Yvonne Grunder

Christopher A Lucas

Paul B J Thompson

Yves Joly

Yvonne Soldo-Oli- Vier

Charge reorganization at the adsorbate covered electrode surface probed through in-situ resonant x-ray diffraction combined with ab initio modelling

Keywords: bond formation, electrochemical interface, interfacial charge, ab-initio calculation, in-situ characterization, x-ray diffraction

Understanding the charge distribution and bonding mechanism at the polarized solid-liquid interface is a fundamental challenge in electrochemistry which impacts on applications ranging from materials processing to renewable energy production. The applied potential, ions in solution and the polarization of the interface all combine to alter the bond formation and the interfacial charge distribution thus techniques must be developed to provide in-situ characterization of the interface.

Here we present a combination of in-situ resonant surface x-ray diffraction studies and self-consistent DFT calculations to assess the charge distribution and bonding mechanism for the adsorption of bromide anions onto a single crystal Cu(001) electrode surface. Comparison between the experimental and modelled data gives detailed information about the charge distribution at the interface and the bonding of specific adsorbates, predicting a charge rearrangement rather than charging of the atoms involved and a surface dipole moment situated at the metal surface.

Introduction

Although electrochemical interfaces play a key role in many day-to day applications ranging from renewable energy production to materials processing, detailed information on the charge distribution at the interface and its influence on reactivity, activity and phase formation is not easy to obtain. Understanding the influence of the applied electrochemical potential, the interfacial atomic arrangement and adsorbate layer formation on the nature of the charge transfer and the interfacial electronic structure is central to developing a fundamental understanding of electrochemical reactions. Adsorbates play a crucial role in electrochemical processes such as phase formation during electrodeposition [START_REF] Krug | In-situ surface x-ray diffraction studies of homoepitaxial electrochemical growth on Au(100)[END_REF][START_REF] Golks | In situ surface x-ray diffraction studies of the influence of the peg-cl-complex on homoepitaxial electrodeposition on Cu(001)[END_REF][START_REF] Golks | In situ surface x-ray diffraction studies of homoepitaxial growth on Cu(001) from aqueous acidic electrolyte[END_REF][START_REF] Golks | Anomalous potential dependence in homoepitaxial Cu(001) electrodeposition: An in situ surface x-ray diffraction study[END_REF] and during electrocatalytic reactions [START_REF] Hermann | Enhanced Electrocatalytic Oxidation of Formic Acid on Au(111) in the Presence of Pyridine[END_REF] , where they can be present at the interface either as adsorbed species from the electrolyte solution or as reaction intermediates. Adsorbates can also affect the electrode stability and result in degradation of the electrode. [START_REF] Grunder | Potential Dependent Structure and Stability of Cu(111) in Neutral Phosphate Electrolyte[END_REF][START_REF] Kolb | Reconstruction phenomena at metal-electrolyte interfaces[END_REF][START_REF] Kaghazchi | Nanoscale-faceting of metal surfaces induced by adsorbates[END_REF][START_REF] Pender | Electrode Degradation in Lithium-Ion Batteries[END_REF][START_REF] Angelucci | The role of adsorbates in electrocatalytic systems: An analysis of model systems with single crystals[END_REF] Insights into the exact mechanism of adsorbate bonding at the electrochemical interface, the polarizability of the bond and its dependence on the applied potential will thus help to better understand and design electrochemical processes. In addition, insight into bond formation and modification by surrounding ions or an electrical field is of more general interest to several areas in Chemistry, such as bio-molecular or complex-formation . [START_REF] Chen | Hydrogen and Halogen Bonding in Homogeneous External Electric Fields: Modulating the Bond Strengths[END_REF][START_REF] Kong | Direct Observation of Amide Bond Formation in a Plasmonic Nanocavity Triggered by Single Nanoparticle Collisions[END_REF] Although recent advances have been made in understanding the atomic scale structure of the electrode-electrolyte interface and helped to elucidate many interfacial electrochemical phenomena, including the ion arrangements close to the electrode surface, [START_REF] Abad | Direct electron transfer to a metalloenzyme redox center coordinated to a monolayer-protected cluster[END_REF][START_REF] Lucas | The structure of the electrochemical double layer: Ag(111) in alkaline electrolyte[END_REF][START_REF] Toney | Electrochemical deposition of copper on a gold electrode in sulfuric acid: resolution of the interfacial structure[END_REF][START_REF] Steinrück | The nanoscale structure of the electrolyte-metal oxide interface[END_REF][START_REF] Zhou | Three-Dimensional Molecular Mapping of Ionic Liquids at Electrified Interfaces[END_REF][START_REF] Tong | Hydrophobic Water Probed Experimentally at the Gold Electrode/Aqueous Interface[END_REF][START_REF] Letchworth-Weaver | Joint density functional theory of the electrode-electrolyte interface: Application to fixed electrode potentials, interfacial capacitances, and potentials of zero charge[END_REF] the exact nature of the charge distribution at the electrochemical interface is not understood. The distribution of charges in the absence and presence of adsorbates has been extensively discussed in terms of partial charge transfer and electrosorption valencies, but up to now without direct measurements of these parameters. [START_REF] Schultze | Bond formation in electrosorbates -I Correlation between the electrosorption valency and Pauling's electronegativity for aqueous solutions[END_REF][21][START_REF] Schmickler | The partial charge transfer[END_REF][START_REF] Schultze | The partial discharge of electrosorbates and its influence in electrocatalysis[END_REF] The situation is complicated by the phenomenon of non-specific adsorption which can alter the potential distribution at the interface. The effect of the double layer structure and composition on catalytic reactions has been suggested and recognized for a number of examples, [START_REF] Stoffelsma | Promotion of the oxidation of carbon monoxide at stepped platinum single-crystal electrodes in alkaline media by lithium and beryllium cations[END_REF][START_REF] Strmcnik | The role of non-covalent interactions in electrocatalytic fuelcell reactions on platinum[END_REF][START_REF] Colic | Influence of the electrolyte composition on the activity and selectivity of electrocatalytic centers[END_REF][27][START_REF] Frumkin | Influence of cation adsorption on the kinetics of electrode processes[END_REF][START_REF] Waegele | How cations affect the electric double layer and the rates and selectivity of electrocatalytic processes[END_REF] but the exact effect on the charge distribution is difficult to probe experimentally. Possible indications about the electron density and potential drop modification near the electrode surface in the presence of chemisorbed adsorbates by non-specifically adsorbed ions can be gained from molecular dynamics simulations, whereas the near range ordering of the ions in solution is accessible through x-ray reflectivity and surface x-ray scattering. [START_REF] Lucas | The structure of the electrochemical double layer: Ag(111) in alkaline electrolyte[END_REF][START_REF] Fenter | Mineral-water interfacial structures revealed by synchrotron X-ray scattering[END_REF][START_REF] Bourg | Stern Layer Structure and Energetics at Mica-Water Interfaces[END_REF] Experimental verification of the theoretical results from molecular dynamics simulations, such as a direct probing of the predicted electron density and modification by the applied potential and electrolyte ordering is still lacking.

Due to the buried nature of the electrochemical interface standard electron spectroscopy techniques which could give details about interfacial electron densities are difficult to employ, although there has been progress recently in developing ambient pressure photoelectron spectroscopy, despite the rather severe experimental limitations required for the technique to be feasible. [START_REF] Saveleva | Insights into electrocatalysis from ambient pressure photoelectron spectroscopy[END_REF] Resonant surface x-ray diffraction has been employed previously to study oxidation state changes during the anodic oxidation of Pt(111) electrodes and to probe the adsorption of carbon monoxide onto the Pt electrode surface. [START_REF] Menzel | Resonance anomalous surface X-ray scattering[END_REF][START_REF] Menzel | Polarization-dependent resonant anomalous surface X-ray scattering of CO/Pt(111)[END_REF] First principles calculations were made to reproduce the essential features in the signal from the specularly reflected x-rays and the modelling was restricted to the forward scattering cross sections, only allowing a qualitative comparison with the experimental data. The authors laid out the necessary computational steps to be taken for a more quantitative analysis of the x-ray diffraction data. Recently we have managed to successfully implement these computational steps into the FDMNES program [START_REF] Bourke | FDMX: Extended X-ray absorption fine structure calculations using the finite difference method[END_REF][START_REF] Bunǎu | Self-consistent aspects of xray absorption calculations[END_REF] to allow the first-principles simulation of resonant surface x-ray diffraction data. [START_REF] Joly | Simulation of Surface Resonant X-ray Diffraction[END_REF][START_REF] Soldo-Olivier | Unraveling the Charge Distribution at the Metal-Electrolyte Interface Coupling in Situ Surface Resonant X-Ray Diffraction with Ab Initio Calculations[END_REF] In this paper we present experimental measurements and first principles calculations of the resonant surface x-ray diffraction from a bromide-covered Cu(001) electrode in the electrochemical environment. As shown in previous measurements [START_REF] Grunder | Probing the charge distribution at the electrochemical interface[END_REF] this system is an ideal test case to develop the methodologies as the c(2x2) ordering of the bromide adlayer enables specific bromide and Cu atoms to be selected by the diffraction conditions for the spectroscopic measurements. Each resonant diffraction spectra measured at a position in reciprocal space has a distinguished contribution from the surface and the bulk atoms. The x-ray scattering amplitude depends on the electron density of the contributing atoms and thus any modification of charge distribution should be observable in a modification of the scattered intensity close to the adsorption edge of the specific atom. [START_REF] Joly | Resonant Xray diffraction: Basic theoretical principles[END_REF] The intensity variation at each position in reciprocal space is thus specific to the spectroscopic response of the atoms contributing to the intensity at that specific positions and to the modification of the atomic form factor due to the change in the electron arrangement at the interface. The effect of the electrochemical potential drop and the near interfacial ions were not included in previous calculations. [START_REF] Joly | Simulation of Surface Resonant X-ray Diffraction[END_REF] These effects can modify the electron density of the surface atoms and subsequently the resonant diffraction signal close to the absorption edge. Here we mimic the electrochemical environment by including the effect of a simple double layer model at the electrochemical interface and this has been implemented into the FDMNES code. The results yield new insights into the underlying bonding and charge distribution, suggesting a charge rearrangement and a surface dipole moment situated at the metal surface.

Methodology 2.1 Theoretical details

The modelling of the data extends our recent developments of the FDMNES code to model resonant surface x-ray diffraction data [START_REF] Joly | Simulation of Surface Resonant X-ray Diffraction[END_REF] by including the effect of the electrochemical interface and applied potential in the form of a Helmholtz potential due to the double layer and ions present at the interface. To account for the electrochemical double layer, we included an additional potential VHelm as would be expected from a three-dimensional Gaussian charge distribution ρ(z)= 𝜌 0 √(2𝜋) 3 𝜎 3 exp( -

(𝑧-𝑧 0 ) 2
2𝜎 2 ) at a distance z0 from the surface, with a width σ and containing the total charge ρ0. The additional potential VHelm then takes the form of a peak centered at z0 and described by the relationship:

VHelm(z)=𝑉 0 √𝜋 2 erf ( (𝑧-𝑧 0 ) √2𝜎 ) (𝑧-𝑧 0 ) √2𝜎
, where 𝜎 = 𝛼∆𝐻 √2 with α=0.285925 is linearly related to ΔH, the FWHM of the potential. This potential accounts for the counter ions plane present close to the interface. The potential is schematically shown together with the surface atomic positions in Figure 1 for the particular system under study, namely bromide anions adsorbed onto a Cu(001) electrode surface. This model is necessary to account for the ion accumulation at the interface and the resulting electrostatic potential, induced by the applied electrochemical potential and the electrolyte. Note that this is a simplistic model to represent a possible counter charge close to the electrode surface and does not necessary represent the 'real' form of the induced potential modification. This work is aiming to elucidate the change of the charge distribution at the electrode surface which is induced by the additional Helmholtz potential representing the ionic counter plane at the position of the specific atom. The resonant x-ray diffraction spectra are calculated using the FDMNES code. Its ab initio DFT approach uses a free shape potential allowing the adding of the Helmholtz barrier potential described above. The resonant form factors, giving finally the intensity of the reflections, depend on the local electronic structure around the resonantly excited atoms. The code can thus deliver at the same time and with the same model, for example with or without the Helmholtz potential, the projected density of states (pDOS) on the atoms and the Crystal Orbital Overlap Population (COOP). [START_REF] Diaz-Lopez | Crystal Orbital Overlap Population and X-ray Absorption Spectroscopy[END_REF] The COOP is a direct measure of the covalency between 2 neighboring atoms. It corresponds to the integral over space of the product of 2 orbitals of the 2 atoms. Its unit is as for the pDOS, the number of electrons per energy unit. Importantly its sign reveals the bonding (>0) or antibonding (<0) character of the bond. Results on the COOP and pDOS are analyzed in the next sections.

2.2 Experimental details X-ray measurements. The resonant x-ray scattering experiments were performed at XMaS (BM28), the UK-CRG beamline at the European Synchrotron Radiation Facility in Grenoble. The samples were aligned using an incident x-ray beam energy below the Cu K-edge (8.979 keV). The Cu(001) crystal was indexed to the conventional fcc unit cell with the (0, 0, L) direction along the surface normal (a* = b* = c* = 2π/a, with a = 3.615 Å) and aligned with the (0, 0, 2) and (1, 1, 1) bulk Bragg reflections. The energy of the incoming xray beam was scanned through the Cu and Br absorption Kedges at 8.919 keV and at 13.474 keV, respectively, while the Q-vector was kept constant at the selected point in reciprocal space. A 4-circle diffraction geometry [START_REF] Grunder | Probing the charge distribution at the electrochemical interface[END_REF][START_REF] Toney | Instrumental effects on measurements of surface X-ray diffraction rods: resolution function and active sample area[END_REF] was used and the spectra were each measured in two different geometries changing the polarization of the incoming x-ray beam by 90° with respect to the surface normal to allow to distinguish charge rearrangement parallel and perpendicular to the surface normal. The change in polarization was achieved by physically rotating the sample by 90° while the polarization of the x-rays was kept constant. In the vertical geometry the Q-vector is perpendicular to the polarization of the x-ray beam and at low L parallel to the surface normal.

In the horizontal geometry the Q-vector is parallel to the polarization of the beam and at low L perpendicular to the surface normal. [START_REF] Paolasini | Resonant and magnetic X-ray diffraction by polarized synchrotron radiation[END_REF] The intensity of the elastically diffracted x-rays was recorded using a Vortex® x-ray detector. This detector has sufficient energy resolution to enable separation of the elastic and the fluorescent x-ray signal from the copper substrate and record both in parallel. The derivative of the recorded fluorescence together with the EXAFS features of the fluorescence was used to define the position of the Cu K-edge and allow exact alignment of the energy spectra measured in the two different geometries. At each particular reciprocal lattice position where energy scans were performed, a background signal was measured by rotating the sample about its surface normal (by ~0.5°) and repeating the energy scan. Each set of scans was repeated at least once to allow for averaging and to acquire sufficiently precise counting statistics.

Sample Preparation and Setup. The Cu(001) single-crystal sample (Mateck, 99.999%, 10 mm diameter, miscut <0.1°) was prepared prior to the experiments by electropolishing in 70% orthophosphoric acid. The sample was covered by a droplet of milli-Q water and mounted into the electrochemical thin layer x-ray cell. [START_REF] Gründer | Probing the halide-metal interaction by monolayer metal deposition at the electrochemical interface[END_REF][START_REF] Tidswell | In situ x-ray-scattering study of the Au(001) reconstruction in alkaline and acidic electrolytes[END_REF][START_REF] Samant | In-situ grazing incidence X-ray diffraction study of electrochemically deposited Pb monolayers on Ag(111)[END_REF] In all experiments 10 mM HClO4 + 10 mM KBr solution was prepared from 99.999% trace metals basis KBr (Aldrich) and 99.999% trace metals basis 70% perchloric acid (Aldrich) electrolyte. Perchloric acid was chosen to keep an acidic pH to stop the formation of surface oxide as perchlorate is strongly solvated and thus does not specifically adsorb on the surface. [START_REF] Magnussen | Ordered anion adlayers on metal electrode surfaces[END_REF] All potentials were measured versus a Ag/AgCl (3 M KCl) reference electrode. The measurements of the x-ray resonant data were performed at OCP (-0.08V versus Ag/AgCl). At this potential the electrode is covered by a full monolayer of bromide. We chose this potential to avoid any possible reference electrode potential shifts as the collection of the full data set took several days. In all measurements, the outer chamber of the xray cell was continuously purged with nitrogen to protect the electrode surface from oxygen.

Results and discussion

The experimental diffraction spectra obtained at specific points in reciprocal space at the Cu K-edge (8.979 keV) are presented in Figure 2 (a-d). Fig 2 (e-g) shows the calculated electron densities of the empty p-orbital states for the 1st two Cu atomic layers (specifically the Cu atoms labelled in Figure 1) calculated both with and without the presence of the counter ions included through the form of the Helmholtz potential. The structural parameters for the Br adatoms and Cu substrate atoms are based on a previous in-situ surface x-ray diffraction study of the Br-Cu(001) -c(2x2) system. [START_REF] Saracino | Surface relaxation phenomena at electrified interfaces: Revealing adsorbate, potential, and solvent effects by combined x-ray diffraction, STM, and DFT studies[END_REF] As can be seen, the inclusion of the counter ions only affects the density of states of the outermost layer, Cu1. We have modelled the Br-Cu(001)c(2x2) interface both with and without the presence of the potential representing the Helmholtz layer. For the choice of the parameters we have taken a distance, z0, of 1.9 Å above the Br adlayer which corresponds to 3.4 Å above the first Cu(001) atomic layer. The potential maximum coming from the electrostatic potential due to cation layering above the adsorbed bromine is 18 eV. The width of the potential has been taken as ΔH=2 Å, which corresponds to the potential of a charge distribution with a Gaussian profile along the surface normal with σ=0.4 Å and thus a FWHM of 0.94 Å. Note that the surface atoms are affected only by the tail of the potential distribution, thus small changes in the parameters will not have a large effect. Figure 1 depicts the potential as used to model the data with respect to the atomic positions of the Cu(001)-Br surface. The structural ion layering parameters used in this study to give a good agreement with the experimental data are close to ion ordering parameters found previously. [START_REF] Lucas | The structure of the electrochemical double layer: Ag(111) in alkaline electrolyte[END_REF][START_REF] Toney | Voltage-dependent ordering of water molecules at an electrode-electrolyte interface[END_REF][START_REF] Bourg | Stern Layer Structure and Energetics at Mica-Water Interfaces[END_REF] Cation-substrate distances were found to be of the order of 2-3 Å for an adsorbate-free aqueous-mica interface and of the order of ~4 Å for an adsorbate-free Ag(111) surface in aqueous solution. [START_REF] Lucas | The structure of the electrochemical double layer: Ag(111) in alkaline electrolyte[END_REF][START_REF] Bourg | Stern Layer Structure and Energetics at Mica-Water Interfaces[END_REF] In the presence of adsorbed hydroxyl anions the cation layer was found to be stabilized closer to the Ag(111) surface at a distance of ~3.6 Å. [START_REF] Lucas | The structure of the electrochemical double layer: Ag(111) in alkaline electrolyte[END_REF] Molecular Dynamics simulations have been able to give a concrete value for the electrostatic potential induced by ion ordering close to a charged interface. A potential of the order of 6 eV has been obtained for a water layer adsorbed close to a Pt(111) surface. [START_REF] Sakong | The electric double layer at metal-water interfaces revisited based on a charge polarization scheme[END_REF][START_REF] Skulason | Density functional theory calculations for the hydrogen evolution reaction in an electrochemical double layer on the Pt(111) electrode[END_REF] In this study we need a larger value to reproduce the experimental data than the one obtained in the above-mentioned study. These values are however not unreasonable as we do consider the case of a specifically adsorbed anion layer for which it is not unreasonable to consider a remaining partial charge on the adsorbate in comparison to a potential induced by the dipole moment of adsorbed water. In addition, for the halide covered copper surface the potential at which the data was measured corresponds to a shift of 1.1 V in potential compared to the work function of a halide covered Cu(001) surface in UHV. [START_REF] Gründer | Reversal of chloride-induced Cu(001) subsurface buckling in the electrochemical environment: An in situ surface x-ray diffraction and density functional theory study[END_REF][START_REF] Trasatti | Structuring of the solvent at metal/solution interfaces and components of the electrode potential[END_REF] The water layer on Pt(111) was found to induce a lower shift of 0.55 V [START_REF] Sakong | The electric double layer at metal-water interfaces revisited based on a charge polarization scheme[END_REF] thus supporting a lower potential due a less ordered charge layer on the solution side of the interface.

The calculated spectra at the points in reciprocal space for which experimental data was recorded, both with and without including the potential representing the counter cations at the interphase, are shown in Figure 2 (a-d). The spectra recorded at the most surface sensitive position, (1 1 0.2), are shown in Figure 2 a. The features of the energy spectra are better reproduced considering the counter ions/Helmholtz layer and the change observed are associated with a modified bonding and shift of the electron density and surface dipole moment as will be discussed below. The (0 1 0.2) and (0 1 0.7) positions (Figure 2b andd, respectively) are sensitive to atoms with the symmetry of the c(2x2) Br adlayer and thus, for the case of the Cu K-edge, have contributions from the second atomic Cu layer only (atoms Cu2 and Cu3 in Figure 1). The differences between the intensity modelled with and without including the Helmholtz layer are not as pronounced as for the (1 1 0.2) position, though the near edge structure is slightly better reproduced. To compare the goodness of fit of the model to the experimental data, a distance reliability factor R1 and a confidence factor D1 were used. [START_REF] Joly | Low-temperature structure of magnetite studied using resonant x-ray scattering[END_REF][START_REF] De Santis | Epitaxial growth and structure of cobalt ferrite thin films with large inversion parameter on Ag(001)[END_REF] The inclusion of the Helmholtz potential in the calculations leads to a reduction in the R1 and D1 values from .0012 to .00069 and from 1.096 to 0.925 respectively. These are the values obtained for the entire data set, a breakdown of the R1 and D1 values for the individual spectra is presented in the supplementary information in table SI1. The results can be better understood by plotting the calculated electron densities associated with each atom and orbital as shown in Figure 3 for the case with and without the presence of the Helmholtz potential. For the Br adatom, a clear shift to lower energies of the pzorbitals combined with a more defined energy range is observed. In addition to the energy spectra at the Cu K-edge, the energy spectra measured at the (0 1 0.2) position, while scanning the energy through Br K-edge, are shown in the supplementary information, Figure SI1.

Localized energy states of the s electrons are also observed in the same lower energy ranges for the 1st two Cu atomic layers. This indicates formation of a stronger bond through orbital overlap, with the charge mostly residing on the Br adatoms. This result is further supported by the COOP which is shown in the supplementary information, Figure S2. The localized energy state at -9 eV with contributions mostly from the Br p-and Cu s-orbitals can be observed. Interestingly sharpening of the energy states can be observed down to the second atomic layer. As discussed previously [START_REF] Ávila | Role of the Partial Charge Transfer on the Chloride Adlayers on Au(100)[END_REF] the assignment of electronic charge belonging to specific atoms is difficult. To distinguish between the amount of charge belonging to the metal and to the adsorbing species is impossible as the charge assigned to a specific atom depends on the area selected for the integration of the electronic density around each atom. Therefore, we only quantitatively discuss the changes in the electron densities and try to further the discussion around charge redistribution by considering differences in charge density maps and potential, as the differences do not rely on a specific radius selected to obtain atomic electron densities. The effect of the electrochemical potential on the electron distribution at the interface is shown in Figure 4, in which the difference of the electron density Δρ= ρ2-ρ1 with (ρ2) and without (ρ1) the Helmholtz potential is shown along (a) the <100> and (b) the <110> directions (bulk coordinates) through the Br adsorbate atom. The charge distribution at the atoms has been altered, not only by adding or subtracting charges, thus resulting in simply a charged atom, but by a rearrangement of the electron densities at different distances from the center of the atom. The Br atom shows an additional negative charge which is concentrated towards the center and in an additional sphere around the atom, whereas in between these spheres a slight positive charge can be seen. The Cu atoms close to the surface show an additional positive charge close to the center and in a sphere ~0.5 Å from the center, whereas the space in between these two positive charge accumulations is slightly negatively charged. This can be understood in terms of a redistribution of the charges between the orbitals and a possible hybridization of the orbitals due to a change in the bonding between the atoms. Note that the electron density as shown in Figure 4 is deduced from the contribution of the atomic orbitals only. To estimate the effect of hybridization and of the associated charge rearrangement, the COOP between the different atoms was integrated up to the Fermi level; the difference with and without applying a Helmholtz potential indicates the shift in the charge arrangement in the free electrons (s and p orbitals) due to bond formation and orbital overlap. This is indicated schematically by the '+' and '-' signs in Figure 4. The modification of the potential energy Vr due to the rearrangement of the electron density is also shown in Figure 4 using a different color scheme (green/grey).

From both the change in the electron density rearrangement and the potential a change in the surface dipole moment can be deduced, with a slightly increased negative charge towards the Br adsorbates. Interestingly the origin of the change in the dipole moment does not lie, as might be expected, between the Br adsorbate and the first Cu atomic layer but is actually in the first Cu atomic layer. The origin of the charge modification thus is between the Br-adsorbate and the 2nd Cu atomic layer. These effects explain the buckling of the 2nd Cu atomic layer [START_REF] Gründer | Reversal of chloride-induced Cu(001) subsurface buckling in the electrochemical environment: An in situ surface x-ray diffraction and density functional theory study[END_REF] which can, with the origin of the dipole moment on the 1st Cu atomic layer, be understood as being due to local Friedel oscillations induced by the Br ad-atoms. It is apparent that the simple approximation of a metal electrode with the jellium model is invalid at least when specific adsorption occurs. The first atomic layers of the metal electrode have to be treated differently from the bulk atoms due to the interaction of the surface atoms with ions in solution and subsequent adsorbate formation. The position of the surface or interface can be assumed as the position of the surface dipole moment (Friedel oscillations can induce additional smaller dipole moments further into the substrate). With the dipole surface moment shifting into the metal electrode, the 1st atomic layer cannot clearly be assigned as part of the substrate or of the electrolyte anymore. This may sound counterintuitive from the perspective of traditional surface science but could well help to explain several aspects of electrochemical processes such as corrosion, electrodeposition, the occurrence of underpotential deposition and the stability of catalysts undergoing electrochemical reactions.

Conclusions

In this article we have shown that a combination of in-situ resonant surface x-ray diffraction studies and self-consistent DFT calculations can suitably assess the charge distribution and bonding mechanism at the electrochemical interface for the adsorption of bromide anions onto a single crystal Cu(001) electrode surface. Comparing the experimental and modelled data gives information about the modification of the charge distribution at the interface and the bonding of specific adsorbates in the presence of a Helmholtz potential. A charge reorganization within the atoms at the interface was observed rather than a charge shift in between atoms. In addition, the dipole surface moment shifts into the metal electrode. With a charge modification found down to the second metallic layer, the results imply that it is important to consider the detailed charge distribution in the metal electrode in the modeling of electrochemical reactions.

Supporting information

(1) Experimental results on the Br-Kedge

In addition to the energy spectra at the Cu K-edge, the energy spectra measured at the (0 1 0.2) position, while scanning the energy through Br K-edge, were measured and are shown in Figure SI1. With the bromine adatoms only occupying one symmetric site, the spectra observed at the Br K-edge resemble typical adsorption spectra.

Figure SI1:

The spectra at the (0 1 0.2) position on the c(2x2) superstructure rod as measured in horizontal and vertical polarisation around the Br K-edge are shown together with the modelled data obtained without applying an additional potential (dotted line) and with applying an additional potential of 18 eV at a distance of 1.9 Å (solid line) as further specified in the text.

Figure 1 .

 1 Figure 1. The Helmholtz potential VHelm(z) is depicted together with the positions of the surface atoms. The different copper atoms (orange) and the bromine atoms (brown) sites are labelled, with Cu1 referring to the Cu in the 1 st atomic layer, Cu2 and Cu3 referring to the Cu atom in the second atomic layer to a position below the Br atom and to a position without a Br atom on top, respectively. The adjustable parameters of the potential are H, the FWHM of the potential Vhelm, the distance z0 of the center of the charge layer from the surface and V0 the maximum value of the potential.

Figure 2 .

 2 Figure 2. The spectra at different points in reciprocal space (a) (1 1 0.2), (b) (0 1 0.2), (c) (1 1 0.8) and (d) (0 1 0.7) measured in horizontal and vertical polarization mode are shown together with the modelled data obtained both with (solid line) and without (dotted line) applying an additional potential of 18 eV at a distance of 1.9 Å as further specified in the text. Error bars on the measured intensity have been obtained as the square-root of the measured counts in the detector. The position of the Cu K-edge is indicated by a vertical dashed line. The changes in the empty states of the electron density of the 4porbitals, which are probed by exciting the Cu K electrons in the Cu atoms in the 1st two atomic layers Cu1 (e), Cu2 (f) and Cu3 (g) are shown to illustrate the changes induced in the spectra. The energy is indicated as the photon energy (against which the data is measured) and as E-EF, with EF the Fermi energy, for the density of states. The scales are aligned to each other to allow for comparison.

Figure 3 .

 3 Figure 3. The s-,p-, and d-electron densities obtained by modelling the resonant data without (a,c,e,g) and with an applied potential of 18 eV at a distance of 1.9 Å above the Br adsorbate (b,d,f,h) are shown. The z-contribution of the p and d-orbitals are separately shown as a purple and blue line, respectively. The electron densities are shown for (a,b) the Br adsorbate atom, (c,d,) the 1st layer of Cu atoms, (e,f) the 2nd layer Cu atom beneath an empty hollow site, (g,h) the 2nd layer Cu atom beneath an adsorbed Br atom.

Figure 4 .

 4 Figure 4. Map of the difference in electron density Δρ and in the potential energy Vr induced by the applied Helmholtz potential and without an applied potential are shown. (a) Shows a cut along the [100] (x) direction (bulk coordinates) through the Br adsorbate. The Br adsorbate atoms and the second copper layer are visible. The '+' and '-' indicate the additional charge rearrangement from the hybridization of the orbitals obtained from the COOP. (b) Shows a cut along the [110] (x=y) direction through the Br atom, the Br adsorbate atom and the 1st copper atoms are visible.

( 2 )Figure SI2 :

 2SI2 Figure SI2: The overlap of the orbitals between the different atoms (COOP) for (a,b)the Br on copper in the 1 st layer, (c,d) 1 st layer copper and 2 nd layer copper atom beneath the bromide adsorbate, (e,f) 1 st layer copper and 2 nd layer copper atom beneath an adsorbate free hollow site and (g,h) between the two copper atoms in the second layer as obtained from the model without (a,c,e,g) and with a potential of 18 eV at a distance of 1.9 Å above the Br adsorbate (b,d,f,h) applied.

  Charge Reorganization at the Adsorbate covered Electrode Surface probed through in-situ resonant x-ray Diffraction combined with ab-initio Modelling Yvonne Grunder, ‡, * Christopher A. Lucas, ‡ Paul B.J. Thompson, ‡, † Yves Joly, § Yvonne Soldo-Olivier § ‡ Oliver Lodge Laboratory, Department of Physics, University of Liverpool, Liverpool L69 72E, UK † XMaS Beamline, European Synchrotron Radiation Facility, F-38043 Grenoble, France § Université Grenoble Alpes, CNRS, Institut Néel, 38042 Grenoble, France

Table SI1 :

 SI1 The individual changes in the R1 and D1 values for each position in reciprocal space are shown. The last two columns show the difference subtracting the values obtained with potential from the ones obtained without potential. It can be seen that overall a better agreement between the data and the model is achieved for the positions (0 1 0.2), (1 1 0.2) and (1 1 0.8). Only the position at (0 1 0.7) does not show an improvement however the changes in D1 and R1 are much smaller than observed for the other positions.

		with potential	without potential	Difference Δ
	position polarisation D1	Rx	D1	Rx	Δ D1	Δ R1
	0 1 0.2 horizontal	0.252	0.0001	0.295	0.0002	0.043	0.0001
	0 1 0.2 vertical	0.336	0.0002	0.440	0.0004	0.104	0.0002
	0 1 0.7 horizontal	0.486	0.0004	0.456	0.0005	-0.030	0.0001
	0 1 0.7 vertical	0.322	0.0002	0.319	0.0003	-0.002	0.0001
	1 1 0.2 horizontal	1.756	0.0046	2.602	0.0121	0.846	0.0074
	1 1 0.2 vertical	2.257	0.0060	2.394	0.0070	0.136	0.0010
	1 1 0.8 horizontal	0.875	0.0010	1.069	0.0016	0.194	0.0006
	1 1 0.8 vertical	1.116	0.0015	1.190	0.0016	0.074	0.0001

AUTHOR INFORMATION

ACKNOWLEDGMENT

YG thanks the Royal Society for funding through a University Research Fellowship (URF\R\180005) and a research grant (RGF\EA\181030). YG, YJ, YSO and CAL thank the Royal Society for support though an International Exchanges grant (IES\R2\202033). RSXRD measurements were performed on the EPSRC-funded XMaS CRG beamline (EP/S020845/1) at the ESRF, Grenoble, France.

Supporting information available:

Resonant diffraction on the superstructure recorded at the Br Kedge, Orbital overlap between the different atoms (COOP) from 1st principle modelling and the individual changes in the distance reliability factor R1 and confidence factor D1 are given.

For table of contents only


Ludivine Kouton

José Boucraut

Jérome Devaux

Yusuf Rajabally

David Adams

Jean Christophe Antoine

Frédéric Bourdain

Alexandre Brodovitch

Jean-Philippe Camdessanché

Cécile Cauquil

Kouton Ludivine

Boucraut José

Devaux Jérome

Rajabally Yusuf

Adams David

Antoine Jean Christophe

Bourdain Frédéric

Brodovitch Alexandre

Camdessanché Jean-Philippe

Cauquil Cécile

Ciron Jonathan

Dubard Thierry

Echaniz-Laguna Andoni

Grapperon Aude-Marie

Juntas-Morales Raul

Kremer Laurent

Kuntzer Thierry

Labeyrie Céline

Lanfranco Luca

Léger Jean-Marc

Maisonobe Thierry

Mavroudakis Nicolas

Mecharles-Darrigol Sylvie

Merle Philippe

Noury Jean- Baptiste

Rouaud Violaine

Tard Céline

Théaudin Marie

Vallat Jean-Michel

Viala Karine

Attarian Shahram

Delmont Emilien
email: emilien.delmont@ap-hm.fr

Electrophysiological features of Chronic Inflammatory Demyelinating Polyradiculoneuropathy associated with IgG4 antibodies targeting Neurofascin 155 or Contactin1 glycoproteins

Keywords: Neurofascin 155, Contactin 1, CIDP, electrophysiology

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

Introduction:

Chronic inflammatory demyelinating polyradiculoneuropathies (CIDP) are chronic auto-immune neuropathies. They have, since their description, been defined by myelin damage resulting in disorders of the nerve conduction and of the nerve excitability (Joint Task Force of the EFNS and the PNS 2010).

The precise antigenic target of the disease remains unknown in most cases, making it difficult to correlate electrophysiological finding with the pathophysiology of the disease and the clinical pattern.

Recently, IgG4 antibodies against the node of Ranvier or paranodes have been associated with subgroups of CIDP. These antibodies target neurofascin 155 (Nfasc155), neurofascin 186 (Nfasc186), contactin 1 (CNTN1) or contactin-associated protein 1 (Caspr1) [START_REF] Ng | Neurofascin as a target for autoantibodies in peripheral neuropathies[END_REF][START_REF] Querol | Antibodies to contactin-1 in chronic inflammatory demyelinating polyneuropathy[END_REF][START_REF] Doppler | Auto-antibodies to contactin-associated protein 1 (Caspr) in two patients with painful inflammatory neuropathy[END_REF][START_REF] Delmont | Autoantibodies to nodal isoforms of neurofascin in chronic inflammatory demyelinating polyneuropathy[END_REF]. The CIDP patients with anti-CNTN1 antibodies have usually an acute disease course, sometimes misdiagnosed as Guillain-Barré syndrome (GBS), are older and may be associated with nephrotic syndrome [START_REF] Querol | Antibodies to contactin-1 in chronic inflammatory demyelinating polyneuropathy[END_REF][START_REF] Miura | Contactin 1 IgG4 associates to chronic inflammatory demyelinating polyneuropathy with sensory ataxia[END_REF][START_REF] Hashimoto | Chronic Inflammatory Demyelinating Polyneuropathy With Concurrent Membranous Nephropathy : An Anti-paranode and Podocyte Protein Antibody Study and Literature Survey[END_REF][START_REF] Taieb | Neuro-renal syndrome" related to anti-contactin-1 antibodies[END_REF]. CIDP patients with anti-Nfasc155 antibodies are younger, have most often distal motor weakness, sensory ataxia, postural tremor and may have combined central and peripheral nervous system involvement [START_REF] Querol | Neurofascin IgG4 antibodies in CIDP associate with disabling tremor and poor response to IVIg[END_REF][START_REF] Ogata | Characterization of IgG4 anti-neurofascin 155 antibody-positive polyneuropathy[END_REF][START_REF] Devaux | Neurofascin-155 IgG4 in chronic inflammatory demyelinating polyneuropathy[END_REF][START_REF] Kadoya | IgG4 anti-neurofascin155 antibodies in chronic inflammatory demyelinating polyradiculoneuropathy: Clinical significance and diagnostic utility of a conventional assay[END_REF].

Intravenous immunoglobulins are mostly ineffective in CIDP with antibodies against paranodal proteins [START_REF] Querol | Antibodies to contactin-1 in chronic inflammatory demyelinating polyneuropathy[END_REF][START_REF] Devaux | Neurofascin-155 IgG4 in chronic inflammatory demyelinating polyneuropathy[END_REF].

The clinical phenotype of CIDP with antibodies against CNTN1 and Nfasc155 tends to be better understood. However, knowledge on the electrophysiological characteristics is still scarce in these patients. CIDP patients with anti-CNTN1 were first described to present mixed axonal and demyelinating features [START_REF] Querol | Antibodies to contactin-1 in chronic inflammatory demyelinating polyneuropathy[END_REF] although, only demyelinating features were described in another study [START_REF] Miura | Contactin 1 IgG4 associates to chronic inflammatory demyelinating polyneuropathy with sensory ataxia[END_REF]. Few studies provided detailed electrophysiological data on patients with anti Nfasc155 antibodies, showing delayed distal motor latencies and prolonged F-waves latencies [START_REF] Ogata | Characterization of IgG4 anti-neurofascin 155 antibody-positive polyneuropathy[END_REF][START_REF] Vallat | Paranodal lesions in chronic inflammatory demyelinating polyneuropathy associated with anti-neurofascin 155 antibodies[END_REF].

The objective of this study was to detail the nerve conduction studies of patients with anti-CNTN1 or anti-Nfasc155 antibodies and to compare these electrophysiological studies to those of CIDP without known antibodies.

Methods

Patients with available electrophysiological data and positive IgG4 antibodies against Nfasc155 or CNTN1 were included in this study. Antibodies were tested in the Aix-Marseille University of Medicine, France. One thousand sera of patients from neurological departments from France, Belgium and Switzerland were screened for this study. A cohort of 40 consecutive CIDP patients without antibodies against the node of Ranvier was enrolled for comparison. These seronegative patients fulfilled the definite CIDP diagnostic criteria according to the EFNS/PNS (Joint Task Force of the EFNS and the PNS 2010) and were followed in the Referral Centre of Neuromuscular Diseases and ALS of Marseille, France.

Data were obtained from available records. Assessments were part of routine evaluation. Patients did not undergo any additional electrophysiological tests, imaging or cerebrospinal fluid examinations as part of the current care. The study was approved by the the ethic committee of the Assistance Publique des Hôpitaux de Marseille (Agreement number PADS19-365).

Epidemiological, clinical and biological data were retrospectively collected: age, sex, clinical impairment, efficacy of IVIg treatment, Overall Neuropathy Limitations Scale (ONLS) [START_REF] Graham | A modified peripheral neuropathy scale: the Overall Neuropathy Limitations Scale[END_REF], and cerebrospinal fluid (CSF) protein level. Good response to IVIg was defined as an improvement of at least one point on the ONLS scale.

Nerve conduction studies were performed bilaterally on the median, ulnar, tibial and fibular nerves at presentation. The following parameters were considered: distal motor latency (DML), distal compound muscle action potential (CMAP) amplitude, distal CMAP duration, sum of the distal CMAP amplitudes (CMAP sum score), motor conduction velocity at the forearm or at the leg, F-waves latencies. Motor conduction block was defined by a ≥50% amplitude reduction of the proximal negative peak of the CMAP relative to the distal negative peak. Temporal dispersion was defined by a >30% duration increase between the proximal and the distal negative peak of the CMAP (Joint Task Force of the EFNS and the PNS 2010). Terminal latency index (TLI) were calculated for median and ulnar nerves using the formula: distal distance in millimetre / forearm conduction velocity / DML. TLI < 0.25 was indicative of excessive distal conduction slowing [START_REF] Attarian | Terminal latency index and modified F ratio in distinction of chronic demyelinating neuropathies[END_REF]. Modified F ratio (MFR) was calculated using the formula:

(F-wave latency + DML -2*PML -1) / (2 * DML), where PML is proximal motor latency measured at the elbow. MFR > 2.5 for median nerve and >3.25 for ulnar nerve were indicative of prominently proximal conduction slowing [START_REF] Attarian | Terminal latency index and modified F ratio in distinction of chronic demyelinating neuropathies[END_REF]. Distal CMAP duration thresholds were 8ms, 9ms, 9.1ms and 8.7ms for the median, ulnar, fibular and tibial nerves respectively [START_REF] Rajabally | A European multicentre reappraisal of distal compound muscle action potential duration in chronic inflammatory demyelinating polyneuropathy[END_REF]. Sensory nerve action potentials (SNAP) amplitudes and sensory conduction velocities were recorded bilaterally on median, ulnar and sural nerves. SNAP sum core was obtained by adding the SNAP amplitude of median, ulnar and sural nerves.

Antibodies against Nfasc155 and CNTN1 were detected with a cell-binding assay or a flow cytometry technique using HEK cells transfected with the respective plasmid of interest [START_REF] Miura | Contactin 1 IgG4 associates to chronic inflammatory demyelinating polyneuropathy with sensory ataxia[END_REF][START_REF] Devaux | Neurofascin-155 IgG4 in chronic inflammatory demyelinating polyneuropathy[END_REF]). IgG4 isotype was determined using a secondary antibody targeting IgG4 human IgG.

Quantitative data were expressed in mean (standard deviation). Quantitative data were compared using a Mann-Whitney test or an ANOVA test with Bonferroni post-test in case of multiple comparisons.

Qualitative data were compared with a Fisher's exact test. Statistical and receiver operating characteristic (ROC) analysis were performed using IBM SPSS statistics software (version 20) and GraphPad Prism 5 (California, USA). Statistical significance was set for a two-sided p-value < 0.05.

Results:

Thirteen CIDP with IgG4 antibodies against Nfasc155, 9 CIDP with IgG4 antibodies against CNTN1 and 40 CIDP negative for these antibodies were enrolled in this study. All fulfilled the EFNS/PNS diagnostic criteria for definite CIDP (Joint Task Force of the EFNS and the PNS 2010). Clinical characteristics are given in table 1. Eleven patients with antibodies against the node of Ranvier and 34 seronegative patients received IVIg treatment before inclusion in this study. CIDP patients with antibodies against paranodal proteins had more frequently sub-acute onset, higher ONLS score and were resistant to IVIg treatment (table1). CIDP with antibodies against CNTN1 had shorter disease duration. CIDP with antibodies against Nfasc155 were more prone to have a postural tremor. Central nervous system involvement was not present in this cohort.

Sural nerve biopsies were performed in 2 patients with antibodies against Nfasc155 and in one patient with antibodies against CNTN1. Semi-thin sections showed a loss of myelinated fibres without inflammatory cells infiltration. Some myelinated axons presented thin myelin sheaths suggestive of a demyelinating process. Electron microscopy showed a selective loss of the septate-like junctions at the paranodes in the patient with antibodies against CNTN1 (Figure 1).

Electrophysiological features of patients with IgG4 antibodies against Nfasc155 or CNTN1

Electrophysiological examination fulfilled the EFNS/PNS electrodiagnostic criteria for definite CIDP(Joint Task Force of the EFNS and the PNS 2010) in all patients. Abnormalities were symmetric and bilateral and heterogeneously distributed along the nerves. Conduction defects were: prolonged F-waves latencies, delayed distal motor latencies, reduced conduction velocities, conduction blocks, temporal dispersion, prolonged distal CMAP duration and normal sural with abnormal median SNAP amplitudes (table2).

Comparison of electrophysiological features of patients with antibodies against Nfasc155 or CNTN1 and seronegative patients:

There was no statistical difference when comparing electrophysiological data from patients with antibodies against Nfasc155 and patients with antibodies against CNTN1 (table 2). However, by comparison to seronegative patients, patients with antibodies against paranodal proteins had more prolonged motor distal latencies on median, ulnar and fibular nerves, more prolonged F-waves latencies on median nerves and motor and more reduced conduction velocities on median and ulnar nerves (table 2 and figure 2). The number of patients with conduction blocks was similar in CIDP with or without associated antibodies. CMAP amplitudes were comparable in both groups but SNAP amplitudes were lower in patients with antibodies namely on median and ulnar nerves.

As nerve conduction analyses were similar between patients with antibodies against Nfasc155 and anti CNTN1, the electrophysiological data of the 22 reactive patients were combined for comparison with the 40 seronegative patients. ROC analyses were performed to determine if electrophysiological data permitted to distinguish CIDP patients with and without antibodies (figure 2). Best discrimination was assessed with minimum motor velocity on median nerve (AUC = 0.76, p < 0.001), minimum motor velocity on ulnar nerve (AUC = 0.80, p < 0.001) and maximum motor distal latency on ulnar nerve (AUC = 0.76, p < 0.001). Motor conduction velocity for median nerve < 24m/s or motor velocity for ulnar nerve < 26m/s or motor distal latency on ulnar nerve > 7.4ms were predictive of positive antibodies against paranodal proteins with a sensitivity of 59% and a specificity of 93%.

Discussion

We report 13 patients with anti-Nfasc155 antibodies and 9 patients with anti-CNTN1 antibodies.

These patients presented with similar clinical characteristics as those previously described: subacute onset, severe disability, resistance to IVIg treatment and postural tremor for patients with anti-Nfasc155 antibodies [START_REF] Ng | Neurofascin as a target for autoantibodies in peripheral neuropathies[END_REF][START_REF] Querol | Antibodies to contactin-1 in chronic inflammatory demyelinating polyneuropathy[END_REF][START_REF] Miura | Contactin 1 IgG4 associates to chronic inflammatory demyelinating polyneuropathy with sensory ataxia[END_REF][START_REF] Devaux | Neurofascin-155 IgG4 in chronic inflammatory demyelinating polyneuropathy[END_REF].

All the patients with anti-CNTN1 or anti-Nfasc155 antibodies had abnormal nerve conduction features that fulfilled the EFNS/PNS electrodiagnostic criteria for definite CIDP. Electrophysiological data were similar between patients with anti Nfasc155 antibodies and anti-CNTN1 antibodies. Analysis of TLI and MFR did not reveal a predominantly distal or proximal pattern. The defects of nerve conduction had a heterogeneous distribution along nerves trunks and roots as in classical CIDP [START_REF] Attarian | Terminal latency index and modified F ratio in distinction of chronic demyelinating neuropathies[END_REF].

Abnormal electrophysiological features included conduction blocks and temporal dispersions, delayed distal motor latencies, delayed F-waves latencies, reduced conduction velocities and increased distal CMAP duration. Normal sural with abnormal median SNAP amplitudes, which is a supportive criterion for CIDP (Joint Task Force of the EFNS and the PNS 2010), was found in 5 patients. Decreased CMAP amplitudes were observed in most patients, due to axonal degeneration or distal conduction blocks.

Few studies have analysed electrophysiological data from patients with anti-Nfasc155 and CNTN1 antibodies. Anti-CNTN1 antibodies has been associated with an early axonal involvement in 3 patients [START_REF] Querol | Antibodies to contactin-1 in chronic inflammatory demyelinating polyneuropathy[END_REF]) and with conduction blocks in 12 other patients [START_REF] Miura | Contactin 1 IgG4 associates to chronic inflammatory demyelinating polyneuropathy with sensory ataxia[END_REF]. A detailed investigation of 21 Japanese patients with anti-Nfasc155 antibodies [START_REF] Ogata | Characterization of IgG4 anti-neurofascin 155 antibody-positive polyneuropathy[END_REF], showed nerve conduction abnormalities in the same range as in our study. TLI were not in favour of a distal process: mean TLI was 0.32 for median nerves and 0.42 for ulnar nerves, compared to 0.30 and 0.38 respectively in our study. Alteration of nerve excitability has been reported in only one patient with anti-Nfasc155 antibodies, it showed an increase in the depolarization threshold and abnormalities compatible with a paranodal dysfunction [START_REF] Garg | Neurofascin-155 IGG4 Neuropathy: Pathophysiological Insights, Spectrum of Clinical Severity and Response To treatment[END_REF]. We compared the electrophysiological data of patients with antibodies against CNTN1 or Nfasc155 with those of seronegative CIDP patients. Conduction blocks and chronic denervation were observed in the same proportion. Axonal involvement can be analysed through CMAP and SNAP sum scores [START_REF] Delmont | Motor unit number index (MUNIX): Is it relevant in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP)?[END_REF]. The motor component was comparable in patients with and without antibodies but axonal loss was greater for sensory nerves in patients with antibodies against paranodal proteins.

One study [START_REF] Ogata | Characterization of IgG4 anti-neurofascin 155 antibody-positive polyneuropathy[END_REF] showed that patients with anti-Nfasc155 antibodies had more marked nerve conduction abnormalities than CIDP patients without antibodies. Our study found that both patients with anti-CNTN1 antibodies and patients with anti-Nfasc155 antibodies have more prominent nerve conduction abnormalities, notably for the distal motor latencies and motor conduction velocities in the median and ulnar nerves. Considering distal motor latency on the ulnar nerves and motor conduction velocities on the median and ulnar nerves, electrophysiology appears valuable to discriminate between patients with and without antibodies against CNTN1 or Nfasc155 with a sensitivity of 59% and a specificity of 93%.

NfascC155 is expressed by the Schwann cell and CNTN1 by the axon, and both help maintaining the cohesion between the myelin sheath and the axon in the paranodal region and maintaining the segregation of the potassium channels of the juxtaparanodal region and the sodium channels of the nodal region. Several studies argue in favour of a pathogenic role for anti-Nfasc155 and CNTN1 antibodies: [START_REF] Ng | Neurofascin as a target for autoantibodies in peripheral neuropathies[END_REF][START_REF] Doppler | Destruction of paranodal architecture in inflammatory neuropathy with anti-contactin-1 autoantibodies[END_REF][START_REF] Manso | Contactin-1 IgG4 antibodies cause paranode dismantling and conduction defects[END_REF][START_REF] Manso | Anti -neurofascin-155 IgG4 antibodies prevent paranodal complex formation in vivo[END_REF][START_REF] Koike | Paranodal dissection in chronic inflammatory demyelinating polyneuropathy with anti-neurofascin-155 and anti-contactin-1 antibodies[END_REF][START_REF] Vallat | Paranodal lesions in chronic inflammatory demyelinating polyneuropathy associated with anti-neurofascin 155 antibodies[END_REF]. Acute motor axonal neuropathies (AMAN) are related to anti-GM1 antibodies that target the node of Ranvier [START_REF] Susuki | Anti-GM1 Antibodies Cause Complement-Mediated Disruption of Sodium Channel Clusters in Peripheral Motor Nerve Fibers[END_REF]. In these neuropathies, the nerve conduction studies show axonal abnormalities in the form of transient conduction block and decreased amplitudes of the distal CMAP [START_REF] Uncini | Nodo-paranodopathy: Beyond the demyelinating and axonal classification in anti-ganglioside antibody-mediated neuropathies[END_REF]. One would expect that the anti-CNTN1 and Nfasc155 antibodies, that also bind the node of Ranvier, would give the same abnormalities. However, CIDP patients with antibodies against the node of Ranvier have clear nerve conduction abnormalities as seen in demyelinating neuropathies [START_REF] Miura | Contactin 1 IgG4 associates to chronic inflammatory demyelinating polyneuropathy with sensory ataxia[END_REF][START_REF] Ogata | Characterization of IgG4 anti-neurofascin 155 antibody-positive polyneuropathy[END_REF][START_REF] Doppler | Auto-antibodies to contactin-associated protein 1 (Caspr) in two patients with painful inflammatory neuropathy[END_REF][START_REF] Delmont | Autoantibodies to nodal isoforms of neurofascin in chronic inflammatory demyelinating polyneuropathy[END_REF]. Nerve biopsies help to interpret this result. Nerve biopsies of CIDP patients with antibodies against Nfasc155 or CNTN1 show too thin myelin sheaths around some axons (figure 1), but no inflammatory infiltration nor onion bulbs formations [START_REF] Doppler | Destruction of paranodal architecture in inflammatory neuropathy with anti-contactin-1 autoantibodies[END_REF][START_REF] Ogata | Characterization of IgG4 anti-neurofascin 155 antibody-positive polyneuropathy[END_REF][START_REF] Koike | Paranodal dissection in chronic inflammatory demyelinating polyneuropathy with anti-neurofascin-155 and anti-contactin-1 antibodies[END_REF][START_REF] Vallat | Paranodal lesions in chronic inflammatory demyelinating polyneuropathy associated with anti-neurofascin 155 antibodies[END_REF]. Electron microscopy examination shows a selective loss of the septate-like junctions at the paranodes (figure 1) and a detachment of the paranodal myelin loops from the axon [START_REF] Koike | Paranodal dissection in chronic inflammatory demyelinating polyneuropathy with anti-neurofascin-155 and anti-contactin-1 antibodies[END_REF][START_REF] Vallat | Paranodal lesions in chronic inflammatory demyelinating polyneuropathy associated with anti-neurofascin 155 antibodies[END_REF]. The detachment of the paranode leads to a widening of the node of Ranvier, to an alteration of the internode distance and to a loss of the segregation of the sodium and potassium channels [START_REF] Boyle | Contactin orchestrates assembly of the septate-like junctions at the paranode in myelinated peripheral nerve[END_REF][START_REF] Sherman | Neurofascins are required to establish axonal domains for saltatory conduction[END_REF]. This disorganization of the nodes of Ranvier disrupts the saltatory conduction of the nerve impulses and causes nerve conduction abnormalities which may be interpreted as a "demyelinating" process, whereas the nerve biopsies do not display major demyelinating features.

This study shows that patients with anti-CNTN1 and anti-Nfasc155 antibodies have abnormal nerve conduction which fulfil the EFNS/PNS electrodiagnostic criteria for CIDP. Electrophysiological abnormalities seem more marked in patients with antibodies than in patients without antibodies against the node of Ranvier and have a propensity to demonstrate greater motor slowing at the forearm for upper limb nerves and greater distal latency prolongation at the wrist for the ulnar nerve. The limitations of this study are that it is a retrospective analysis, that disease durations were different between seronegative patients and CIDP with anti-CNTN1 antibodies and that most of conduction studies were performed on IVIg-treated patients. Our results need thus to be confirmed in a larger and prospective cohort.

Acknowledgement and funding

JD was supported by the Agence Nationale pour la Recherche and the Association Française contre les Myopathies (21532).

Conflict of Interest Statement

None of the authors have potential conflicts of interest to be disclosed


Sebastian Fedden

Switch-reference and temporal reference in Mian

Introduction

This paper gives a descriptive account of the typologically unusual behaviour of switchreference (henceforth abbreviated 'S/R') marking in the Papuan language Mian by explaining this behaviour in the light of the origin of the S/R markers. I will argue that these derive from tense, aspect, and mood (TAM) markers occurring elsewhere in the language. This process of functional change is typologically not well documented.

Mian is a Papuan language of the Ok family [START_REF] Healey | A survey of the Ok family of languages, reconstructing Proto-Ok[END_REF], which itself is part of the larger Trans-New Guinea (TNG) family (cf. [START_REF] Pawley | The chequered career of the Trans New Guinea hypothesis: Recent research and its implications[END_REF][START_REF] Ross | Pronouns as a preliminary diagnostic for grouping Papuan languages[END_REF][START_REF] Wurm | Papuan Languages of Oceania[END_REF]). Mian is spoken in Telefomin district of Sandaun province in Papua New Guinea. The Eastern dialect of Mian [START_REF] Fedden | A grammar of Mian, a Papuan language of New Guinea[END_REF] has about 1,500 speakers. Mian speakers up into their mid-seventies also speak the Papua New Guinea-variety of Neo-Melanesian Pidgin, Tok Pisin, and some of the older men (about 50 years and older) also speak or at least understand the neighbouring, closely related Ok language Telefol. All Mian data presented in this paper were obtained by the author during a total of eleven months in the field. Examples from the spontaneous corpus are listed with a source reference. Examples without a source reference have been elicited by the author.

The formal and functional features of S/R systems have received considerable attention in the literature (cf. Haiman and Munro 1983[START_REF] Roberts | Switch reference in PNG[END_REF][START_REF] Stirling | Switch-reference and Discourse Representation[END_REF]. S/R is usually described as a discourse tracking device, whose main function is to monitor the subject, i.e. to indicate through verbal morphology whether the subject of some other clause is the same (SS) or different (DS). In Papuan languages, S/R typically occurs in clause chaining constructions [START_REF] Longacre | Hierarchy and universality of discourse constituents in New Guinea languages: discussion[END_REF], Stirling 1993: 16), and S/R is typically calculated with respect to the subject of the succeeding clause [START_REF] Foley | The Papuan Languages of New Guinea[END_REF][START_REF] Foley | The languages of New Guinea[END_REF], Foley and Van Valin 1984). I will use the term 'marked clause' for the medial clause in which the verb is marked for S/R and the term 'reference clause' for the succeeding medial or final clause in reference to which S/R is marked. In adopting this terminology I follow Munro (1980). The marked clause is marked for co-/disjoint reference of the subject with respect to the reference clause.

S/R systems in Trans-New Guinea eastern Highlands languages

In most TNG of the eastern Highlands languages, we find a robust, grammaticalized S/R system. There is a difference in morphological complexity of medial verbs compared to sentence-final verbs, which appear as the predicate of the last clause in a clause chain. See [START_REF] Roberts | Switch reference in PNG[END_REF] for a typological survey of S/R systems in New Guinea based on a sample of 169 languages, 122 of which have such a system.

The simplest case is a medial verb only marked for co-reference of subject, but not for tense or person/number of the subject. The following example is from Usan (Numugenan family, Madang Province):

(1) Usan (Reesink 1983: 217 and pers. comm.) ye nam su-ab is-omei I tree cut-ss go_down-1SG.FPST 'I cut the tree and (I) went down.'

Often the marker has additional temporal or aspectual meaning, which is fused into the same formative, e.g. sequentiality or simultaneity of events. This is the case in Fasu (West Kutubuan family, Southern Highlands Province; cf. [START_REF] Loeweke | General grammar of Fasu (Námo Mē)[END_REF] and Kalam (Kalam family, Madang Province; described in [START_REF] Pawley | The structure of Kalam: A grammar of a New Guinea highlands language[END_REF][START_REF] Pawley | Encoding events in Kalam and English: Different logics for reporting experience[END_REF][START_REF] Pawley | A language which defies description by ordinary means[END_REF]):

(2) Kalam (Pawley 1993: 101) kwt d-y nwp pk-p-yn stick hold-SS.PRIOR him hit-PFV-2SG 'You hit him with a stick.' (lit. 'You took a stick and hit him') DS-forms of medial verbs are usually more complex than SS-forms. They can carry a subject marker, which indicates DS while the SS form is zero, e.g. in Fore (Gorokan family, Eastern Highlands; cf. [START_REF] Scott | The Fore Language of New Guinea[END_REF], or they can have an anticipatory subject marker which indicates person and number of the following subject, as in Hua (Gorokan family, Eastern Highlands; cf. [START_REF] Haiman | Hua: A Papuan Language of the Eastern Highlands of New Guinea[END_REF].

In other languages, one finds portmanteau DS markers, for example in Kobon [START_REF] Davies | The syntax of the simple sentence in Kobon[END_REF] and Usan, where the DS marker also contains information on subject person and number without these categories being segmentable as distinct morphemes:

(3) Usan (Reesink 1983: 218 and pers. comm.) ye nam su-ine is-orei I tree cut-1SG.DS go_down-3SG.FPST 'I cut the tree (and it went) down.' Medial verbs can have separate suffixes for disjoint reference and for subject person and number, as in ( 4):

(4) Kalam (Pawley 1993: 94) an ag-e-k g-a-k? who say-DS.PRIOR-3SG do-3SG-PST 'Who told him to do it?' (lit. 'Who said and he did it?') So in these typical examples of S/R systems in TNG languages medial verbs are different from final verbs in several respects: not all medial verbs are marked for subject person and/or subject number (e.g. SS medial verbs often occur without an affix crossreferencing the subject); they often cannot be marked for various categories such as irrealis mood or absolute tense, i.e. a tense category which has the moment of speaking as its deictic centre (cf. [START_REF] Comrie | Tense[END_REF]; and the formatives that medial and final verbs allow are formally and functionally distinct.

Switch-reference in Mian

Like the TNG languages spoken in the eastern Highlands, Mian makes very frequent use of clause chaining in discourse, but here the situation is different in that medial verbs resemble final verbs much more than in the languages discussed above. Mian has a less grammaticalized S/R system, which is characteristic of languages of the West Papuan Highlands, e.g. Dani [START_REF] Bromley | A Grammar of Lower Grand Valley Dani[END_REF] and Kapauku, also known as Ekagi (William Foley pers. comm.). There are differences between medial and final verbs in Mian, but these are few: medial verbs cannot be marked for negative polarity nor the modal categories irrealis and hortative. But apart from these restrictions, all medial verbs -always index the subject and any additional argument (i.e. the object), depending on the argument structure of the verb, in the same way as if the verb occurred in an independent sentence or the final clause of a clause chain; -take a range of markers which convey temporal and S/R information; these latter suffixes occur in the slot before the subject suffix and are homophonous with TAM suffixes found in the same slot in final verbs; -can be marked for several tense categories to locate an event at different points in the past.

The basic affix slot template for final and medial verbs in Mian is given in (5):

(5) (OBJECT-)Vstem-SR/TAM-SUBJECT(-TENSE)=CLITIC Objects are only indexed for a subset of verbs. Whether a verb indexes its object is lexically determined. Tense suffixes in the post-subject slot are optional. Final verbs have an enclitic indicating illocutionary force, such as =be 'declarative', =bo 'quotative', or =e 'interrogative', while medial verbs invariably end in a conjunctive suffix =(t)a.

The template given in ( 5) is illustrated for a final verb in (6) and a medial verb in (7):

(6) afín bi-n-ib-bio=be friend be_there-REAL-2/3AN.PL-GPST=DECL 'they were friends ' [Mianmin and Telefomin] (7) máam=e a-nâ'-n-i=a mosquito=SG.M 3SG.M.O-kill.PFV-SS.SEQ-1SG.SBJ=MED 'I killed a mosquito and then I …'

The S/R suffixes indicate co-reference or disjoint reference and always provide information about the temporal structure of events as well (namely whether they occur sequentially or simultaneously). These are:

-n '(SS,) Sequential' -s 'DS, sequential, close link' -Ø 'DS, sequential' -b
'DS, simultaneous'

Although not the focus of this paper, I will also mention the auxiliary biaan here, which is used to express the fourth logical possibility, namely 'SS, simultaneous'. The suffix -n is more complicated because it only unequivocally indicates SS if the subject of the marked clause is first person singular. Hence, I will deal with it in some detail below. The other S/R markers are more straightforward and are introduced in the following sections. The DS markers -s and -Ø, both 'DS, sequential', require the subject in the reference clause to have disjoint reference. While -Ø only indicates DS and event sequentiality, -s also highlights that the event in the marked clause is a necessary condition for the event in the reference clause to take place, or that the two events occur in close temporal succession, hence 'close link' in the gloss for -s. This contrast between -Ø and -s is illustrated in the following example (8): There are three events (e1 to e3), all of which occur sequentially. In the clauses describing e1 and e3, the subject is the speaker (hence, 1 st singular subject marking on the verb). The second medial clause (e2) has a different subject from the rest of this text excerpt, namely the tobacco leaf. This referent has been introduced at the beginning of the text from which (8) was taken and has already been referred to anaphorically in the first clause of (8) with the object prefix tob-'3 rd singular long object'. In e2 a necessary condition is specified for the event in the succeeding clause to take place, namely the tobacco having dried (in e2) is a necessary prerequisite for the speaker to break it (in e3). In general, -s, rather than -Ø, is used if the speaker wants to highlight the fact that the event in the marked clause is necessary for the event in the reference clause to take place or that the two events are closely linked temporally. Compare also the subtle difference in meaning in the following two elicited examples: In (9), -s-marking indicates that the arrival of the hearer expressed in a medial clause is a necessary condition for the event expressed in the following clause to take place. In (10), where the medial verb is zero-marked, the relation between the events is purely sequential.

-b 'DS, simultaneous'

The marker -b 'DS, simultaneous' is used to describe situations in which the event in the marked clause is on-going when the event of the reference clause commences. Subject reference is disjoint. The reference clause event can be a bounded event which is properly contained within the event described by the marked clause, as in ( 11):

(11) ngaan-b-e=a call.IPFV-DS.SIM-3SG.M.SBJ=MED naka=i wentê-n-ib=a man=PL.AN hear.PFV-SEQ-2/3PL.AN.SBJ=MED 'While he was calling, the men heard (him), and then …' [Dafinau] The suffix -b 'DS, simultaneous' is also used to describe a situation in which the event in the first reference clause (e2) is itself unbounded and not or not necessarily properly containted within (e1), as in (): If both e1 and e2 are expressed by medial verbs inflected with -b, as in ( 12), the only restrictions regarding the temporal interpretation of the events with respect to each other is that e2 must not commence before e1 does. In that sense, even a sequence of two medial clauses with simultaneous events is iconic. Events can be temporally co-extensive but they do not have to be.

biaan 'SS, simultaneous'

Simultaneity of events without a switch in subject reference is expressed by employing a special verb stem biaan in the marked clause. This stem belongs to the paradigm of the existential verb bi. Example () illustrates the use of biaan as an independent verb. It is used as an auxiliary in serialization with a main verb in ():

( Having discussed the S/R forms -s, -Ø, -b, and biaan, I will now turn to the unusual behaviour of -n. There is an interesting typological quirk in the Mian S/R system. The suffix -n only unequivocally marks SS and event sequentiality if the subject is first person singular. Consider ( 15 In ( 15), the substitution of -n 'SS, sequential' in tob-ò-n-i=a with any of the DS markers, viz. -s 'DS, sequential, close link' or -Ø 'DS, sequential', i.e. tob-ò-s-i=a or tob-ò-Ø-i=a, yields ungrammatical results. On the other hand, -n functions just as an indicator of event sequentiality in all other person-number combinations regardless of the reference of the subject in the reference clause. Except in first person singular, -n does not provide S/R information.

In the following, I will illustrate this point further with several elicited examples. The phenomenon under discussion is attested in the spontaneous corpus, but I choose to use elicited sentential minimal pairs here to make the point clear. In a clause chain with coreferent non-first-singular subjects, marking with the suffix -n is what one would expect given the behaviour of -n with first person singular subjects. Thus: One could of course object that all of these examples are elicited and that it is important, and indeed crucial, to study S/R behaviour in coherent discourse rather than in isolated, and even worse, elicited, examples (cf. Reesink 1983: 241-2). It is well-known that SS marking in languages with a S/R system can be apparently inconsistent (Stirling 1993: 60-114). This can for instance be the case if the reference clause provides background information (e.g. in temporal clauses, in clauses that describe meteorological conditions or physiological/psychological states). S/R marking proceeds with respect to topicality hierarchies (Reesink 1983: 240), rather than mechanically monitoring the subject of the reference clause. Consequently, one clause or even several clauses with less topical subjects can be "skipped" by the reference monitoring system and SS marking is calculated with respect to the clause in which the topic re-occurs (also cf. [START_REF] Farr | The Interface between Syntax and Discourse in Korafe, a Papuan Language of Papua New Guinea[END_REF]: 228ff., Healey 1966: 15-16).

In Mian, such apparently inconsistent marking of SS can be observed before clauses with subjects of low topicality and before clauses providing background information. These clauses are effectively skipped by the S/R mechanism in that SS marking is calculated with respect to the clause in which the topic is mentioned again, as in (20). The clause containing the low topicality subject teng 'tiredness' appears in square brackets: The bracketed medial clause in (20) has a different subject from the surrounding clauses but because teng 'tiredness' is low in topicality, it would be plausible to assume that -n is used in (20a) with respect to the re-occurring topic in (20c).

However, Mian also allows -n before clauses providing background information, even if the topic of the first clause is never mentioned again in the rest of the text (the backgrounded temporal clause is enclosed in square brackets): In this text excerpt the subject of the first medial clause yoma-n-e=ta 'he begot (children) and then ...' is the male protagonist and it would be plausible to assume that the bracketed adverbial clause naka=i utl-Ø-ib=o 'when the people grew up' was skipped and SS marking was interpreted with respect to the clause following this adverbial, if the man reoccurred as the topic. Yet, the begetting of children was the last action he performed in this story. He drops out as a topic and is never mentioned again. Nonetheless, the suffix -n is possible in the first medial clause. This variable behaviour of -n indicates that S/R meaning in Mian is less strongly grammaticalized compared to Usan or Kalam (cf. Section 2 above). The suffix -n in medial verbs is a more general marker of sequentiality except where the subject of the marked clause is first person singular. In this case, -n unequivocally indicates event sequentiality and SS.

Prima facie, this situation creates a functional paradox because "[switch reference] is redundant where either subject [i.e. in the marked or the reference clause -SF] is first or second person, and necessary where both subjects are third person. We may therefore expect to find languages in which switch-reference is limited to the third person." (Haiman and Munro 1983: ix). 2 In the third person, on the other hand, one would expect full functionality of the S/R system because here the system is crucial for referent identification.

These expectation, which derive from the function of S/R systems as reference trackers, are not fulfilled in Mian. However, the language can "afford" such a quirk because it marks gender in the third person cross-referencing affixes. As all medial verbs have suffixes cross-referencing the subject, this helps disambiguating in many cases (as in example [22] repeated from [17] above, subject-indexing suffixes appear in boldface): In cases where subject reference is to be disjoint but gender is the same, marking with -n is nonetheless allowed but here the disjoint-reference interpretation is only available if an overt noun phrase is used in the reference clause, e.g.:

(24) mēn=e do-fâ-n-o=a child=SG.M 3SG.MASC.O-put.PFV-SEQ-3SG.F.SBJ=MED unáng mak=o me-b-o=be woman another=SG.F cry.IPFV-IPFV-3SG.F.SBJ=DECL 'She gave birth to a boy and another woman is crying.'

In example (24), the overt noun phrase unáng mako 'another woman' makes it obvious that subject reference is disjoint. Without such an overt noun phrase, subjects are invariably interpreted as co-referent:

(25) mēn=e do-fâ-n-o=a child=SG.M 3SG.MASC.O-put.PFV-SEQ-3SG.F.SBJ=MED me-b-o=be cry.IPFV-IPFV-3SG.F.SBJ=DECL 'She gave birth to a boy and is crying.' BUT: *'Shej gave birth to a boy and shek is crying.' To sum up this section, Mian S/R markers combine a S/R-related meaning with information on the temporal structure of events. The marker -n only unequivocally means sequentiality of events and same subject if the subject is first person singular. In all other cases, -n just signals event sequentiality.

DS -b -s -Ø

As Mian obligatorily indexes all subjects in final and medial verbs with indicating person, number, and (in the third person) gender, the subject reference issue is in most cases resolved in a straightforward way.

Homophonous TAM formatives in final verbs

Mian S/R markers in medial verbs are homophonous with TAM markers found in final verbs and I will propose in this section that the former have been derived from the latter. Table 2 juxtaposes S/R and TAM formatives and their respective meanings. Both sets occur in the same slot in the verb, namely immediately before the subject marker slot. On the apparent semantic mismatch of -Ø as a mood marker and as a S/R marker, see below. S/R markers having their origin in the TAM morphology of a language does not seem to be a very common development in Papuan languages. Existing theories of the origin of S/R markers in Papuan languages trace them back either to gapping under identity, which gives rise to zero marking for SS [START_REF] Haiman | On some origins of switch reference marking[END_REF], or to a pronominal or deictic origin [START_REF] Givón | Switch reference and topic continuity[END_REF]. In general, homophony between TAM and S/R markers does not seem to be well attested typologically, but see [START_REF] Jacobson | Typological and genetic notes on switch-reference systems in North American Indian languages[END_REF] on some formal similarities of S/R and aspectual morphology in languages of North America. Yet, the formal parallels between the Mian TAM markers in final verbs and S/R markers in medial verbs are too striking to be mere coincidence, especially as the semantics of the former make them a very likely historical source for the latter. I will explore this in some detail in the following sections.

-n ~ -Ø 'realis' and -n '(SS,) sequential'

The suffix -n 'realis' signals that an event has been actualized. There is a strong correlation between realis mood marking and past time reference. Realis forms have a default temporal interpretation of 'immediate past', e.g:

(26) ilem=e te=ta na-(û)t'-ne-n-e=be blood=SG.N1 come=MED do-give.PFV-1SG.D-REAL-SG.N1.SBJ=DECL '(Some) Blood has come and does (this) to me.' [Afoksitgabaam fruit] One also finds realis forms interspersed in discourse about various stages of the past. The event described in ( 27) happened more than half a century ago:

(27) yē ya-l-Ø-io=be there AN.PL.O-kill.PFV-REAL-2/3AN.PL=DECL 'they killed them there' [Mianmin and Telefomin] The S/R marker -n '(SS,) sequential' originates in the homophonous realis mood marker. So what it does is simply to mark the real mood or status (cf. Foley and Van Valin 1984) of an event. It indicates that an event has actually taken place and remains noncommittal with respect to conjoint or disjoint subject reference in the verb of the reference clause, which can be either SS or DS, with the added twist that -n indeed functions as a fully developed S/R marker if the subject is first person singular.

Because of the strong correlation between realis mood and past time reference (actualized events typically lie in the past), the temporal meaning of -n in medial verbs is 'sequential'. In final verbs, -n 'realis' alternates with -Ø mainly for phonological reasons 3 . In medial verbs, -Ø does not mean '(SS,) sequential' like -n but rather 'DS, sequential'. However, like -n '(SS,) sequential', -Ø 'DS, sequential' always indicates event sequentiality, which is not surprising given that their common origin is the realis marker -n ~ -Ø.

-b 'imperfective' and -b 'DS, simultaneous'

The suffix -b 'imperfective' has a default temporal interpretation of 'present' but can also be used for events in the past, as for instance in the following excerpt from an account of traditional initiation rituals: In medial verbs, -b means 'DS, simultaneous' and in final verbs 'imperfective'. Apart from the homophony there is also an obvious semantic connection to be found here. In final verbs, marking for imperfective aspect with -b normally indicates that the action is on-going at the moment of speaking. In medial verbs, -b indicates that an action is ongoing when the next takes place, in other words, that the two actions are overlapping and at least partially simultaneous. Looking at it from the other end, an action or event which is marked as simultaneous with the one described in the following clause is very likely to be temporally extended. There is a strong association between imperfective aspect and simultaneity of events. [START_REF] Longacre | Switch Reference systems in two distinct linguistc areas: Wojokeso (Papua New Guinea) and Guanano (Northern South America)[END_REF] formulates a 'naturalness assumption', which is essentially a converse pair of implicatures. This assumption says that: "[…] we can normally expect that actions in succession are performed by the same person, while actions that overlap are performed by different people " (p. 198).

In the Tucanoan language Guanano, for instance, on which Longacre based his argument, information on temporal structure is more basic than information on S/R. Referential information is derived from temporal information by a (cancellable) implicature based on the assumption that the use of the marker indicating sequentiality or simultaneity of actions implicates that these actions are performed by the same or a different individual, respectively. However, subjects in Guanano can be disjoint in reference even though events take place sequentially or co-referent even though events occur simultaneously.

If simultaneous actions are expected to be performed by different individuals, it is not surprising that the marker -b in Mian has DS as an additional meaning in medial verbs. The suffix -n, on the other hand, which derives from the homophonous realis marker, generally only signals sequentiality of events but if it indicates a S/R-related meaning, i.e. with first person singular subjects, it will always be SS. Hence, the behaviour of -n is also consistent with the prediction of Longacre's naturalness assumption.

Apparently, Mian exemplifies a development from temporal information (sequential vs. simultaneous) to referential information (SS vs. DS) in medial verbs. However, the referential system is only fully explicit in the first person singular. Why this is the case remains unclear. 5.3. -s 'remote past' and -s 'DS, sequential' The suffix -s 'remote past' locates events in the remote or ancient past, i.e. many decades ago or in mythical times. Therefore, it is mainly found in myths and actual historical accounts, e.g.:

(29) yōle éil=e a-nâ'-s-ib=e? well pig=SG.M 3SG.M.O-kill.PFV-RPST-2/3PL.AN.SBJ=INT 'Well, did they kill the pig?' [History of Mianmin and Telefomin] As the marker -s is derived from a deictic tense category, namely 'remote past', the temporal meaning is expected to be sequential. However, it might at first seem counterintuitive that a S/R marker meaning 'DS, sequential' should be derived from the remote past tense suffix. After all, -s in medial verbs does not have 'remote past' as a meaning component but rather has an additional meaning of temporal closeness or necessary condition apart from 'DS, sequential', which is indeed quite unrelated to the notion 'remote past'. However, the only TAM suffix occurring in final verbs that is not also used in medial verbs is -b 'non-hodiernal past'. It is used for events in the past excluding the day containing the moment of speaking. This form is segmentally homophonous with -b 'imperfective' in final verbs and wouldif chosenalso be homophonous with -b 'DS, simultaneous' in medial verbs. 4 Hence, if one assumes that the language filled out its S/R paradigm by deriving forms from the TAM suffixes used in final verbs, it seems that -s was chosen in the absence of a better option.

Further indications for a lesser degree of grammaticaliztion of S/R in Mian

Above I have pointed out that the variable behaviour of -n suggests that S/R meaning is not as strongly grammaticalized in Mian as it is for example in Usan or Kalam. Further evidence for this comes from the fact that for all other S/R markers in medial verbs we find instances, albeit very rarely, where the S/R specification in the marked clause is apparently violated, i.e. we get same subject and sequentiality with -s and -Ø, same subject and simultaneity with -b, and also different subject and simultaneity with biaan, in other words, S/R marking in the marked clause and actual subject reference in the succeeding clause can be at variance as long as the basic temporal structure, i.e. sequentiality or simultaneity, indicated by the markers is consistent. In that respect, the Mian system is more reminiscent of Guanano than of Usan.

Two selected examples from the spontaneous Mian corpus are given below to illustrate this further. Although the male protagonist is the actor and subject in all three clauses in (30), the verb in the first marked clause is onsea inflected with -s '(DS,) sequential, close link': In example (31), S/R is calculated with respect to the verb gabeta, which follows the embedded question nakai unīta tlaaibe? 'Who's about to come?' (enclosed in square brackets). Such embedded clauses are always skipped by the S/R mechanism of the language and S/R is calculated with respect to the next verb on the same syntactic level, in this case the verbum dicendi. Contrary to -n which can have DS following (except in the first person singular) as long as events proceed sequentially, cases such as those illustrated in ( 30) and ( 31) above are very rare. A similar situation can be found in Dani. Bromley (1981: 255-61) analyses a verb suffix -tik which generally expresses SS and sequentiality of events and a verb suffix -ma ~ -mo which expresses DS and sequentiality of events. While the former also occurs with DS following, occasionally in the spontaneous corpus and quite frequently in daily conversation (thus similar to Mian -n), the latter only very rarely also occurs with SS following (thus similar to Mian -s and -Ø). Although it might be possible to discount the Mian examples in ( 30) and (31) as unrepaired errors, especially since they are generally judged incorrect in elicitation, while -n with DS following is generally accepted, the fact that such examples do occur in the spontaneous corpus is at least tentative evidence for the lesser degree of grammaticalization of S/R in Mian.

Conclusion

In contrast to the robust, grammaticalized S/R systems of the PNG Highlands languages, the Mian S/R system is less strongly grammaticalized and thus reminiscent of the systems found in languages of the West Papuan Highlands. Medial verbs are not formally differentiated in any crucial way. They basically look like final verbs and the forms indicating S/R and temporal structure of events are homophonous with TAM suffixes found in the same slot in final verbs. Mian S/R markers show some unexpected behaviour. Tracing them back to their origin, namely the homophonous forms in final verbs, sheds light on the semantic development of the S/R markers within the Mian system and the semantics of the TAM markers help explain and understand the function of the S/R markers.

The process of language change we observe in Mian broadens our understanding of how S/R systems evolve. While existing theories of the origin of S/R markers in Papuan languages assume gapping or a pronominal or deictic origin, the Mian case illustrates a development where markers that provide TAM information in final verbs (remote past, imperfective, or realis) assume temporal information (sequential vs. simultaneous) in medial verbs and subsequently also referential information.

3. 1 .

 1 -s 'DS, sequential, close link' and -Ø 'DS, sequential'

( 8 )

 8 e1 tob-kimà-Ø-i=a 3SG.LONG.O-put_in_fire.PFV-DS.SEQ-1SG.SBJ=MED e2 haangān-s-e=a get_dry-DS.SEQ.CL-SG.N1.SBJ=MED e3 hā-n-i=a break.PFV-SS.SEQ-1SG.SBJ=MED 'I put it (the tobacco leaf) in the fire, as it gets dry, I break it, and then I …' [Rolling smokes]

( 9 )

 9 kōbō te-s-eb=ta you.SG.M come.PFV-DS.SEQ.CL-2SG.SBJ=MED okok ke-n-omab-bio=be work do-AUX.PFV-IRR.PL.AN.SBJ-1PL.SBJ=DECL 'Only when you come, will we work (i.e. the speaker cannot do the work alone).' (10) kōbō tl-Ø-eb=ta you.SG.M come.PFV-DS.SEQ-2SG.SBJ=MED okok ke-n-omab-bio=be work do-AUX.PFV-IRR.PL.AN.SBJ-1PL.SBJ=DECL 'When you come we'll work.'

(

  12) e1 tóm=e fuo fuo ga-b-ib=a stone=SG.N1 blow blow say.IPFV-DS.SIM-2/3PL.AN.SBJ=MED e2 ya-temê'-b-e=a PL.AN.O-see.IPFV-DS.SIM-3SG.M.SBJ=MED e3 tóm=ebelâ-s-e=ta stone=SG.N1 break.PFV-DS.SEQ.CL-SG.N1.SBJ=MED 'While they were blowing (at) the stone, 1 he was watching them and the stone opened (and then they…)'[Danenok]

  ): (15) nē memālo fút=e tob-ò-n-i=a I now tobacco=N1.SG 3SG.LONG.O-take.PFV-SS.SEQ-1SG.SBJ=MED futâan=o om-ò-na-n-i=a tobacco_paper=N2 3SG.FEM.O-take.PFV-do_too-SS.SEQ-1SG.SBJ=MED 'Now I take the tobacco, I also take the cigarette paper, and then I…' [Rolling smokes]

(

  16) mēn=e do-fâ-n-o=a child=SG.M 3SG.MASC.O-put.PFV-SEQ-3SG.F.SBJ=MED me-b-o=be cry.IPFV-IPFV-3SG.F.SBJ=DECL 'Shek gave birth to a boy and shek is crying.' But the following utterance is equally acceptable. In example (17), -n can be used although the subject of the marked clause and that of the reference clause are disjoint in reference: (17) mēn=e do-fâ-n-o=a child=SG.M 3SG.MASC.O-put.PFV-SEQ-3SG.M.SBJ=MED me-b-e=be cry.IPFV-IPFV-3SG.M.SBJ=DECL 'She gave birth to a boy and he is crying.' Now, if one imagines that the mother of the infant is talking, marking with -n requires the subject of the succeeding clause to be co-referent (cf. [18]), while -s requires disjoint reference in (19). Non-observance of these restrictions yields ungrammatical results: (18) mēn=e do-fâ-n-i=a child=SG.M 3SG.MASC.O-put.PFV-SS.SEQ-1SG.SBJ=MED me-b-i=be / *me-b-e=be cry.IPFV-IPFV-1SG.SBJ=DECL / cry.IPFV-IPFV-3SG.M.SBJ=DECL 'I gave birth to a boy and I am crying.' (19) mēn=e do-fâ-s-i=a child=SG.M 3SG.MASC.O-put.PFV-DS.SEQ.CL-1SG.SBJ=MED me-b-e=be / *me-b-i=be cry.IPFV-IPFV-3SG.M.SBJ=DECL / cry.IPFV-IPFV-1SG.SBJ=DECL 'I gave birth to a boy so he is crying.'

(

  20) a. ina-bina bi-n-ib=a do_thus-AUX.HAB stay-SEQ-2/3PL.AN.SBJ=MED b. [teng ya-l-Ø-o=a] tiredness PL.AN.O-hit.PFV-DS.SEQ-N2.SBJ=MED c. yē temdei-(û)b'-aun-Ø-ib=ta there leave-give.PFV-3SG.M.D go.PFV-DS.SEQ-2/3PL.AN.SBJ=MED 'they used to do thus until they got fed up, left him there and went away, and then he ...' [Dafinau]

( 21 )

 21 yoma-n-e=ta beget.PFV-SEQ-3SG.M.SBJ=MED [naka=i utl-Ø-ib=o] man=PL.AN come_up.PFV-REAL-2/3PL.AN.SBJ=ADV ī ninín=o dl-â-n-ib=a they name=N2 PL.FEM.O-put.PFV-SEQ-2/3PL.AN.SBJ=MED 'he begot (children), when the people grew up, they assumed names and then…' [Genesis of the Mianmin people]

(

  22) mēn=e do-fâ-n-o=a child=SG.M 3SG.MASC.O-put.PFV-SEQ-3SG.F.SBJ=MED me-b-e=be cry.IPFV-IPFV-3SG.M.SBJ=DECL 'She gave birth to a boy and he is crying.' In (23), a disjoint reading is forced due to the gender difference in the subject suffixes. The next example illustrates the same point but is from the spontaneous corpus (subjectindexing suffixes again in boldface): (23) wat-n-e=a al=baka yē across-SEQ-3SG.M.SBJ=MED colon=with there klaa-(û)b'-a-n-ang gena-b-o=ta rot-give.PFV-3SG.M.D-REAL-IMM.SG say-DS.SIM-3SG.F.SBJ=MED 'it (a wound) went across, it (his belly) was about to rot with his colon while ...' [Pineapples]

  upriver come.PFV initiation-house=N2 gen-b-io=be build.IPFV-IPFV-2/3PL.AN.SBJ=DECL 'They came upriver and were building the initiation house.' [Initiation rituals]

(

  30) amgolim ut on-s-e=a roof up(on) go.PFV-(DS.)SEQ.CL-3SG.M.SBJ=MED dei-(û)b'-a-n-e=a leave-give.PFV-SG.N1.D-SEQ-3SG.M.SBJ=MED daak-te-n-e=a down-come.PFV-SEQ-3SG.M.SBJ=MED 'he went up onto the roof and left it (a bag) there and came down and then ...' [The flood]

( 31 )

 31 temê'-b-e=to look.IPFV-(DS.)SIM-3SG.M.SBJ=MED [naka=i unīta tl-aa-ib=e?] man=PL.AN who.PL.AN come.PFV-IRR-2/3PL.AN.SBJ=INT ga-b-e=ta say.IPFV-DS.SIM-3SG.M.SBJ=MED 'while looking around, he was asking (himself): "Who's about to come?", while ... ' [Danenok]

Table 1 :

 1 Function of Mian S/R formatives

		Sequential		Simultaneous
		If SBJ is 1SG If SBJ is not 1SG
	SS	-n	-n	biaan

Table 2 :

 2 S/R markers in medial verbs and TAM markers in final verbs

	Medial verb	Final verb
	S/R markers	Meaning	TAM markers	Meaning
	-n	(SS,) sequential	-n ~ -Ø	Realis
	-b	DS, simultaneous	-b	Imperfective
	-s	DS, sequential,	-s	Remote past
		close link
	-Ø	DS, sequential

Abbreviations: 1 -1 st person, 2 -2 nd person, 3 -3 rd person, ADV -adverbial clause, AN -animate, AUX -auxiliary, CL -close link (necessary condition, temporal closeness), D -dative, DECL -declarative, DS -different subject, GPST -general past, F -female, FEM -feminine, FPST -far past, IMM -immediate action, INT -interrogative, IPFV -imperfective, M -male, MASC -masculine, MED -medial, N1 -neuter 1, N2 -neuter 2, O -object, PFV -perfective, PL -plural, REAL -realis, RESID -residue class, RPST -remote past, SBJ -subject, SEQ -sequential, SG -singular, SIM -simultaneous, SS -same subject.