars[END_REF] and to show the placement of Wezmeh 1 compared to Neanderthals, MPMH and Holocene humans.

For the GM assessment of the EDJ morphology, Wezmeh 1 was projected a posteriori in the bgPCA analysis.

Results

The virtual reconstructions of the outer enamel surface (OES) and of the internal aspect of Wezmeh 1 in different views are comparatively shown in Figure 3. The specimen exhibits excellent preservation of its outer and inner morphology. It also lacks sedimentary infill and displays high enamel-dentine contrast in all µCT-based reconstructions.

On the lateral and occlusal aspects, the undamaged and unworn OES shows several small and shallow pits representing minor developmental enamel defects. However, these hypoplastic defects are not visible on the EDJ (Fig. 3). Otherwise, the OES and the dentine surface morphology match perfectly. Specifically, in occlusal view, they share: a low elevation of the buccal segment of the mesial marginal ridge, with a short crest running into the mesial fovea; two buccal and two lingual essential segments of the bifurcated transverse crest; a short distal crest running from the lingual cusp; and a small cusp-like elevation visible on the lingual part of the distal marginal ridge (Fig. 3).

Near the cervix, a shallow subcircular enamel depression is noticeable on the mesiobuccal aspect of the paracone. The virtual rendering of the coronal dentine shows that its imprint is also slightly expressed beneath the enamel (Fig. 3). Approximately 2.0 mm from the cervix, this feature also extends to the root dentine [START_REF] Trinkaus | Late Pleistocene human remains from Wezmeh Cave, Western Iran[END_REF], where it appears as a larger and deeper depression (Fig. 3; SOM Fig. S1). Apparently, it does not affect the pulp morphology, the inner dentine wall being straight and flat at the level of the depression, even if the dentine at this spot is thinner than measured at other points of the growing root (SOM Fig. S1).

2D and 3D measures of crown tissue proportions and enamel thickness in Wezmeh 1 are reported in Tables 3 and4, respectively, together with the comparative estimates for each premolar type of the NEA, MPMH, and HH samples used in this study.

For a large majority of the measured/calculated parameters, the values (absolute and proportions) displayed by the P 3 and P 4 crowns overlap within each sample, or are close. A greater variation characterizes the MPMH sample, especially for b/a, 2D RET, Vcdp/Vc, SEDJ, and 3D RET. Whatever its metameric position, Wezmeh 1 shows absolutely higher values for most of the 2D and 3D tissue proportion parameters. It fits better with the Neanderthal P 3 and, to a slightly lesser extent, the Neanderthal P 4 . The variables that align the Iranian specimen and the Neanderthals and best discriminate both from the fossil and recent modern human figures are: c, b, e, Ve, Vcdp, and SEDJ (Tables 3 and4). For these parameters, Wezmeh 1 is clearly apart from the HH condition and approaches the NEA pattern, while the MPMH tend to occupy an intermediate position between the HH and NEA values. However, no substantial differences exist between Wezmeh 1 and the comparative samples for b/a and Vcdp/Vc, notably among the P 3 . Whatever the extent of the differences shown in Tables 3 and4, all of the comparisons with the MPMH and HH representatives show that the whole inner structural signature of Wezmeh 1 better fits those expressed by the P 3 , not the P 4 .

A closer affinity of Wezmeh 1 with the Neanderthals is revealed by the adjusted Z-scores of the absolute estimates of nearly all 2D and 3D variables (SOM Figs. S2 andS3). However, this is not the case for the enamel thickness. In fact, for both 2D and 3D AET, Wezmeh 1 is compatible with the HH values. Nonetheless, for both size-free 2D and 3D RET indices, the Iranian tooth crown is slightly closer to the NEA estimates (Tables 3 and4). Additionally, the affinity of Wezmeh 1 with NEA is shown by the bivariate graph of log 2D AET against log b (Fig. 4), which also includes two specimens from Qafzeh and where Wezmeh 1 plots among the NEA specimens and apart from the HH by having larger coronal dentine and pulp areas.

The cartographies of enamel thickness distribution in Wezmeh 1 and in representatives of the comparative samples (NEA, MPMH and HH) are shown in Figures 5 and6, separately for the P 3 and P 4 crowns imaged in different projections. As a whole, they reveal only minor differences among the investigated specimens, including between NEA and HH. At least in the limited comparative assemblage of premolar crowns available, it appears that the relatively thicker Qafzeh 4 specimen, similar to the HH P 3 , exhibits thicker enamel distributed over the occlusal basin and at the cusp apices, especially the protocone, whereas in the relatively thinner Qafzeh 10, thicker enamel is mostly localized on the protocone, as in NEA. Nonetheless, Wezmeh 1 shares with Qafzeh 4 and NEA P 3 , a relatively thicker enamel on the buccal aspect.

A more detailed picture illustrating topographic enamel thickness variation is shown by the sitespecific standardized values comparatively assessed from the cervix towards the apex across the entire lingual (protocone) and buccal (paracone) mesiodistal virtual sections, respectively (SOM Figs. S4 andS5). The enamel distribution in Wezmeh 1 follows the NEA pattern revealed by the pooled P 3 and P 4 , especially for the lingual cross-section (SOM Fig. S4). Conversely, for the buccal section, its trajectory is less distinct, including when compared to the MPMH and the HH pattern.

In this respect, Wezmeh 1's profile follows a modern-like trajectory along approximately the first half of its buccal section and a Neanderthal-like trajectory nearing the paracone (SOM Fig. S5).

The results of the bgPCA of the geometric morphometric analysis of the EDJ in the shape space of the P 3 and P 4 are displayed in Fig. 7. The first two components, which account for 88.6% of the total variation (59.8% for bgPC1 and 31.5% for bgPC2), show a weak allometric signal (p < 0.05; R 2 = 0.34) and no size-dependent shape variation (p > 0.05; R 2 = 0.06). The GM analysis combining P 3 and P 4 thus mostly represents shape variation. By displaying a relatively symmetrical outline of the occlusal basin and a large distal part of the occlusal basin, Wezmeh 1 falls within the NEA P 3 variation space. It is also relatively close to the NEA P 4 shape, a space characterized by a slightly more reduced talon, as well as by an acute mesiobuccal angle of the occlusal basin. In this context, the Iranian tooth differs from the upper premolars from Qafzeh by having a more symmetrical outline of the EDJ, and is also well separated from the HH P 3 and P 4 by having a more mesiodistally extended talon and a more marked distolingual extension of the latter. These results are confirmed by the CVA, in which the predictive accuracy between human groups is robust (100% correct classification for NEA, 83.3% for MPMH, and 96.3% for HH), and the Iranian specimen is classified with 100% posterior probability as NEA. We also ran a CVA to assess metameric variation in the comparative sample (predictive accuracy: 88.5% and 87.5% of correct classification for P 3 and P 4 , respectively). This analysis indicates a 100% posterior probability of Wezmeh 1 representing a P 3 .

We also analyzed Wezmeh 1 separately with the P 3 (SOM Fig. S6) and the P 4 (SOM Fig. S7).

Again, only a weak allometric signal is found along the bgPC1 (R 2 = 0.48 and 0.34 for the P 3 and the P 4 analyses, respectively), while no allometry is recorded along the bgPC2 for both analyses (p > 0.05; R 2 < 0.03). When compared with the P 3 , Wezmeh 1 is much closer to the NEA condition than to the MPMH and HH groups. More specifically, the Iranian premolar exhibits the typical Neanderthal square outline of the occlusal basin, whereas the other groups have a more ovoid pattern (SOM Fig. S6). Compared with the P 4 (SOM Fig. S7), Wezmeh 1 again fits the NEA shape and sets apart from both the three MPMH (displaying a more rounded outline of the occlusal basin) and the HH sample (characterized by a shorter talon and no angulation of the distolingual corner).

Discussion and conclusions

A number of sites in Central and Southern Zagros have yielded variably rich Mousterian lithic assemblages (e.g., [START_REF] Biglari | Approvisionnement et utilisation des matières premières au Paléolithique moyen dans la plaine de Kermanshah (Iran): le cas de la Grotte Do-Ashkaft[END_REF][START_REF] Otte | Iran Palaeolithic[END_REF][START_REF] Scott | Paleolithic hominin remains from Eshkaft-e Gavi (southern Zagros Mountains, Iran): description, affinities, and evidence for butchery[END_REF][START_REF] Bazgir | Test excavations and initial results at the Middle and Upper Paleolithic sites of Gilvaran, Kaldar, Ghamari caves and Gar Arjene Rockshelter, Khorramabad Valley, western Iran[END_REF][START_REF] Bazgir | Understanding the emergence of modern humans and the disappearance of Neanderthals: Insights from Kaldar Cave (Khorramabad Valley, Western Iran)[END_REF][START_REF] Shidrang | Continuity and change in the Late Pleistocene lithic industries of the Central Zagros: A typo-technological analysis of lithic assemblages from Ghar-e Khar Cave, Bisotun, Iran[END_REF][START_REF] Becerra-Valdivia | Chronometric investigations of the Middle to Upper Paleolithic transition in the Zagros Mountains using AMS radiocarbon dating and Bayesian age modelling[END_REF][START_REF] Heydari-Guran | The MUP Zagros Project: Tracking the Middle-Upper Palaeolithic transition in the Kermanshah region, west-central Zagros, Iran[END_REF].

Within an approximate 30 km radius of Wezmeh Cave, 13 Middle Paleolithic sites have been recorded; among them, the nearest sites (cluster of Cham Souran and Cham Barzeh) are located about 10 km to the northwest [START_REF] Biglari | Paleolithic artifacts from Cham-e Souran, the Islamabad Plain, Central Western Zagros Mountains, Iran[END_REF]. Surface collected lithic artifacts from these cave, rockshelter and open-air sites generally resemble excavated Zagros Mousterian assemblages from sites situated in the neighboring intermountain plain of Kermanshah. However, the physical record of their producers remains elusive (review in [START_REF] Trinkaus | Pleistocene human remains from Iran[END_REF].

The first description of the isolated immature human upper premolar Wezmeh 1 suggested it more likely represents a P 3 [START_REF] Trinkaus | Late Pleistocene human remains from Wezmeh Cave, Western Iran[END_REF]. Our comparative observations combining outer morphology and inner structural organization corroborate this attribution. Importantly, the new analyses add relevant information to more confidently assess the taxonomic status of this specimen.

Even if the possibility that Wezmeh 1 "could derive from an earlier OIS 3 Neandertal" (Trinkaus et al., 2008: 376) was evoked because of its large buccolingual crown diameter, based solely on its outer crown characteristics, its attribution to a late archaic or an early modern human was unclear [START_REF] Trinkaus | Late Pleistocene human remains from Wezmeh Cave, Western Iran[END_REF]. However, our analyses of the internal structure confirm that Wezmeh 1 belongs to a Neanderthal.

Because of its discriminant value, quantitative information on the inner tooth structural organization and tissue proportions is increasingly used in paleoanthropological research as a reliable tool of taxonomic significance, including among species within the genus Homo (e.g., [START_REF] Macchiarelli | How Neanderthal molar teeth grew[END_REF][START_REF] Olejniczak | Dental tissue proportions and enamel thickness in Neandertal and modern human molars[END_REF][START_REF] Skinner | Dental trait expression at the enamel-dentine junction of lower molars in extant and fossil hominoids[END_REF][START_REF] Smith | Taxonomic assessment of the Trinil molars using nondestructive 3D structural and development analysis[END_REF]Bayle et al., 2010;[START_REF] Benazzi | Early dispersal of modern humans in Europe and implications for Neanderthal behaviour[END_REF][START_REF] Liu | Late Middle Pleistocene hominin teeth from Panxian Dadong, South China[END_REF][START_REF] Crevecoeur | First early hominin from Central Africa (Ishango, Democratic Republic of Congo)[END_REF][START_REF] Martinón-Torres | Talonid crests expression at the enamel-dentine junction of hominin lower permanent and deciduous molars[END_REF][START_REF] Weber | The Qesem Cave hominin material (part 1): A morphometric analysis of the mandibular premolars and molar[END_REF][START_REF] Hublin | New fossils from Jebel Irhoud, Morocco and the pan-African origin of Homo sapiens[END_REF][START_REF] Martin | The morphology of the enamel-dentine junction in Neanderthal molars: Gross morphology, non-metric traits, and temporal trends[END_REF][START_REF] Martínez De Pinillos | Comparative analysis of the trigonid crests patterns in Homo antecessor molars at the enamel and dentine surfaces[END_REF][START_REF] Pan | Intraindividual metameric variation expressed at the enamel-dentine junction of lower post-canine dentition of South African fossil hominins and modern humans[END_REF][START_REF] Hershkovitz | The earliest modern humans outside Africa[END_REF][START_REF] Xing | The fossil teeth of the Peking Man[END_REF]Zanolli et al., 2018a).

Our µCT-based analyses indicate that the Iranian premolar most likely derives from a Neanderthal-like individual to a degree of reliability comparable to that usually accepted for discriminating Neanderthal from extant and fossil modern human molar teeth (e.g., Olejniczak et al., 2008;[START_REF] Smith | Variation in enamel thickness within the genus Homo[END_REF][START_REF] Weber | The Qesem Cave hominin material (part 1): A morphometric analysis of the mandibular premolars and molar[END_REF][START_REF] Bayle | The dental remains: Enamel thickness, and tissue proportions[END_REF][START_REF] Martin | The morphology of the enamel-dentine junction in Neanderthal molars: Gross morphology, non-metric traits, and temporal trends[END_REF]Zanolli et al., 2018a). In this context, the results of the 3D geometric morphometric analysis of the EDJ conformation are of special value, as EDJ topography approximates the inner enamel epithelium of the developing tooth and provides useful information about taxon-specific processes underlying crown growth (e.g., [START_REF] Skinner | Dental trait expression at the enamel-dentine junction of lower molars in extant and fossil hominoids[END_REF][START_REF] Crevecoeur | First early hominin from Central Africa (Ishango, Democratic Republic of Congo)[END_REF][START_REF] Zanolli | Molar crown inner structural organization in Javanese Homo erectus[END_REF][START_REF] Martin | The morphology of the enamel-dentine junction in Neanderthal molars: Gross morphology, non-metric traits, and temporal trends[END_REF][START_REF] Zanolli | Evidence for increased hominid diversity in the Early-Middle Pleistocene of Indonesia[END_REF].

Studies of the outer crown morphology indicate that there are no particular non-metric features enabling to diagnostically attribute an isolated upper premolar to a Neanderthal or to an early modern human (e.g., [START_REF] Bailey | Beyond shovel-shaped incisors: Neandertal dental morphology in a comparative context[END_REF][START_REF] Bailey | What does it mean to be dentally "modern[END_REF]. However, the bifurcated essential crest slightly visible in Wezmeh 1, notably at the EDJ level, is generally present in Neanderthals but, to the best of our knowledge, has not been recorded in early modern humans thus far [START_REF] Bailey | Beyond shovel-shaped incisors: Neandertal dental morphology in a comparative context[END_REF], evidence that strengthens our endostructurally-based attribution of Wezmeh 1 to a Neanderthal individual.

Besides the limited size of the available comparative fossil samples, information currently is rare for coronal tissue proportions and enamel thickness variation of upper premolar crowns, including in recent humans [START_REF] Feeney | Enamel thickness in Asian human canines and premolars[END_REF][START_REF] Luyer | Roman Empire Bosco Pontini (BP 5, BP 6, BP 7) Bosco Pontini (BP 5, BP 6, BP 7, BP 8, BP 9)[END_REF]. Importantly for this study, no data suitable for direct comparisons have been published so far on tooth tissue proportions of fossil modern human P 3 . The µCT record of an unpublished right P 3 (specimen H3) from the Magdalenian (OIS 2) site of La Piscine, in Montmorillon, Central-Western France [START_REF] Henry-Gambier | Les fossiles humains du Paléolithique supérieur de Poitou-Charentes[END_REF][START_REF] Delage | Le Site Préhistorique de La Piscine[END_REF], allowed us to use this otherwise worn tooth (stage 4) for the GMbased comparative analysis of the EDJ. Following a virtual integration of its slightly eroded protocone (SOM Fig. S8a)-according to the geometric method described in Zanolli et al. (2018a)-the bgPCA results (SOM Fig. S8b) show that this latest Pleistocene tooth plots together with the MPMH P 3 , distant from Wezmeh 1.

In our analyses, a Neanderthal vs. modern human-like signature is less distinct for the enamel thickness, whatever the parameter used. The absolute enamel volume of Neanderthal upper and lower molars is comparable to that of modern humans, but in Neanderthals it is deposited over a larger volume of coronal dentine, thus resulting in lower average and relative enamel thickness values [START_REF] Olejniczak | Dental tissue proportions and enamel thickness in Neandertal and modern human molars[END_REF]. However, the possibility exists that enamel thickness in upper premolars is not as powerful to discriminate between Neanderthals and modern humans. This hypothesis should be tested in larger samples. Unfortunately, no directly comparable quantitative information on the inner tooth structural organization and topographic enamel thickness variation is available so far on Neanderthal premolars from Southeast Asia.

An additional limiting aspect in our study is the fact that Wezmeh 1 is from a still growing individual bearing a modest portion of radicular dentine [START_REF] Trinkaus | Late Pleistocene human remains from Wezmeh Cave, Western Iran[END_REF]. This prevents the possibility of virtually unrolling and projecting it into a standardized morphometric map for assessing its topographic thickness variation, a feature which tends to discriminate Neanderthal from non-Neanderthal teeth (e.g., [START_REF] Bayle | Dental maturational pattern and dental tissue proportions in the early Upper Paleolithic child from Abrigo do Lagar Velho, Portugal[END_REF][START_REF] Bondioli | Morphometric maps of long bone shafts and dental roots for imaging topographic thickness variation[END_REF][START_REF] Macchiarelli | From outer to inner structural morphology in dental anthropology. The integration of the third dimension in the visualization and quantitative analysis of fossil remains[END_REF][START_REF] Zanolli | The late Early Pleistocene human dental remains from Uadi Aalad and Mulhuli-Amo (Buia), Eritrean Danakil: Macromorphology and microstructure[END_REF]Zanolli et al., , 2018a)).

As first noted by [START_REF] Trinkaus | Late Pleistocene human remains from Wezmeh Cave, Western Iran[END_REF], an interesting feature in Wezmeh 1 is represented by a distinct depression located on its mesial root aspect. In this study we investigated its occurrence in two comparative P 3 and P 4 samples representing 20 Western European Neanderthals [START_REF] Rougier | Etude descriptive et comparative de Biache-Saint-Vaast 1 (Biache-Saint-Vaast[END_REF][START_REF] Toussaint | The dentition of the Scladina 1-4A juvenile Neandertal[END_REF]NESPOS Database, 2019) and 48 Western European Holocene humans ( LeLuyer, 2016 and original data; SOM Table S2). According to our record, a similar and topographically homologous feature occurs more frequently on the P 3 , either in Neanderthals (7/12) or extant humans (7/28), whereas it is rarely present on the P 4 (1/8 and 0/20, respectively).

Additionally, at least among the samples available to us, this radicular depression is more frequent in European Neanderthals (8/18) than in Holocene humans (7/48). However, further studies are necessary to elucidate its nature and significance as a possible taxon/population-related feature.

Along with the Shanidar remains from further north, the Wezmeh 1 maxillary premolar, from the Central Western Zagros of Iran, provides solid evidence for the presence of Neanderthals through the Zagros Mountains in the Late Pleistocene. Although undated other than to at least OIS 3, or OIS 2 [START_REF] Trinkaus | Late Pleistocene human remains from Wezmeh Cave, Western Iran[END_REF], its morphological affinities and the presence of a Late Pleistocene faunal assemblage from Wezmeh Cave [START_REF] Mashkour | Carnivores and their prey in the Wezmeh Cave (Kermanshah, Iran): A Late Pleistocene refuge in the Zagros[END_REF] strongly imply that it derives from the earlier OIS 3 and therefore relates to the increasingly documented Middle Paleolithic of the region. As such, it adds to the limited evidence for Neanderthals in Southwest Asia east of the Mediterranean region. 1 and2 and SOM Table S1 for sample composition. The extremes of morphological variation are illustrated at the ends of each bgPC axis in occlusal (mesial aspect, upward) and buccal views (mesial aspect, rightward).

Table 1

Number of specimens (n) and degree of occlusal wear (range; [START_REF] Smith | Patterns of molar wear in hunter-gatherers and agriculturalists[END_REF]

  of both P 3 and P 4 representing: European Neanderthals (NEA) from Krapina, Sima de las Palomas, and Spy (Bayle et al., 2017, In press; NESPOS Database, 2019); Middle Paleolithic modern humans (MPMH) from Qafzeh (Qafzeh 10 and 15 [ESRF, 2019] and Qafzeh 4 [original data]), and Holocene humans (HH) of Western European origin (Le Luyer, 2016 and original data).

Figure captions Figure 1

 captions1 Figure captions

Figure 2 .

 2 Figure 2. a) The Wezmeh cave (white arrow) at the western foothills of the Qazivand Mountain, Kermanshah region of Central Western Zagros. b) Entrance of the cave and original location of the Late Pleistocene skeletal remains redeposited at the front of the cave mouth. c) Plan of the WezmehCave with the areas of excavation (after[START_REF] Abdi | Islamabad Project 2001. Test excavations at Wezmeh cave[END_REF]; the area of the clandestine excavation is indicated at the top (black arrow). d) The Wezmeh 1 maxillary premolar in mesial (M), distal (D), buccal (B), lingual (L) and occlusal views (after[START_REF] Trinkaus | Late Pleistocene human remains from Wezmeh Cave, Western Iran[END_REF].

Figure 3 .

 3 Figure 3. µCT-based 3D virtual rendering of Wezmeh 1 a) external morphology; b) the specimen imaged with the virtually removed enamel cap revealing the morphology of the coronal dentine.The correspondence between the outer enamel and the inner dentine morphology can be appreciated in occlusal view for (1) the elevation of the mesial marginal ridge attached to a low short crest running into the mesial fovea and (2) a cusp-like relief on the distal marginal ridge. The arrows show the segments of the bifurcated transverse crest and the asterisks indicate the position of small pits representing minor enamel defects.

Figure 4 .

 4 Figure 4. Plot of log 2D average enamel thickness (2D AET) against log coronal dentine and pulp area (b) for Wezmeh 1 and the P 3 and P 4 forming the Neanderthal (NEA), Middle Paleolithic

Figure 5 .

 5 Figure 5. Enamel thickness cartographies of Wezmeh 1 in occlusal (O), buccal (B), mesial (M), lingual (L), and distal (D) views compared with selected P 3 from the Neanderthal (NEA), Middle Paleolithic modern human (MPMH), and Western European Holocene human (HH) samples.Topographic thickness variation is rendered by a pseudocolor scale ranging from thinner dark blue to thicker red; isolated spots correspond to wear-related enamel removal. Independently from their original side, all crowns are shown as right.

Figure 6 .

 6 Figure 6. Enamel thickness cartographies of Wezmeh 1 in occlusal (O), buccal (B), mesial (M), lingual (L), and distal (D) views compared with selected P 4 from the Neanderthal (NEA), Middle Paleolithic modern human (MPMH), and Western European Holocene human (HH) samples.Topographic thickness variation is rendered by a pseudocolor scale ranging from thinner dark blue to thicker red; isolated spots correspond to wear-related enamel removal. Independently from their original side, all crowns are shown as right.

Figure 7 .

 7 Figure 7. Between-group principal component analysis (bgPCA) of the Procrustes shape coordinates of the enamel-dentine junction (EDJ) of Wezmeh 1 compared with the P 3 and P 4 forming the Neanderthal (NEA), Middle Paleolithic modern human (MPMH), and Western European Holocene human (HH) samples. See Tables1 and 2and SOM TableS1for sample

  of the P 3 and P 4 forming the three comparative samples used in the 2-3D analyses. The samples represent Neanderthals (NEA), Middle Paleolithic modern humans (MPMH) and Western European Holocene humans (

  GM EDJ = geometric morphometrics of the enamel-dentine junction shape; DTP dental tissue proportions; ET = enamel thickness; TOPO ET: standardized enamel thickness topographic variation.

								ESRF Paleontological
		MPMH 3	1	3	2	2	3	Microtomographic Database, 2019;
								original data
		HH	13 1-4	13	13	11	13	Le Luyer, 2016; original data
	Abbreviations:
		HH	14 1-4	14	13	11	13	Le Luyer, 2016; original data
								Bayle et al., 2017, 2019; NESPOS
	P 4	NEA	8 1-4	8	8	4	8
								Database, 2019

Acknowledgments

Research funded by the French CNRS. We thank the National Museum of Iran for research support, contribution and generous collaboration; the UMR 7209 for funding the surface scans at the SURFAçUS Platform of the MNHN Paris and the 3D print of Wezmeh 1; the PLATeforme INstrumentale d'Analyse of the IC2MP laboratory of the University of Poitiers for access to the µCT device; and the LabEx "Sciences archéologiques" of the University of Bordeaux for the analysis of comparative materials. We also thank the Editor, the Associate Editor, and the two anonymous reviewers for their helpful comments on the manuscript.

1 and the P 3 forming the Neanderthal (NEA), Middle Paleolithic modern human (MPMH), and Western European Holocene human (HH) samples.

SOM Table S1

List of P 3 and P 4 forming the Western European Holocene human sample (HH) used in the comparative analyses.

UP3

UP4

List of the P 3 and P 4 forming the Neanderthal (NEA) and the Middle Paleolithic modern human (MPMH) samples used in the comparative analyses.

See Table 1 for the data sources. 2D dental tissue proportions and enamel thickness in Wezmeh 1 compared to the values of the P 3 and P 4 forming the Neanderthal (NEA), Middle Paleolithic modern human (MPMH), and Western European Holocene human (HH) samples. See Tables 1 and2 for sample composition. Abbreviations: 2D AET = 2D average enamel thickness; 2D RET = 2D relative enamel thickness; a = total crown area; b =: coronal dentine and pulp area; b/a = percentage of crown area that is dentine and pulp; c = enamel area; e = enamel-dentine junction length; BCD = bicervical diameter.

Table 4

3D dental tissue proportions and enamel thickness in Wezmeh 1 compared to the values of the P 3 and P 4 forming the Neanderthal (NEA), Middle Paleolithic modern human (MPMH), and Western European Holocene human (HH) samples. See Tables 1 and2 Abbreviations: 3D AET = 3D average enamel thickness; 3D RET = 3D relative enamel thickness; SEDJ = EDJ surface area; Vc = total crown volume; Vcdp = coronal dentine and pulp volume; Vcdp/Vc = percentage of crown volume that is dentine and pulp; Ve = enamel volume.

Supplementary Online Material (SOM):

A Holocene human (HH) samples. The line passing through zero represents the mean; the dotted lines correspond to the estimated 95% limit of variation expressed by the comparative samples. See Materials and methods for variable abbreviations and Tables 1 and2 and SOM Table S1 for sample composition.