Nina Eleni Christou

Isabel Ayala

Karine Giandoreggio-Barranco

Martin Byrdin

Virgile Adam

Dominique Bourgeois

Bernhard Brutscher

NMR Reveals Light-Induced Changes in the Dynamics of a Photoswitchable Fluorescent Protein

Keywords: GFP, chromophore, photoswitching, conformational dynamics, NMR spectroscopy, laser illumination Significance

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Introduction

The discovery and engineering of photo-transformable fluorescent proteins (PTFPs), derived either from Aequora victoria green fluorescent protein (GFP) or other fluorescent proteins from Anthozoa species (corals and anemones), has triggered exciting new modalities in fluorescence microscopy [START_REF] Schermelleh | Super-resolution microscopy demystified[END_REF] and biotechnology [START_REF] Adam | Data storage based on photochromic and photoconvertible fluorescent proteins[END_REF]. In particular, PTFPs have become indispensable tools for modern super-resolution light microscopy techniques that achieve resolution beyond the diffraction limit [START_REF] Betzig | Imaging Intracellular Fluorescent Proteins at Nanometer Resolution[END_REF][START_REF] Hess | Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy[END_REF]. At present, a large number of PTFPs have been discovered or engineered that differ in their specific response to light (photoactivation, photoconversion, or reversible photoswitching), in the kinetics of this response, in their photostability and general fluorescence characteristics (brightness, emission color, Stokes shift, …), as well as in their oligomerization state, folding stability, and maturation efficiency.

Furthermore, these properties are generally dependent on environmental conditions [START_REF] Endesfelder | Chemically Induced Photoswitching of Fluorescent Probes-A General Concept for Super-Resolution Microscopy[END_REF]. The requirements for an optimal PTFP marker are different for specific applications, which calls for further expansion and improvements of the PTFP toolbox.

Understanding at near-atomic resolution the mechanistic features responsible for the observed photochemical and photophysical properties of PTFPs is a key step to envision the rational design of new variants optimized for a particular application or experimental context.

Most mechanistic studies on PTFPs reported in the literature have used X-ray crystallography as the method of choice to determine structures of their different ground-state conformations.

Such structures are essentially static and, as data are typically collected at cryogenic temperature, they are representative of frozen states. Kinetic crystallography has also been used in combination with optical spectroscopy and/or molecular dynamics simulations to decipher the structures of short-lived ground state species such as intermittent dark states [START_REF] Adam | Structural Basis of X-ray-Induced Transient Photobleaching in a Photoactivatable Green Fluorescent Protein[END_REF][START_REF] De Zitter | Mechanistsic investigation of mEos4b reveals a strategy to reduce track interruption in sptPALM[END_REF]. Recently, ultrafast time-resolved crystallography using an XFEL source has been able to image an electronically excited short-lived intermediate state in rsEGFP2 that becomes populated during photoswitching between a nonfluorescent ("off") and a fluorescent ("on") state [START_REF] Coquelle | Chromophore twisting in the excited state of a photoswitchable fl uorescent protein captured by time-resolved serial femtosecond crystallography[END_REF], shedding some light on the switching dynamics of a PTFP at the picosecond timescale.

While FPs are generally quite resistant to their environment, an increasing number of studies have shown that PTFP photophysics are affected by external factors such as pH, redox potential, oxygen level, viscosity, temperature, etc [START_REF] Endesfelder | Chemically Induced Photoswitching of Fluorescent Probes-A General Concept for Super-Resolution Microscopy[END_REF][START_REF] Duan | Structural Basis of Photoswitching in Fluorescent Proteins[END_REF][START_REF] Avilov | In cellulo Evaluation of Phototransformation Quantum Yields in Fluorescent Proteins Used As Markers for Single-Molecule Localization Microscopy[END_REF][START_REF] Cloin | Efficient switching of mCherry fluorescence using chemical caging[END_REF][START_REF] Dedecker | Optimizing the fluorescent protein toolbox and its use[END_REF][START_REF] Kao | Protein-flexibility mediated coupling between photoswitching kinetics and surrounding viscosity of a photochromic fluorescent protein[END_REF]. These environmental effects are difficult to study by X-ray crystallography where structural data are collected in conditions imposed by crystallogenesis that may differ substantially from the conditions found during insitu fluorescence imaging. Moreover, little is known about the conformational dynamics of PTFPs at physiological temperatures, and their relevance for the observed fluorescence and phototransformation properties. To address these issues, multidimensional liquid-state NMR spectroscopy may provide a complementary tool to investigate at atomic resolution the conformational dynamics of PTFPs in different states, and over a wide range of temperatures and buffer conditions. However, quite surprisingly, so far only little information on PTFPs has been obtained from NMR [START_REF] Cloin | Efficient switching of mCherry fluorescence using chemical caging[END_REF][START_REF] Mizuno | Light-dependent regulation of structural flexibility in a photochromic fluorescent protein[END_REF][START_REF] Mizuno | Molecular basis of photochromism of a fluorescent protein revealed by direct 13C detection under laser illumination[END_REF], while a few more studies on chromophore dynamics in conventional GFPs can be found in the literature [START_REF] Seifert | Backbone Dynamics of Green Fluorescent Protein and the Effect of Histidine 148[END_REF][START_REF] Seifert | Slow Exchange in the Chromophore of a Green Fluorescent Protein Variant[END_REF][START_REF] Oltrogge | Ground-State Proton Transfer Kinetics in Green Fluorescent Protein[END_REF].

Here, we introduce a toolbox of multidimensional and multinuclear ( 1 H, 13 C, 15 N) NMR experiments specifically tailored to investigate PTFPs structural dynamics. We apply this toolbox to probe the conformational dynamics of rsFolder, a PTFP that can be reversibly switched from a stable fluorescent "on" state to a meta-stable nonfluorescent "off" state, and vice versa, upon illumination with cyan or violet light, respectively. Such reversibly photoswitchable fluorescent proteins (RSFP) are required for a variety of applications, ranging from super-resolution microscopy (RESOLFT, NL-SIM, pcSOFI, and others) [START_REF] Grotjohann | Diffraction-unlimited all-optical imaging and writing with a photochromic GFP[END_REF][START_REF] Rego | Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution[END_REF][START_REF] Dedecker | Widely accessible method for superresolution fl uorescence imaging of living systems[END_REF], optical lockin detection (OLID) [START_REF] Marriott | Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells[END_REF], multiplexed imaging (OPIOM) [START_REF] Querard | Photoswitching Kinetics and Phase-Sensitive Detection Add Discriminative Dimensions for Selective Fluorescence Imaging[END_REF], up to the development of highdensity optical storage media [START_REF] Adam | Data storage based on photochromic and photoconvertible fluorescent proteins[END_REF]. rsFolder has been genetically engineered as a chimera between rsEGFP2 [START_REF] Grotjohann | rsEGFP2 enables fast RESOLFT nanoscopy of living cells[END_REF] and superfolder GFP [START_REF] Tran | Engineering and characterization of a superfolder green fluorescent protein[END_REF] to facilitate proper folding in highly reducing environments such as the bacterial periplasm [START_REF] El Khatib | Rational design of ultrastable and reversibly photoswitchable fluorescent proteins for super-resolution imaging of the bacterial periplasm[END_REF]. The photophysical properties of rsFolder have been characterized by optical spectroscopy, and high resolution crystallographic structures are available for both the "on" and "off" states [START_REF] El Khatib | Rational design of ultrastable and reversibly photoswitchable fluorescent proteins for super-resolution imaging of the bacterial periplasm[END_REF]. These structures reveal the typical 11-stranded β-barrel fold, enclosing an endogenous p-HBI chromophore, formed by maturation of an Ala-Tyr-Gly tripeptide. Upon light illumination at 488 nm (405 nm), the chromophore undergoes a cis to trans (trans to cis) isomerization, and protonation (deprotonation) at the ζ position of the phenol ring. While the β-barrel structures for the "on" and "off" states almost perfectly superpose (C  rmsd = 0.21 Å), slight structural rearrangements are observed for a few side chains located in the close vicinity of the chromophore (Figure 1). Our NMR data complement this static picture by adding information on the changes in local conformational sampling after photoswitching, providing some new insight toward the long-term goal of better understanding how the photophysical properties of PTFPs are influenced by different types of local molecular flexibility. This work opens the door to further studies aiming at evaluating how PTFPs conformational dynamics respond to mutational or environmental changes.

Materials and Methods

Protein samples

Purification of rsFolder was performed as described previously [START_REF] El Khatib | Rational design of ultrastable and reversibly photoswitchable fluorescent proteins for super-resolution imaging of the bacterial periplasm[END_REF]. The full-length rsFolder cDNA was cloned in the ampicillin-resistant expression plasmid pET15b (Addgene, Teddington, UK), and constructs were designed to bear a six-residue N-terminal His-tag linked to the protein coding region via a Thrombin cleavage sequence. When considered necessary, the His-tag was cleaved using a Thrombin protease. Escherichia coli BL21(DE3) cells were transformed with the plasmid described above for protein expression. An isotopically enriched minimal medium, containing 15 NH 4 Cl (1 g/L) and 13 C-glucose (2 g/L), was used to grow the cells until the cultures reached an OD at 600 nm between 0.6 and 0.8, at which point protein expression was induced by adding IPTG (isopropyl β-D-1thiogalactopyranoside). For uniformely 15 N/ 13 C labeled samples, 1mM IPTG was added and induction was performed overnight at 20°C. The cells were harvested by centrifugation and subsequently lysed by sonication in lysis buffer (50 mM HEPES, 100 mM NaCl, pH 7.5). The lysate was centrifuged at 4°C for 30 min at 46 000g. The protein present in the supernatant was purified using a Ni-NTA column (Qiagen). The protein was eluted by increasing the concentration of imidazole. The resulting protein was further purified using size exclusion chromatography with a Superdex 75 column (GE) equilibrated with the NMR buffer (50 mM HEPES pH 7.5).

In-situ laser illumination inside the NMR magnet

An in-situ laser illumination device similar to that described by Mizuno et al. [START_REF] Mizuno | Molecular basis of photochromism of a fluorescent protein revealed by direct 13C detection under laser illumination[END_REF] was built. It comprises an Oxxius laser box (wavelength combiner L4Cc, Oxxius S.A., Lannion, France) hosting two lasers of wavelengths 405 nm (LBX-405-180-CSB-OE) and 488 nm (LBX-488-200-CSB-OE), and an optical fiber (Thorlabs FG105UCA, 0.22NA, 105 μm core diameter, 2 mm diameter protective cover, 5 m length). The fiber end was left naked and routinely cleaved when necessary, using a Ceramic Fiber Scribe (Thorlabs CSW12-5). The fiber was inserted into the plunger of a 5 mm diameter Shigemi tube (Shigemi Co LTD, Tokyo, Japan). Importantly the Shigemi plunger was filled with water before inserting the fiber to avoid a sudden change in the refractive index at the air-glass interface, thus reducing the divergence of the laser beam. The laser power was set and monitored using the Oxxius Software from a laptop connected to the laser combiner, and the laser output was controlled either manually using a shutter, or by digital on/off modulation from the NMR console connected to the laser box via TTL lines. The difference between nominal laser power as displayed by the Oxxius software and the measured output power at the fiber tip was about 30%, suggesting a coupling efficiency of about 70%. For short illumination times (less than 1 minute), the full laser power was used (200 mW nominal), while for longer exposure times (>> 1 minute) the output was set to 30% of the maximal power to limit the effect of photobleaching. In order to allow smooth insertion of the fiber-coupled Shigemi tube into the NMR magnet, the use of 2 Bruker sample holders put on top of each other proved to be helpful.

NMR measurements

All NMR experiments were performed on Bruker Avance IIIHD spectrometers, at the high-field NMR facility of IBS, operating at magnetic field strengths of 600 MHz (H/D exchange), 700-MHz ( 15 N relaxation, NMR assignment), and 850-MHz (LD-EXSY, 15 N CPMG, H/D exchange, methyl side chain, aromatics and chromophore NMR experiments), and equipped with cryogenically cooled triple-resonance probes and pulsed z-field gradients.

NMR measurements were performed at 40°C on NMR samples containing 100 -200 μM [U-13 C, 15 N]-enriched rsFolder in 50 mM HEPES (pH 7.5), and 5% (v/v) D 2 O. The samples were stored at 4 °C between data recordings, and the sample quality was routinely checked for possible aggregation and/or degradation by recording 1 H-15 N correlation spectra. "On" state data were recorded after short (5 s) illumination at 405 nm in the absence of laser illumination, while 'off" state data were acquired after a short (10 s) high-power illumination at 488 nm, under continuous illumination (488 nm) using reduced (30%) laser power to prevent on-state recovery by thermal relaxation. Most NMR experiments used in this study are implemented in the NMRlib pulse sequence library for Bruker spectrometers [START_REF] Favier | NMRlib: user-friendly pulse sequence tools for Bruker NMR spectrometers[END_REF] that can be freely downloaded from our web site (http://www.ibs.fr/research/scientificoutput/software/pulse-sequence-tools). NMR data processing was performed using Topspin 3.5 (Bruker BioSpin), while data analysis was done using the CCPNMR software [START_REF] Vranken | The CCPN data model for NMR spectroscopy: Development of a software pipeline[END_REF].

For sequential resonance assignment of the "on" state, a set of five triple-resonance 3D BEST-TROSY experiments [START_REF] Favier | Recovering lost magnetization: polarization enhancement in biomolecular NMR[END_REF][START_REF] Solyom | BEST-TROSY experiments for time-efficient sequential resonance assignment of large disordered proteins[END_REF], HNCO, HNcoCA, HNCA, HNcoCACB, and HNCACB, was recorded. For the "off" state, two LD-EXSY data sets ( 15 N, 15 N, 1 H) and ( 1 H, 15 N, 1 H) were recorded to transfer the "on"-state 1 H, 15 N assignments to the "off" state, and complemented by 3D BEST-TROSY HNcaCO, HNCACB to extend the assignment to CO, CA, and CB.

Additional side chain assignments of aromatic tyrosine and histidine residues, including the tyrosine moiety of the p-HBI chromophore, was achieved using a series of specifically tailored 2D correlation experiments as described recently [START_REF] Christou | BEST and SOFAST experiments for resonance assignment of histidine and tyrosine side chains in 13C/15N labeled proteins[END_REF]. Partial methyl ( 1 H, 13 C) assignments were obtained by a combination of 3D CcoNH-TOCSY, 3D HccoNH-TOCSY, and 3D H met C met C experiments, complemented by amino-acid-edited SOFAST 1 H- 13 C correlation spectra [START_REF] Schanda | SOFAST-HMQC experiments for recording two-dimensional heteronuclear correlation spectra of proteins within a few seconds[END_REF][START_REF] Van Melckebeke | Amino acid-type edited NMR experiments for methyl-methyl distance measurement in 13 C-labeled proteins[END_REF] 15 N relaxation experiments (T 1 , T 2 , HETNOE) were performed using standard pulse schemes implemented in NMRlib [START_REF] Favier | NMRlib: user-friendly pulse sequence tools for Bruker NMR spectrometers[END_REF]. Relaxation data for 195 "on"-state and 175 "off"-state backbone amide sites could be obtained at 40 °C, from cross peaks that were well resolved and of sufficient signal to noise in the recorded 2D 1 H- 15 N spectra, while all others (overlapping or weak) peaks were excluded from further analysis. The overall correlation time has been estimated from the "plateau" T 1 and T 2 values [START_REF] Fushman | Backbone dynamics of ribonuclease T1 and its complex with 2' GMP studied by two-dimensional heteronuclear NMR spectroscopy[END_REF] as

𝜏 𝑐 = (√ 6𝑇 1 𝑇 2 -7) 4𝜋𝜈 𝑁 ⁄ .
For H/D exchange measurements, lyophilized samples of protonated 13 were recorded for both "on" and "off" states (at 600 and 850 MHz). In order to obtain more quantitative data for amide sites that are slowly exchanging in the "on" state, but much faster in the "off" state, we performed an additional H/D exchange experiment. In this experiment, the exchange measurement starts with the "on" state, and the sample is switched after a few minutes to the "off" state using high-power laser (488 nm) illumination.

Results

Laser-driven photoswitching

In order to be able to perform a set of time-consuming multi-dimensional (2D and 3D) NMR experiments for the metastable "off" state of rsFolder, we have adapted a setup similar to the one proposed by [START_REF] Mizuno | Molecular basis of photochromism of a fluorescent protein revealed by direct 13C detection under laser illumination[END_REF] that allows to illuminate the sample in the NMR magnet during data collection with 488 nm or 405 nm laser light via an optical fiber introduced into the glass insert of a standard Shigemi tube (figure 2a). This illumination device is portable, can be used with any high-field NMR spectrometer (figure 2b), and can be synchronized with an NMR pulse sequence to switch the sample between the "on" and "off" states on demand. In order to characterize the efficiency of this illumination device to switch rsFolder between the "on" and the "off" states, we have performed real-time 1D 1 H NMR experiments under laser illumination. The spectra plotted in Figure 2d and 2e show a decrease of the "on" state population, and an increase of the "off" state population of rsFolder over time when illuminating the sample at 488 nm. The two NMR resonances visible in this spectral window correspond to a methyl 1 H that is spectroscopically resolved in both states, and thus presents a convenient probe for such 1D NMR-detected switching kinetics. As expected, the switching kinetics depend on the chosen laser power, with a measured on-off switching half time of about 2 s at 140 mW (maximal laser power output, Figure 2e). The lower limit of ~2 s results from the high optical density of the sample at 488 nm, substantially increasing the time it takes for the laser light to reach all molecules in the NMR sample. Note that the inverse switching reaction, from "off" to "on" under 405 nm light illumination (120 mW) is about twice faster (data not shown).

A superposition of the 1 H-15 N fingerprint spectra recorded in the absence ("on" state) and presence of 488 nm light illumination ("off" state) is plotted in Figure S1. Many residues (amide groups) show significant peak shifts in these spectra, indicating that the change in chromophore geometry induced by photon absorption has a widespread effect on the amide 1 H and 15 N chemical shifts in the β-barrel (see below). The laser-induced peak shifts are reversible as illustrated in Figure S2 by the superposition of "on"-state and "off"-state spectra recorded before and after 1000 switching cycles using alternate sample illumination by cyan and violet light. We found that photobleaching resulted in a progressive loss of peak intensity (about 25% after 1000 cycles under the chosen experimental conditions), but did not induce any noticeable peak shifts in the spectra, indicating that the NMR-observable conformational "on" and "off" states are not affected by photobleaching. The absence of additional peaks in our NMR spectra developing over time suggests that photo-damaged rsFolder may form large aggregates not observable by liquid-state NMR.

The observed peak shifts between rsFolder "on" and "off" states offer the possibility to quantify a "switching efficiency" that can be related to the fluorescence switching contrast of importance in microscopy applications. Quantification of the residual peak intensity of "on" state peaks in the "off" state spectrum provides a measure of the relative state populations.

For rsFolder, we have measured this residual "on" state population to be ~ 3.5 % (Figure S1), independent of the sample temperature in the range 20° -40°C. This NMR-derived value closely matches results from fluorescence spectroscopy [START_REF] El Khatib | Rational design of ultrastable and reversibly photoswitchable fluorescent proteins for super-resolution imaging of the bacterial periplasm[END_REF], where a residual fluorescence intensity of ~4% in the "off" state at room temperature was obtained, in line with the notion that the rsFolder "off" state is fully nonfluorescent. NMR also enables the measurement of the "off" to "on" back-switching efficiency, which in the case of rsFolder turns out be close to 100% (Figure S1).

Laser-aided backbone resonance assignments and secondary structure 1 H N , 13 C α , 13 C β , 13 CO, and 15 N resonance assignment of rsFolder at 40°C in the "on" state was obtained from a series of 3D BEST-TROSY HNC correlation experiments [START_REF] Solyom | BEST-TROSY experiments for time-efficient sequential resonance assignment of large disordered proteins[END_REF].

Almost complete (95%) assignment was obtained for residues 2 -230, corresponding to the β-barrel structure. Only 12 non-proline residues (W58, V69, D77, H78, E133, Y144, V151, K167, I168, K210, D211, and K215) could not be assigned from these spectra. Of those, V69, V151 and K167 are in direct contact with the chromophore based on crystallographic data.

The 1 H- 15 N correlation spectrum, annotated with the assigned residue number, is shown in Figure S3.

Recording and analyzing such a set of 3D assignment spectra is a time-consuming task (typically 1 week of data recording, and another week of data analysis). In order to speed up the assignment process for the "off" state, we have adapted a different strategy that makes use of our laser-induced in-situ switching capabilities. The "on" state assignment can be directly transferred to the "off" state by performing a laser-driven exchange experiment (LD-EXSY), conceptually similar to SCOTCH experiments [START_REF] Pouwels | Diagonal peak suppression in the SCOTCH experiment, a 2D technique for studying photochemical reactions[END_REF], proposed in the past to monitor lightinduced chemical reactions. The basic scheme of a LD-EXSY experiment is shown in Figure 3a. During the magnetization recovery time in the beginning of the pulse sequence (typically a few seconds) a laser illumination (488 nm) switches the proteins in the sample to the "off" state, allowing to edit either the 1 H or 15 N chemical shift for this state. After chemical shift editing, the magnetization is stored as longitudinal spin order (N z ), and a second laser pulse, this time at 405 nm, converts the proteins back to the "on" state during a mixing period (exchange time) of typically a few hundred milliseconds. The remainder of the sequence allows to edit the 1 H and 15 N frequencies in the "on" state. The resulting 3D data set thus comprises one "off" state ( 1 H or 15 N), and two "on" state ( 1 H and 15 N) chemical shift dimensions. Figure 3b shows annotated 2D projections of two 3D data sets recorded for rsFolder, with either the 1 H or 15 N chemical shift labeled in the "off"-state spectral dimension.

Off-diagonal peaks detected in these spectra originate from protein residues with amide chemical shifts ( 1 H or 15 N) that differ between the two states, while diagonal peaks are either due to incomplete switching, or chemical shifts that are not affected by photoswitching.

These LD-EXSY spectra thus provide a straightforward and time-efficient way for amide 1 H, 15 N assignments in the "off" state. Additional 13 C chemical shift information in the "off" state was then extracted from 3D BT-HNcaCO and BT-HNCACB spectra. This resulted in an assignment completeness of 93% (residues 2 -230), with a total of 16 unassigned non-proline residues (W58, E133, Y144, S148, N150, V151, K157, Q158, N165, K167, I168, R169, L202, K210, D211, and K215), of which S148, V151 and K167 are in direct contact with the chromophore based on crystallographic data. Note that in the case of severely line-broadened amide resonances (see below) no cross peaks were observed in the triple-resonance data, and assignment was only possible with the LD-EXSY experiment. The assigned NMR frequencies, and in particular the 13 C chemical shifts, provide useful information on the secondary structural elements (α-helix, β-strand) present in rsFolder under our experimental conditions. In Figure S4, the secondary structural features derived from our measured chemical shifts using the TALOS-N program are plotted [START_REF] Shen | Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks[END_REF]. A comparison with the X-ray structures of rsFolder (PDB files 5DTZ and 5DU0) shows that the -barrel structure in solution is almost identical to that observed in the crystal at cryogenic temperature.

Nevertheless, a few slight differences are observed for strand 7 and the two helices (H2 and H3) inside the barrel to which the chromophore is connected. The 7 strand in solution is extended by 3 residues at the N-terminal end, helix H2 is shifted by 3 residues towards the Cterminus, and helix H3 has a non-canonical backbone geometry that is not identified as an helix by TALOS. Interestingly, it has been concluded from the hydrogen bonding patterns observed in the high resolution crystal structure of EGFP [START_REF] Arpino | Crystal Structure of Enhanced Green Fluorescent Protein to 1.35 A Resolution Reveals Alternative Conformations for Glu222[END_REF], an "ancestor" protein of rsFolder, that helix H2 is composed of a 3 10 helix (P57 -L61 in rsFolder) followed by a short regular -helix (L61-L65 in rsFolder), in agreement with our NMR data.

Backbone dynamics in the "on" and "off" states

In order to obtain information on the local backbone dynamics we have performed 15 N relaxation measurements of rsFolder in both the "on" and "off" states at 40°C. The measured T 1 , T 2 , and HETNOE data are plotted in Figure 4a as a function of the protein sequence, with the secondary structural elements as identified from TALOS-N plotted on top of each graph.

These data indicate an overall rigid monomeric globular protein, with a tumbling correlation time of about 10 ns at 40°C. Increased flexibility on the ps to ns time scale (longer T 2 and reduced HETNOE values) is detected in some loop regions, especially the loops connecting the first helix (H1) and strand β1, and the loop between β-strands 9 and 10. Most interestingly, there is almost no significant difference in the fast time scale dynamics, as seen from these 15 N relaxation data, between the "on" and "off" states. However, some differences are observed that can be ascribed to increased conformational dynamics on the µs to ms time scale in the "off" state of rsFolder. A first observation is the lack of peak intensity for some amides in β-strands 7 and 8 in the 15 N relaxation spectra (turquoise bars in Figure 4b), as well as reduced T 2 values measured for residues C71 and S73 in the non-canonical helix H3. The reduction in peak intensities for some residues in the "off" state can also be observed in standard 2D 1 H-15 N BEST-TROSY spectra, as illustrated in Figure 4b. In order to further characterize these conformational dynamics we have performed CPMG-type relaxationdispersion experiments. Unfortunately, the signal to noise ratio obtained for the abovementioned residues was too low to perform a quantitative analysis. Only S73 yielded data of reasonable quality (Figure 4c) that confirm the presence of a conformational exchange process in the "off" state that leads to a modulation of the chemical shift during the relaxation period, while no relaxation dispersion is observed for this residue in the "on" state. Due to the limited experimental sensitivity, we did not attempt to extract kinetic (K ex ) or thermodynamic (state populations) information from these dispersion curves. As the exchange contribution to the transverse relaxation rate is not completely quenched at the highest CPMG frequency of 1000 Hz, we can reasonably assume that the exchange process is faster than 1000 s -1 . Note that the reported chemical shift changes are not a direct evidence of changes in structure or conformational dynamics, as part of it may be due to long-range ring-current-induced chemical shift effects that follow from the change in orientation of the chromophore.

Interestingly, all of these NMR data point towards significant changes in conformational dynamics in a particular β-barrel region. The missing "on" state peaks for a few residues in β7 (V151) and β 8 (K167, I168) indicates some possible dynamics already present in the fluorescent state, highlighting a well-known defect in the β-barrel structure and a hotspot for backbone fluctuations [START_REF] Seifert | Backbone Dynamics of Green Fluorescent Protein and the Effect of Histidine 148[END_REF][START_REF] Lelimousin | Intrinsic Dynamics in ECFP and Cerulean Control Fluorescence Quantum Yield[END_REF][START_REF] Shinobu | The Hole in the Barrel : Water Exchange at the GFP Chromophore[END_REF]. After the cis-trans isomerization of the chromophore, the conformational dynamics occurring on a s to ms time scale are expanded to a larger region close to the phenol ring of the chromophore, comprising strands 7, 8, 10, and eventually 11, as well as helix H3. (iv) amide 1H-15N with large chemical shift changes between "on" and "off" states (orange). In addition, proline residues lacking an amide proton are color coded in dark grey.

Fold stability and water accessibility

Complementary information on the conformational dynamics of backbone amides in the "on" and "off" states of rsFolder has been obtained from hydrogen-deuterium (H/D) exchange measurements. For amide hydrogens engaged in an intramolecular H-bond, H/D exchange is assumed to proceed via transient opening of the hydrogen bond followed by interaction with a solvent exchange catalyst [START_REF] Dempsey | Hydrogen exchange in peptides and proteins using NMR spectroscopy[END_REF]. Therefore, NMR derived H/D exchange rates provide an indirect measure of the dynamic fluctuations of the protein backbone leading to transient breakage of hydrogen bonds. At neutral or acidic pH, the formation of the H-bond after transient opening is generally much faster than the intrinsic chemical exchange process. In this so-called EX2 exchange regime, the exchange rate constants provide a measure of the exchange-competent state population. A second structural parameter that needs to be considered is water accessibility. Typically, an amide hydrogen surrounded by water has a higher exchange probability than the same amide hydrogen buried in the interior of a globular protein. For rsFolder, the available crystal structures for the "on" and "off" states do not indicate any major change in water-accessible cavities inside the β-barrel as illustrated in Figure S6 for the "on" and "off" states. Examples of intensity decays extracted from the realtime NMR data for individual amide sites in the "on" and "off" states are shown in Figure 6a.

A first observation from these H/D exchange data is that, while overall the amide protons in the β-barrel of the "on" state are well protected from solvent exchange, strand β7 is much less protected due to the lack in hydrogen bonding with the adjacent β8 strand. In the "off" state, a significant increase in the measured solvent exchange rates is detected for amides in the neighboring strands β8 and β10, further emphasizing the increased dynamics in this part of the β-barrel structure upon light illumination, leading to a destabilization of the hydrogen bonding network. Some local destabilization is also observed for amide hydrogens in strands β4 and β5, while no significant changes are observed for the remainder of the β-barrel (β1-3, β6, β11) as illustrated in Figure 6b. The most pronounced changes in protection from solvent exchange, however, are observed for the two helices (H2 and H3) flanking the chromophore in the interior of the β-barrel (see Figure 6b), with a decrease in solvent protection ranging from a factor of 5 up to a factor of 100. These data clearly indicate the presence of conformational dynamics affecting these helical elements induced by the chromophore cistrans isomerization. Interestingly, while 15 residues for which the hydrogen exchange rate is increased by more than a factor of 20 (red); (iv) residues without a significant change in exchange rate between "on" and "off" states (grey); (v) no information due to peak overlap, proline, or unassigned residues in one of the two states (dark grey).

Sidechain and chromophore dynamics in the "on" and "off" states

Line broadening effects similar to those already discussed in the context of backbone dynamics (Figure 4b) are also observed in the 1 H- 13 C correlation spectra of rsFolder in the "off" state, indicating that a number of protein side chains undergo conformational exchange.

Examples of methyl groups (I168and) and an aromatic side chain (H149) that become severely line broadened in the "off" state are shown in Figure 7a. In order to identify the protein regions that are involved in conformational side chain dynamics, we have performed 1 H, 13 C resonance assignment of methyl and aromatic side chain moieties in the "on" and "off" states of rsFolder as further explained in the Materials and Methods section. Figure S8 highlights 1 H- 13 C spectral regions displaying NMR signals that experience either large chemical shift changes or severe line broadening in the "off" state. The side chains involved in conformational dynamics are all located in close proximity to the chromophore (Figure 7b), attached to β-strands 7, 8, 10, and 11 or helices 2 and 3, the same backbone regions that have been shown to experience conformational exchange in the "off"-state. These data further support the conclusion that the chromophore itself and/or the surrounding environment experience significant conformational dynamics in the "off" state leading to chemical shift modulation on the s to ms time scale. NMR also provides information on the dynamics of the chromophore, and its stabilization in the β-barrel by hydrogen bonding. The exceptional thermal stability of rsFolder in the "off" state was tentatively attributed to the H-bond that H149 maintains with the phenol ring of the chromophore in both 'on' and 'off' states [START_REF] El Khatib | Rational design of ultrastable and reversibly photoswitchable fluorescent proteins for super-resolution imaging of the bacterial periplasm[END_REF], as illustrated in Figure 8a. In order to validate or contradict this hypothesis, we first performed NMR experiments that reveal the protonation state of H149. In the "on" state, the imidazole ring of H149 is predominantly in a neutral tautomeric state, protonated at the Nδ 1 position (31). In the "off" state, as discussed in the previous paragraph, the 1 H-13 C correlation peaks of H149 (Hδ 1 -Cδ 1 and Hε 2 -Cε 2 ) are severely line broadened, and no correlation peaks are detected in aromatic HCC-type experiments. Therefore, no conclusions can be drawn on the protonation state of H149 in the "off" state. Further information on the involvement of H149 in hydrogen bonding interactions was obtained by a 1 H-15 N SOFAST-HMQC experiment [START_REF] Schanda | SOFAST-HMQC experiments for recording two-dimensional heteronuclear correlation spectra of proteins within a few seconds[END_REF] tailored to the chemical shifts of histidine side chains. The presence of a hydrogen bond protects the "labile" imidazole hydrogen(s) from solvent exchange. Therefore, a correlation peak for the protonated histidine ring nitrogens is expected if the lifetime of the hydrogen is at least a few hundred milliseconds. Figure 8b shows such correlation spectra recorded for the "on" and "off" states at different temperatures (15°C, 25°C, and 40°C). At low temperature (15°C), a total of 6 out of the 10 histidines of rsFolder give rise to a cross peak in the "on" state indicating their involvement in a hydrogen bond. While 4 of them (H26, H182, H200, and H218) remain visible also at higher temperature (40°C), no correlation peak is detected for H149 and H232 at this temperature. Taking H218 as an internal reference, the relative H-bond stability (peak intensity) decreases by a factor of 2 when increasing the temperature from 15° to 25°C. Our NMR data validate the crystal-structure-derived hypothesis of H-bond formation between the Nδ 1 of H149 and the phenol ring of the chromophore. However, we can also conclude that this H-bond is relatively unstable, as compared to other H-bonds involving histidines H26, H182, H200, and H218. In the "off" state, the same peak patterns and Tdependence is observed, except for H149 that does not give rise to a correlation peak at any temperature. This observation, however, does not allow to conclude on the absence of a Hbond between H149 and the chromophore in the "off" state, as the protonated phenol ring has to interact with an unprotonated imidazole ring nitrogen in order to form an H-bond, that is undetectable in our NMR spectra. Nevertheless, the observed line broadening of the nonexchangeable ring hydrogens of H149, resulting from conformational exchange dynamics, makes the hypothesis of a stable H-bond in the "off" state very unlikely. We also attempted to obtain some information on the NMR signature of the chromophore itself. Figure 8d shows the aromatic region of the 1 H- 13 C correlation spectra of rsFolder in the "on" state (blue spectrum). The chromophore contains 5 C-H pairs (Figure 8c)

in the tyrosine (Y) part of the chromophore that is made up from the A66-Y67-G68 tripeptide in the rsFolder sequence. The 5 corresponding correlation peaks detected in the spectrum of Figure 8d were identified and unambiguously assigned by a set of 2D correlation spectra shown in Figure S9, recorded with pulse sequences that were specifically tailored for tyrosine side chains [START_REF] Christou | BEST and SOFAST experiments for resonance assignment of histidine and tyrosine side chains in 13C/15N labeled proteins[END_REF]. These NMR data allowed us to assign all 1 H and 13 C resonances (except for CO) of the tyrosine moiety of the chromophore (Table 1). The upfield shifted CZ chemical shift (178 ppm) is characteristic of an anionic phenol group as expected for a deprotonated chromophore in the fluorescent "on" state. The observation of distinct correlation peaks for the symmetric CD and CE sites of the phenol ring indicates that the chromophore is structurally stabilized in the β-barrel with slow ring flip dynamics (k ex < 500 s -1 ) at 40°C.

These slow ring flips are in contrast to all other tyrosine and phenylalanine side chains of rsFolder for which a single correlation peak was detected for the symmetric δ1 and δ2 (ε1 and ε2) sites, indicative of fast ring flip dynamics leading to chemical shift averaging. In the nonfluorescent "off" state (red spectrum in Figure 8d), the 4 correlation peaks of the chromophore's phenol group disappear, indicative of a significant change in chromophore dynamics. Only the signal of the HB-CB in the methine bridge of the chromophore remains detectable in the dark state, with the CB resonance experiencing an impressive 7.6 ppm upfield shift after cis-trans isomerization of the chromophore. The NMR assignment of the chromophore in the "off" state could be further extended to the CA and CG (Figure S9b) that also shift upfield by 3.3 and 4.4 ppm, respectively (see table 1). Our NMR data indicate a destabilization of the chromophore in the "off" state leading to motion of the phenol ring on the millisecond time scale. However, whether these motions correspond to increased ring flip dynamics, local fluctuations around the methine bridge, or both cannot be distinguished from these data.

Discussion

In-situ NMR illumination and time-efficient NMR experiments

Our portable laser illumination device allowed us to perform a large set of 2D and 3D correlation experiments on a single rsFolder sample. Due to its portability, the setup is compatible with any high-field NMR instrument and probe, provided that the optical fiber is long enough for the sample to reach its position in the magnet. Illumination from the top (fiber placed in the Shigemi plunger) ensures minimal perturbation of the magnetic field homogeneity, and thus results in close to optimal NMR line shapes. However, relatively high laser power is required to ensure fast "on" to "off" (or "off" to "on" ) switching, in particular due to nonuniform illumination and high optical density of the sample. The LD-EXSY experiment that makes use of fast switching capabilities provides a convenient and timeefficient way to transfer NMR assignments of fast-switching PTFPs from one conformational state to another. This is of importance as NMR resonance assignment is the most timeconsuming step for investigating the dynamic properties of PTFPs. Once a sufficient level of resonance assignment has been achieved, NMR provides a unique tool to access local dynamics in the protein backbone, side chains, and chromophore on a broad range of time scales, as will be discussed further in the following paragraph(s).

Conformational dynamics in the fluorescent state of rsFolder

The β-barrel scaffold of fluorescent proteins stabilizes the anionic cis conformation of the chromophore via hydrogen bonding, π-stacking and cation-π interactions, as well as steric hindrance. This restricts the chromophore's internal motion observed in free solution, and prevents radiationless decay upon light excitation, a prerequisite for a high fluorescence quantum yield. However, this conformational rigidity is somehow in contradiction with the capability of RSFPs to switch efficiently between distinct conformations, implying local structural rearrangements, as well as changes in the hydrogen bonding network upon light excitation. Therefore, a detailed experimental characterization of the structural heterogeneity and conformational flexibility of the chromophore and its protein environment is important for understanding and improving the photochromic properties of RSFPs. Such dynamic information is typically not available from crystallographic structures. In the case of rsFolder, the available X-ray structures show a well-defined β-barrel fold, and a single chromophore conformation in both the "on" and "off"-states. Here, we have applied liquid-state NMR spectroscopy to complement this static picture. Information on the local dynamics over time scales ranging from picoseconds to about a few hundred milliseconds have been obtained from a combination of 15 N spin relaxation measurements, NMR line width analysis, and H/D exchange data. Our NMR data point towards a well-known defect in the β-barrel structure of GFP-derived fluorescent proteins around strand β7 [START_REF] Seifert | Backbone Dynamics of Green Fluorescent Protein and the Effect of Histidine 148[END_REF][START_REF] Oltrogge | Ground-State Proton Transfer Kinetics in Green Fluorescent Protein[END_REF][START_REF] Lelimousin | Intrinsic Dynamics in ECFP and Cerulean Control Fluorescence Quantum Yield[END_REF][START_REF] Wachter | Structural basis of spectral shifts in the yellow-emission variants of green fluorescent protein[END_REF] where the H-bonds formed with the neighboring strands (especially β8), typical of an anti-parallel β-sheet conformation, are weakened by longer distances and other structural irregularities. This leads to backbone fluctuations and transient opening of the β-barrel in the fluorescent "on" state of rsFolder as evidenced by a few unobservable amide resonances (V151, K167, and I168), and fast H/D exchange rates measured for amide hydrogens in strand β7. Similar observations have been made previously for conventional GFPs [START_REF] Seifert | Backbone Dynamics of Green Fluorescent Protein and the Effect of Histidine 148[END_REF][START_REF] Oltrogge | Ground-State Proton Transfer Kinetics in Green Fluorescent Protein[END_REF]. The dynamic "holes" in the β-barrel between β7 and the neighboring strands have also been identified by MD simulations as major entry points of water molecules into the interior of the β-barrel [START_REF] Shinobu | The Hole in the Barrel : Water Exchange at the GFP Chromophore[END_REF]. Although certainly not a sufficient condition, such conformational plasticity is likely required for efficient photoswitching.

The data show clear evidence of an anionic chromophore, with the phenolate oxygen hydrogen-bonded to the Nδ1 of H149. This finding is in agreement with the crystal structure (26) that indicates that the chromophore's phenolate oxygen is involved in 3 hydrogen-bond interactions with H149, T204, and a water molecule. However, our NMR data show that the H-bond between the chromophore and H149 is thermally unstable. At physiological temperature, the bond is constantly broken and reformed on a sub-millisecond time scale. It has been shown by QM/MM simulations of the RSFP Dronpa (42) that two or three H-bonds stabilizing the chromophore are required for high fluorescence quantum yields, while less hydrogen bonding favors photo-switching of the chromophore by a hula-twist mechanism.

We speculate that the weak H-bond observed with H149 in rsFolder is a characteristic property of RSFPs that have been engineered to obtain reasonable fluorescence quantum yields combined with efficient switching capability.

Light-induced conformational dynamics in the chromophore and its environment

As for a majority of RSFPs, on-off photoswitching in rsFolder involves a cis-trans isomerization of the chromophore coupled to deprotonation at the hydroxy group of the phenol moiety. In our NMR spectra, this photoinduced isomerization results in large upfield 13 C shifts of carbons in the methine bridge (CA, CB, and CG) of the chromophore. As 13 C chemical shifts are particularly sensitive to the local geometry (torsion angles) of the chemical bonds [START_REF] Dios | Secondary and Tertiary Structural Effects on Protein NMR Chemical Shifts : An ab Initio Approach[END_REF], the methine bridge 13 C chemical shifts can be considered a spectral signature of p-HBI being in cis or trans configuration. In principle, the protonation state at the chromophore's phenol moiety can be inferred from the 13 C Z chemical shift, which is expected to be 175-180 ppm for a phenolate, and 155-160 ppm for a phenol. However, for rsFolder in the "off" state no NMR signals could be detected for the phenol moiety in the aromatic 1 H- 13 C spectra, indicative of a chromophore undergoing conformational exchange dynamics on the s to ms time scale. Similarly, NMR signals of nuclei in the protein side chains H149 and T204, forming hydrogen bonds with the phenolate oxygen in the "on" state", become strongly line broadened in the "off" state. These NMR observations clearly indicate a destabilization of the chromophore, and the absence of any stable hydrogen bonding interaction with the βbarrel. In the crystal structures of rsFolder, the observed distances between the histidine δ1 nitrogen and the phenol oxygen are only little altered (from 2.8 Å to 2.9 Å) upon photoisomerization of the chromophore (Figure 7). Our findings thus contradict the conclusion drawn from these structures that H149 remains hydrogen bonded to the hydroxyl group of the phenol in the "off" state. Most likely, the crystal structure observed at cryogenic temperatures represents the lowest energy conformation in the "off" state, which at higher temperature is in exchange with alternate conformations that are separated by low energy barriers. A loss of hydrogen bonding of the chromophore phenolate with the β-barrel scaffold has also been observed for other RSFPs, e. g. Dronpa, rsEGFP2, and rsGreen [START_REF] Coquelle | Chromophore twisting in the excited state of a photoswitchable fl uorescent protein captured by time-resolved serial femtosecond crystallography[END_REF][START_REF] Morozov | Photochromic Proteins Hydrogen Bond Fluctuations Control Photochromism in a Reversibly Photo-Switchable Fluorescent Protein[END_REF][START_REF] Duwe | Expression-Enhanced Fluorescent Proteins Based on Enhanced Green Fluorescent Protein for Superresolution Microscopy[END_REF][START_REF] Andresen | Structural basis for reversible photoswitching in Dronpa[END_REF]. Of note, this has also been observed recently in the case of a long-lived dark state in the photoconvertible fluorescent protein mEos4b [START_REF] De Zitter | Mechanistsic investigation of mEos4b reveals a strategy to reduce track interruption in sptPALM[END_REF].

The chromophore cis-trans isomerization also induces increased structural dynamics at the β-barrel side (β7 -β10) facing the chromophore's phenol group, the two helical regions (H2 and H3) connecting the chromophore with the rest of the protein, and a number of side chains attached to these peptide regions, and pointing towards the chromophore. In particular, all side chains that undergo a structural rearrangement in the "off" state (T63, F146, H149, V151, and I168), as seen by X-ray crystallography (Figure 1), are heavily line-broadened in our NMR data (Figure 6) except for F146 that shows large chemical shift changes. These observations further consolidate the hypothesis that photo-isomerization in rsFolder leads to a polymorphic and highly dynamic chromophore environment with multiple sub-states (conformations) of comparable free energy that interconvert on the micro-to-millisecond time scale. Most likely, the structural fluctuations of the chromophore and surrounding side chains also effects the backbone regions to which they are attached, thus explaining the observed amide 1 H line broadening and increased solvent exchange rates. These data are fully in line with the notion of a fully nonfluorescent dark state unable to undergo radiative de-excitation upon photon absorption. Similar findings on changes in backbone dynamics upon photoswitching have previously been reported for the anthozoan-derived RSFP Dronpa [START_REF] Mizuno | Light-dependent regulation of structural flexibility in a photochromic fluorescent protein[END_REF].

So far, the chromophore connecting helical elements, H2 and H3, have found only little attention in the field of GFP engineering. Concerning the helical region H3, we know that L70 is an important residue for the photo-switching properties of rsEGFP2 [START_REF] Grotjohann | rsEGFP2 enables fast RESOLFT nanoscopy of living cells[END_REF] and rsFolder [START_REF] El Khatib | Rational design of ultrastable and reversibly photoswitchable fluorescent proteins for super-resolution imaging of the bacterial periplasm[END_REF], while S72 (S73 in rsFolder) has been suggested as one of the 'hopping' points of protons when entering from the solvent onto their way to the chromophore [START_REF] Shinobu | The Hole in the Barrel : Water Exchange at the GFP Chromophore[END_REF]. In our NMR data, the H3 region of rsFolder shows increased conformational dynamics in the "off" state, as evidenced by line broadening (C71, F72, S73) and faster H/D exchange (L70, C71). It has also been reported recently using time-resolved serial crystallography at an XFEL that during photo-switching in rsEGFP2 an intermediate state becomes populated, in which the chromophore is twisted, and helix H2 is moved downward along the β-barrel axis [START_REF] Coquelle | Chromophore twisting in the excited state of a photoswitchable fl uorescent protein captured by time-resolved serial femtosecond crystallography[END_REF]. Here, we have shown that water exchange for several amide hydrogens (V62, T63, and T64) in H2 is increased by up to 2 orders of magnitude. We may therefore speculate that conformations similar to those seen in the excited state may be also transiently occupied under equilibrium conditions at ambient temperature, possibly involved in the thermally induced relaxation mechanism of rsFolder from the "off" state to the "on" state.

Conclusions

In this work, we have used multidimensional liquid-state NMR spectroscopy coupled to in-situ sample illumination as a complementary high-resolution tool to study light-induced changes in the conformational dynamics of the photo-transformable fluorescent protein rsFolder. Our results add a dynamic dimension to the static view provided by X-ray crystallographic protein structures. In particular, we identified dynamic hotspots in the chromophore environment and the β-barrel structure that may suggest future mutation sites to be probed by e.g. saturation mutagenesis in order to alter the photophysical properties of RSFPs. Furthermore, NMR spectroscopy provides a unique tool to investigate the influence of environmental conditions on the conformational dynamics of PTFPs, and correlate them with their photophysical and photochemical properties. Therefore, including NMR spectroscopy into the toolbox used for rational fluorescent protein engineering will provide new

Figure 1 .

 1 Figure 1. Crystallographic structures of rsFolder in the fluorescent "on" state (green) and the non-fluorescent "off" state (grey). (a) Superposition of the main chain coordinates highlighting that the overall β-barrel structure of the protein is identical in the two states. (b) Zoom on the chromophore region of rsFolder showing the change in chromophore geometry from "cis" to "trans", as well as some side chain rearrangements in the vicinity of the chromophore. The most pronounced structural changes upon light illumination are observed for F146, H149 and V151.

Figure 2 .

 2 Figure 2. In-situ NMR sample illumination device. (a) Schematic representation of the insertion of the optical fiber into the water-filled plunger of a Shigemi NMR tube. The NMR sample is illuminated from the top in a conic geometry. (b) Photograph of the high-field NMR platform showing the portable setup of the in-situ illumination device; a laptop is connected to the laser box which is mounted on the mezzanine of the magnet. (c) Sample of rsFolder: picture with and without light illumination. (d-e) 2D plots of 1D 1 H time series. Uponcontinuous laser illumination at 488 nm and power levels of (d) 70 mW and (e) 140 mW, the NMR signal of a methyl 1 H in the "on" state decreases while the signal of the corresponding "off" state peak increases.

Figure 3 .

 3 Figure 3. Laser-driven magnetization exchange (LD-EXSY) experiment. (a) Schematic drawing of LD-EXSY pulse sequence including light illumination at 2 different wavelengths; at 405 nm during the recycle delay (magnetization recovery, 2s), and at 488 nm during the EXSY mixing time (magnetization storage, 600 ms). The laser sources are coupled to the NMR console via TTL lines, and switched on and off by specific commands in the NMR pulse sequence code. The resulting 3D spectra have 2 "on" state and 1 "off" state chemical shift dimensions. (b) 2D projection of a 3D 15 N-edited LD-EXSY spectrum. Off-diagonal peaks correspond to rsFolder residues (annotated) which show 15 N chemical shift differences between "on" and "off" states. (c) Same as (b) but for 1 H-edited LD-EXSY.

Figure 4 .

 4 Figure 4. Backbone dynamics of rsFolder in "on" and "off" states. (a) 15 N relaxation data (T 1 , T 2 and HETNOE) measured at 700 MHz and 40°C, plotted as a function of protein sequence. Secondary structural elements as identified from the 13 C chemical shifts by the program TALOS-N are plotted on top of each graph. Cyan bars indicate peptide region for which no peak is detected in the "off" state. (b) 1 H-15 N BEST-TROSY spectralregions (red: "on" state, blue: "off" state) zooming on line-broadened "off" state peaks. (c) CPMG-RD data recorded for residue S73 at 850 MHz in the "on" (green) and "off" (black) states.

Figure 5

 5 Figure 5 summarizes the main changes in NMR observables after light illumination. The following NMR data have been color-coded on the structure of rsFolder in an open-sheet and a 3-dimensional representation: (i) unobserved amide resonances in both the "on" and "off" states (dark blue); (ii) amide resonances undetected in the "off" state only (blue); (iii) amide resonances with significantly reduced peak intensity in the "off" state as extracted from the graph of figure S5b, and/or decreased 15 N T 2 values (light blue); (iv) significant 1 H-15 N chemical shift changes (Δ CS > 2 ppm) as extracted from the graph of figure S5a (orange).

Figure 5 .

 5 Figure 5. Increased conformational dynamics on the μs-ms timescale in the "off" state of rsFolder. The NMRderived information is color-coded on the (a) open-sheet and (b) 3-dimensional representation of the rsFolder structure as revealed by X-ray crystallopgraphy and NMR (A '*' indicates structural elements only identified by NMR): (i) undetected/unassigned amides in both states (dark blue), (ii) undetected/unassigned amide peaks in the "off" state only (middle blue), (iii) strongly line-broadened amide NMR signals in the "off" state (light blue),

Figure S7 .

 S7 Figure S7. Therefore we consider that changes in the observed H/D exchange rates are mainly due to a population increase (or decrease) of exchange competent, unprotected conformational sub-states.

  N relaxation and line broadening data already identified increased dynamics in helix H3, they did not point towards changes in helix H2 dynamics. Our findings highlight the complementarity of NMR relaxation and H/D exchange data in providing information on local protein dynamics, the latter being sensitive to much longer time scales and smaller chemical shift changes.

Figure 6 .

 6 Figure 6. H/D exchange NMR data. (a) Representative intensity decay for individual amide hydrogens extracted from real-time 2D NMR data recorded for the "on" (green) and "off" (black) states of rsFolder at 40°C and 850 MHz 1 H frequency. (b) The derived H/D exchange information is color-coded on the crystal structure of rsFolder: (i) residues with amide hydrogen exchange time constants Tex < 5 min in both the "on" and off" states (light blue); (ii) residues for which the hydrogen exchange rate is increased by a factor of 4 -20 (orange); (iii)

Figure 7 .

 7 Figure 7. rsFolder side chains involved in conformational exchange processes in the "off" state as deduced from extensive line broadening. Examples of peak line broadening observed in the 1 H-13 C correlation spectra of rsFolder: on-state (blue), "off"-state (red). (b) Zoom on the chromophore environment highlighting the side chains which show NMR line broadening in the "off" state (red). In addition, F146 is plotted as it experiences large chemical shift changes upon light illumination.

Figure 8 .

 8 Figure 8. NMR information of the chromophore. (a) Chromophore and His149 side chain conformations extracted from the available crystal structures of rsFolder in the "on" (cis configuration) and "off" (trans configuration) states. (b) Temperature series of 1 H-15 N SOFAST-HMQC spectra of histidine side chains in the "on" and the "off" states recorded at 850 MHz 1 H frequency. The detected cross peaks are annotated by the residue number. (c) Schematic representation of the chemical structure of the chromophore. Nuclei giving rise to NMR signals are highlighted by blue letters, and their NMR frequencies are provided in Table1. (d) Overlay of 2D aromatic 1 H-13 C Best-HSQC spectrum (31) of rsFolder, recorded without CT13 C editing to achieve highest experimental sensitivity, in the "on" (blue) and "off" (red) state. NMR signals from the tyrosine moiety (Y63) of the chromophore are annotated.

Table 1 .

 1 Chromophore 1 H and13 C chemical shifts

	Tables
	Y67	ON STATE OFF STATE
	NMR SHIFTS
	(PPM)
	CA	131.1	134.4
	CB (HB)	137.9 (6.79)	145.5 (7.05)
	CG	121.2	125.6
	CD1 (HD1)	138.2 (8.34)	n.d.*
	CD2 (HD2)	141.3 (6.54)	n.d.
	CE1 (HE1)	125.5 (6.48)	n.d.
	CE2 (HE2)	124.6 (5.84)	n.d.
	CZ	179.2	n.d.
	* not detected

opportunities for further improving advanced fluorescent markers for a wide range of microscopy and biotechnological applications.

Acknowledgements

We thank Dr Paul Schanda for many stimulating discussions. Financial support from the CNRS (Défis Instrumentation 2018) is acknowledged. This work used the NMR and isotope labeling platforms of the Grenoble Instruct-ERIC center (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) within the Grenoble Partnership for Structural Biology (PSB). Platform access was supported by FRISBI (ANR-10-INBS-05-02) and GRAL, a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA).

Author Contributions

DB and BB conceived and directed the research; IA and KG expressed the protein and prepared the samples; NC and BB performed the NMR experiments and analyzed the data;

VA and MB contributed plasmids, and helped with the experimental setup; NC, DB and BB wrote the paper. All authors reviewed the results and approved the final version of the manuscript.


domain: sdv sde

text:
Sylvain Hugel
email: hugels@inci-cnrs.unistra.fr

First record of shield-backed katydids in Madagascar with the description of a new genus and three new species

Keywords: Arytropteridini) Orthoptera, endemism, South Western Indian Ocean islands, Madagascar, bush cricket

Shield-backed katydids of tribe Arytropteridini Caudell, 1908 are recorded for the first time in Madagascar. The new genus Toliaridectes n. gen. is proposed to include three new species from the south west of the island: Toliaridectes meridionalis n. gen. n. sp., Toliaridectes wendenbaumi n. gen. n. sp. and Toliaridectes antsycurvis n. gen. n. sp.. Elements of biology of Toliaridectes n. gen. are given

Introduction

Whereas Malagasy grasshoppers are quite well known, particularly the day-active acridoids, Malagasy crickets and bush crickets have been relatively neglected until recently. Indeed, over the last decade, several groups have reviewed and/or described new bush cricket species from Madagascar (e.g. [START_REF] Heller | Paraplangia sinespeculo, a new genus and species of bush-cricket, with notes on its biology and a key to the genera of Phaneropterinae (Orthoptera: Tettigonioidea) from Madagascar[END_REF], 2019[START_REF] Gorochov | New taxa of the subfamily meconematinae (Orthoptera: Tettigoniidae) from Africa and adjacent islands[END_REF][START_REF] Hugel | New and little known Phisidini from Madagascar, Comoro and Seychelles (Ensifera: Meconematinae)[END_REF][START_REF] Ünal | Revision of the Madagascan genera Oncodopus Brongniart and Colossopus Saussure (Orthoptera: Tettigoniidae: Conocephalinae; Euconchophorini), with description of Malagasopus gen[END_REF].

Interestingly, as for many other groups in Madagascar, Orthoptera display a relatively low overlap in species composition across sampling sites [START_REF] Krištín | Assemblages of orthopteroid insects along environmental gradients in central and southern Madagascar[END_REF] and during our numerous surveys, we recorded 3 times more morphospecies of Ensifera than the total amount of species considered occurring in the island, suggesting that most of Madagascar's Orthopteran diversity remains to be described.

In the present article, I record for the first time shield-backed katydids of tribe Arytropteridini [START_REF] Caudell | Orthoptera Fam. Locustidae Subfam. Decticinae[END_REF] from Madagascar. Until now, all Arytropteridini were only known from Southern Africa [START_REF] Rentz | The shield-backed katylids of Southern Africa: their taxonomy, ecology and relationships to the faunas of Australia and South America (Orthoptera: Tettigoniidae: Tettigoniinae)[END_REF][START_REF] Naskrecki | A new genus and species of South African Tettigoniinae (Orthoptera: Tettigoniidae)[END_REF]. We propose the new genus Toliaridectes n. gen. to include Toliaridectes meridionalis n. gen. n. sp., Toliaridectes wendenbaumi n. gen. n. sp. and Toliaridectes antsycurvis n. gen. n. sp..

Material and methods

The new taxa described in the present paper are based on specimens recently collected in Madagascar by the author and Nicolas Cliquennois (Antsirabe). All samples were collected by sight after dark, using a headlamp. The number following the name of the collector corresponds to field pad number of the specimen, and is printed in the first label pinned on the specimen. Additional specimens were found in non-identified material of MNHN.

Measurements. The measurements have been performed on dry specimens following Rentz (1988).

Acoustics. Sounds were acquired with an Audiotechnica AT822 stereo microphone, on a NIKON D5200 camera (sampling rate: 96 kHz) with a red illumination (to follow the insect behavior). Song analysis has been performed with Clampfit 10.2 software. Song recordings are deposited in both coll. S.H. and MNHN acoustic databases: https://sonotheque.mnhn.fr/.

Acoustic terminology is from [START_REF] Stumpner | True katydids (Pseudophyllinae) from Guadeloupe: Acoustic signals and functional considerations of song production[END_REF]. Arytropteridini Caudell, 1908genera: Arytropteris Herman, 1874 [Southern-Africa] Thoracistus Pictet, 1888 [Southern-Africa] Anarytropteris Uvarov, 1924 [Southern-Africa] Ceresia Uvarov, 1928 [Southern-Africa] Alfredectes Rentz, 1988 [Southern-Africa] Zuludectes Rentz, 1988 [Southern-Africa] Transkeidectes Naskrecki, 1992 [Southern-Africa] Toliaridectes n. gen. Head. Head in frontal view 1.4-1.8 times higher than wide, narrower than pronotum in dorsal view (Fig. 123). Fastigium verticis narrower than scapus (Fig. 1-3), triangular in frontal view (Fig. 789), no fastigial gap between fastigium verticis and fastigium frontis, fastigium frontis contacting the fastigium verticis in a narrow ridge (Fig. 456789). Median ocellus very distinct, in line with ventral part of antennal scrobus (Fig. 789). Genae with no carinae. Scapus unarmed.

Results

List of

Thorax. Pronotum little modified, slightly different in males and females, in both cases not exceeding first abdominal segment, not producing posteriorly, without median carina, without lateral carinae, rims of lateral lobe and disk not enlarged; disk flat in side view, arched in cross section; anterior margin inconspicuously concave; posterior margin broadly rounded; surface of disk smooth, faintly shining; disk prozona traversed by a transverse sulcus shaped as a curly bracket (Fig. 123). Prosternum armed with a pair of broadly spaced blunt erected spines; meso and metasternum with a pair of more robust processes.

Wings. Forewings present in males, absent in females.

Legs. Leg surface covered with numerous minute imprints, often pigmented and bearing a small seta. Fore coxa with one median antero lateral spine, relatively flattened (Fig. 123). Fore femora with 1-4 minute spinules in anterior and posterior ventral margins (rarely none on posterior margin of T. antsycurvis n. gen. n. sp.); fore femora genicular lobes with 2 very small spinules on both sides. Fore tibia (Fig. 10-15): with 6 ventral inner spurs (including apical spur); with 6 ventral outer spurs (including apical spur); without dorsal apical and subapical spurs.

Auditory foramen narrow, slit shaped, visible in side view (Fig. 101112131415). Mid coxa with a short ventral blunt process. Mid femora with 2-5 minute spinules in anterior and posterior ventral margins (rarely absent on posterior margin of T. antsycurvis n. gen. n. sp.); mid femora genicular lobes with 2 very small spinules on both sides. Mid tibia (Fig. 16-21): with 3 dorsal posterior subapical spurs; without dorsal apical spurs; with 6 ventral inner spurs (including apical spur); with 6 ventral outer spurs (including apical spur). Hind coxa with a short ventral blunt process. Hind femora with 6-10 spines on ventral inner and ventral outer carinae (Fig.

22-24)

; hind femora genicular lobes with a minute spine on each side.

Abdomen. Tergites with inconspicuous longitudinal carina (Fig. 123).

Male. Pronotum (Fig. 123456) reaching the middle of the first abdominal segment; disk more arched in cross section than in females, leaving room for the wings. [25][26][27][28][29][30][31][32][33] slightly exceeding hind margin of pronotum, reaching the distal margin of the first abdominal segment; visible part of the forewing white or light green; file with 38-42 teeth, largest on the middle. X th abdominal tergite posterior end with a median quadrangular notch of variable width separating two pointing lobes (Fig. 36,43,50). Paraprocts very small, with a ventral-median projection variable in size (Fig. 35,42,49). Subgenital plate (Fig. 34, 41, 48): broadly triangular in ventral view, with a shallow median notch between styli; with distinct styli. [44][45][46][47][51][52][53][54] Derivation of name: Named after the Malagasy province "Toliara" where all known species of Toliaradectes n. gen. occur (Fig. 72).

Toliaridectes meridionalis n. gen. n. sp. Fig. 1,4,[START_REF] Krištín | Assemblages of orthopteroid insects along environmental gradients in central and southern Madagascar[END_REF]10,13,16,19,22,[25][26][27][34][35][36][37][38][39][40]55,[58][59]72,Tab

Diagnosis.

Color: hind femur with distinct shiny black longitudinal pattern on external (outer) field (Fig. 22, similar pattern in T. wendenbaumi n. gen. n. sp., pattern weaker in T. antsycurvis n. gen. n. sp.). Male. X th abdominal tergite emargination as deep as wide (Fig. 36; similar in T.

wendenbaumi n. gen. n. sp., wider in T. antsycurvis n. gen. n. sp.); paraprocts with a long digitiform projection (Fig. 35; wide and short projection in T. wendenbaumi n. gen. n. sp., shorter digitiform projection in T. antsycurvis n. gen. n. sp.). Cercus median projection long (Fig. 37; as long as in T. antsycurvis n. gen. n. sp., shorter in T. wendenbaumi n. gen. n. sp.). Subgenital plate wider than long (Fig. 34; similar in T. antsycurvis n. gen. n. sp., longer than wide in T. wendenbaumi n. gen. n. sp.). Titillators distal end pointing (Fig. 55; similar in T.

wendenbaumi n. gen. n. sp., enlarged distally in T. antsycurvis n. gen. n. sp.). Female.

Subgenital plate wider than long, with U shaped emargination (Fig. 58; wider than long in T.

antsycurvis n. gen. n. sp., about as long as wide in T. wendenbaumi n. gen. n. sp.). Ovipositor very long for the genus (Fig. 59, Table 1).

Description. Size average for the genus (Table 1). Color (male holotype and females only; the male paratype is discolored): head genae without distinct black pattern; hind femur with distinct shiny black longitudinal pattern on external (outer) field (Fig. 22). Male. X th abdominal tergite emargination as deep as wide (Fig. 36); paraprocts with a long digitiform projection (Fig. 35). Cercus dorsal lobe with angulose tip; median projection long, bilobate (Fig. 37383940).

Subgenital plate wider than long; with shallow but distinct emargination; styli separated by less than styli length (Fig. 34). Titillators distal end pointing; internal part shaped as inverted narrow Y (Fig. 55). File with ca. 38 teeth (Fig. 27). Female. Subgenital plate wider than long, with U shaped emargination (Fig. 58). Ovipositor very long for the genus, ventral margin with a long strait area (Fig. 59, Table 1).

Deriviatio nominis.

After the distribution of the species, in the South of Madagascar.

Toliaridectes wendenbaumi n. gen. n. sp. Fig. 2,5,8,11,14,17,20,23,[28][29][30][41][42][43][44][45][46][47]56,[60][61][68][69]72,Tab. 2. Toliaridectes wendenbaumi n. gen. n. sp., here described. Diagnosis. Color: hind femur with distinct shiny black longitudinal pattern on external (outer) field (Fig. 23, similar pattern in T. meridionalis n. gen. n. sp., pattern weaker in T. antsycurvis n. gen. n. sp.). Male. X th abdominal tergite emargination as deep as wide (Fig. 43; similar in T. meridionalis n. gen. n. sp., wider in T. antsycurvis n. gen. n. sp.); paraprocts with a short wide projection (Fig. 42; long digitiform projection in T. meridionalis n. gen. n. sp., shorter digitiform projection in T. antsycurvis n. gen. n. sp.). Cercus median projection relatively short (Fig. 44; longer in T. antsycurvis n. gen. n. sp. and T. meridionalis n. gen. n. sp.). Subgenital plate longer than wide (Fig. 41; wider than long in T. meridionalis n. gen. n. sp. and T. antsycurvis n. gen. n. sp.). Titillators distal end pointing (Fig. 56; similar in T. meridionalis n. gen. n. sp., enlarged distally in T. antsycurvis n. gen. n. sp.). Female. Subgenital plate about as wide as long, with Vshaped emargination (Fig. 60; wider than long in T. meridionalis n. gen. n. sp. and T. antsycurvis n. gen. n. sp.). Ovipositor average for the genus (Fig. 61, Table 2).

Description. Size average for the genus (Table 2). Color: head with genae without distinct black pattern; pronotum folds with usually a light band edging the black band; hind femur with distinct shiny black longitudinal pattern on external (outer) field (Fig. 23). Male. X th abdominal tergite emargination as deep as wide (Fig. 43); paraprocts with a shirt wide projection (Fig. 42). Cercus dorsal lobe broadly rounded; median projection relatively short, bilobate (Fig. 44-47). Subgenital plate longer than wide; with indistinct emargination; styli separated by about styli length (Fig. 41). Titillators distal end pointing; internal part shaped as inverted wide Y (Fig. 56). File with ca. 42 teeth (Fig. 30). Female. Subgenital plate longer than wide, with V shaped emargination (Fig. 60). Ovipositor average for the genus, ventral margin with a relatively long strait area (Fig. 61,Table 2).

Derivation of name. Named after Evrard Wendenbaum, founder of Naturevolution NGO who organized multiple field surveys in Makay.

Toliaridectes antsycurvis n. gen. n. sp. Fig. 3,6,9,12,15,18,21,24,[31][32][33][48][49][50][51][52][53][54]57,[62][63][64][65][66][67][70][71][72]Tab. 3.

Toliaridectes antsycurvis n. gen. n. sp., here described. Holotype male. Madagascar, province Toliara, région Atsimo-Andrefana, Tulear II, Manombo-Sud, Andrevo-Bas, 23.1524°S, 43.6270°E, 15-16.ii.2019, S. Hugel leg. (2019MADASH 043),

MNHN (MNHN-EO-ENSIF10802).

Paratypes

Diagnosis.

Color: hind femur with weak dark longitudinal pattern on external (outer) field (Fig. 24, shiny black pattern in T. meridionalis n. gen. n. sp. and T. wendenbaumi n. gen. n. sp.).

Male. X th abdominal tergite emargination wider than deep (Fig. 50; narrower in T.

wendenbaumi n. gen. n. sp. and T. meridionalis n. gen. n. sp.); paraprocts with a short narrow projection (Fig. 49; long digitiform projection in T. meridionalis n. gen. n. sp., short and wide in T. wendenbaumi n. gen. n. sp.). Cercus median projection long (Fig. 51; shorter in T.

wendenbaumi n. gen. n. sp.). Subgenital plate wider than long (Fig. 48; wider than long in T. meridionalis n. gen. n. sp., longer than wide in T. wendenbaumi n. gen. n. sp.). Titillators enlarged distally (Fig. 57; pointing in T. meridionalis n. gen. n. sp. and T. wendenbaumi n. gen. n. sp.). Female. Subgenital plate wider than long, with shallow rounded emargination (Fig. 62; longer than wide in T. wendenbaumi n. gen. n. sp.). Ovipositor short for the genus (Fig. 63, Table 3).

Description. Size rather small for the genus (Table 3). Color: head genae with distinct black pattern and dots; pronotum folds with usually without a light band edging the black band; hind femur with weak dark longitudinal pattern on external (outer) field (Fig. 24). Male. X th abdominal tergite emargination wider than deep (Fig. 50); paraprocts with a short narrow projection (Fig. 49). Cercus dorsal lobe angulose; median projection long, bilobate (Fig. 51525354).

Subgenital plate wider than long; with distinct emargination; styli separated by more than styli length (Fig. 48). Titillators enlarged distally; internal part shaped as inverted wide T (Fig. 57).

File with ca. 39 teeth (Fig. 33). Female. Subgenital plate with a shallow rounded emargination (Fig. 62). Ovipositor short for the genus, straight area of ventral margin short (Fig. 63, Table 3). 70). Males of Toliaridectes antsycurvis n. gen. n. sp. sing at night; we have not recorded calls by day hours. Males adopt a peculiar position while singing, the abdomen bent downwards allowing to release the forewings from the pronotum (Fig. 70).

Acoustic behavior

I recorded one male by night at 0h30 a.m. and 30°C. This call was mostly in the ultrasound range (peaking above 20 kHz). It was made of very long repetitions (minutes) of short phonatomes repeated every 46.5 ± 0.3 ms (corresponding to 21.7 Hz). Phonatomes were made of 5-7 pulses of increasing amplitude (Fig. 67; small pulses may have been missed in the noise). Phonatome duration is 29.0 ± 0.4 ms. Derivation of name. Antsy (Malagasy): knife; curvis (latin): curved. After the shape of ovipositor.

Discussion

Distribution and Endemism. Although endemism in Madagascar is very high, this island harbors many cosmotropical ensifera species such as Natula longipennis (Serville, 1838), Gryllodes sigillatus (Walker; 1869), and many others. The endemic/non endemic status of the new species described in the present article may therefore be questioned, particularly for T.

antsycurvis n. gen. n. sp. and T. meridionalis n. gen. n. sp. living near the coast and near villages. Nevertheless, all three species are close from each other and distinct enough from all other Arytropteridini to group them in a distinct new genus. This strongly suggests that this genus as well as the three species included are endemic to Madagascar.

Interestingly, other groups of Orthoptera originally described as having a localized distribution in Southern Africa have been later recorded in South Western Indian Ocean islands, such as the genus Taciturna Otte, 1987 [START_REF] Hugel | Burrowing crickets endemic to summits in Mauritius (Orthoptera, Gryllidae): occupation of similar niches by species possibly derived from Australasian and African colonists[END_REF].

Biology. As the vast majority of Malagasy endemic Ensifera, Toliaridectes n. gen. is mostly nocturnal and virtually impossible to observe by day hours. All three species occur in sandy areas harboring spiny thickets. Some specimens were observed eating other insects such as crickets or caterpillars (Fig. 69). One female of T. antsycurvis n. gen. n. sp. was observed while ovipositing in sand (Fig. 71). All specimens were close to the ground (less than 50 cm), usually at the heart of dense bushes (Fig. 70), making them difficult to catch. All specimens were present in relatively well preserved spots; none was observed within cultures.

Taxonomic position of Toliaridectes n. gen. In this new genus, fore tibia are dorsally nonarmed whereas fore tibia of all previously described Arytropteridini display 2 (rarely 1) dorsal outer subapical spurs. All other characters of this new genus correspond to those of Arytropteridini listed by [START_REF] Storozhenko | Revision of the Tribe Onconotini with a Description of a New Tribe (Orthoptera, Tettigoniidae, Tettigoniinae)[END_REF]. I therefore propose to place Toliaridectes n. gen. within Arytropteridini.

Taxonomic position of Arytropteridini. Taxonomic history of shield-backed katydids has been detailed by [START_REF] Rentz | The shield-backed katylids of Southern Africa: their taxonomy, ecology and relationships to the faunas of Australia and South America (Orthoptera: Tettigoniidae: Tettigoniinae)[END_REF] even before [START_REF] Storozhenko | Revision of the Tribe Onconotini with a Description of a New Tribe (Orthoptera, Tettigoniidae, Tettigoniinae)[END_REF] formally exhumed Arytropteridini from Caudell's "Arytropterees" (1908). Arytropteridini are currently under Tettigoniinae [START_REF] Cigliano | Orthoptera Species File[END_REF]. Interestingly, following Beier's key (1962), in Tettigoniinae as well as in Mecopodinae and Phyllophorinae fore tibia are dorsally armed with at least one dorsal outer spur. Arytropteridini are devoid of dorsal apical spurs on fore tibia and, therefore, do not comply with Beier's definition of Tettigoniinae. The opinion that Arytropteridini do not belong to Tettigoniinae is further reinforced by molecular data suggesting that Arytropteridini reveal the Tettigoniinae paraphyletic [START_REF] Mugleston | A century of paraphyly: a molecular phylogeny of katydids (Orthoptera: Tettigoniidae) supports multiple origins of leaf-like wings[END_REF]. Following strictly Beier's key would place Arytropteridini in Listroscelidinae, but this subfamily is most likely paraphyletic in its current definition [START_REF] Mugleston | A century of paraphyly: a molecular phylogeny of katydids (Orthoptera: Tettigoniidae) supports multiple origins of leaf-like wings[END_REF]. Should data from [START_REF] Mugleston | A century of paraphyly: a molecular phylogeny of katydids (Orthoptera: Tettigoniidae) supports multiple origins of leaf-like wings[END_REF] be confirmed, a taxon including [START_REF] Rentz | Tettigoniidae of Australia. Volume 3. The Listroscelidinae, Tympanophorinae, Meconematinae and Microtettigoniinae[END_REF]) Listroscelidinae Redtenbacher, 1891(i.e. Terpandrini Gorochov, 1990;Hexacentrini Karny, 1925;Requenini Rentz, 2001;Conocephalomimini Rentz, 2001;Phisidini Jin, 1987)

  Specimen repository. CAS, California Academy of Sciences (CA, USA); MNHN, Muséum national d'Histoire naturelle (France); coll. SH, Collection Sylvain Hugel, Strasbourg, France.

  [Madagascar] Toliaridectes meridionalis n. gen. n. sp. [Madagascar, Anôsy and Androy regions] Toliaridectes wendenbaumi n. gen. n. sp. [Madagascar, Atsimo-Andrefana region] Toliaridectes antsycurvis n. gen. n. sp. [Madagascar, Atsimo-Andrefana region] Genus Toliaridectes n. gen. Type species. Toliaridectes antsycurvis n. gen., n. sp, here designated. Distribution. South-Eastern Madagascar. Diagnosis. Toliaridectes n. gen. is characterized by: Fastigium of vertex narrower than antennal scape (Fig. 1-3; as broad or broader in Zuludectes, Anarytropteris, Namaquadectes, Thoracistus, Alfredectes), no fastigial gap (Fig. 4-6; with a gap in Arytropteris). Pronotum little modified (Fig. 1-6; elongate in males of Thoracistus, Alfredectes, some Arytropteris), not exceeding first abdominal segment. Ventral surface of fore and mid femora armed with minute teeth (Fig. 1; unarmed in Zuludectes). Fore tibia unarmed dorsally (Fig. 10-15; armed in all other Arytropteridini). Mid tibia armed with 3 dorsal posterior spurs, not armed on dorsal anterior surface (Fig. 16-21; armed in all other Arytropteridini). Male forewings with fewer than 45 teeth (Fig. 27, 30, 33; when known, >49 in all Arytropteridini except Zuludectes). Description. Body size average for the tribe, form robust. Color. General coloration brown with dark and light patterns (Fig. 68-71). Face with sagittal black pattern from antennal sockets to labrum tip. Black retro-ocular patterns often edged with dorsal light pattern. Scapus and pedicel with black patterns. Pronotum folds (see Rentz 1988) with black pattern in line with retroocular black lines, often edged dorsally with light stripes; ventral and posterior rims of lateral lobes black. Abdomen with black patterns laterally, often edged dorsally with light stripes. Hind femur outer field with a dark or black longitudinal pattern.

  elongate, proximal half broad, distal half narrow, with two projections on the middle: one dorsal lobe pointing toward midline, one median projection pointing toward midline. Titillators well sclerotized, external (distal) part pointing (Fig. 55, 56) or enlarged (Fig. 57) distally in dorsal view, with minute denticles; internal (proximal) part with two branches forming an inverted Y or T (Fig. 55-57).

Female.

  Pronotum hardly reaching metathoracic posterior margin, disk more flattened posteriorly than in males. Wings absent. Suranal plate small, triangular or rounded, with or without a longitudinal sulcus. Subgenital plate (Fig.58, 60, 62) of variable shape, posterior end with a median emargination of variable width and depth separating two pointing lobes.Ovipositor(Fig. 59, 61, 63) flattened proximally, upcurved distally; the flattened part of variable length.

  Holotype male. Madagascar, province Toliara, région Atsimo-Andrefana, Ambalamanga, confluence Ankolisky/Mangoky, ExpéditionMakay 2011 -camp 3, 21.675°S, 44.9933°E, 19- 22.i.2011, S. Hugel leg. (2011MADASH 429), MNHN (MNHN-EO-ENSIF10800).Paratypes. Same as holotype (2011MADASH 373, ♀ Allotype), MNHN (MNHN-EO-ENSIF10801). Same as holotype (2011MADASH428 ♂, 430 ♀, 431 ♀), coll. SH. Same as holotype (2011MADASH371 ♀), CAS (CASENT0872622). Madagascar, province Toliara, région Atsimo-Andrefana, Antsoa, rivière Androsi, Expédition Makay 2011 -camp 1, 21.596°S, 45.1131°E, 19-22.i.2011, S. Hugel leg. (2011MADASH 190 ♀). [Madagascar, province Toliara, region Mandabe,] Besely (Mandabe), 11.v.1964, Madagascar Sud, D. Wintrebert Rec., ♂, MNHN. [Madagascar, province Toliara, region Atsimo-Andrefana,] Tulear, Manombo, 10-v-56, ♀, MNHN. [Madagascar, province Toliara, region Atsimo-Andrefana,] Sakaraha, Lambomakandro, III-56 A.R., ♀, MNHN.

  . Madagascar, province Toliara, région Atsimo-Andrefana, Tulear II, Manombo-Sud, Ranobe, 23.0033°S, 43.6233°E, 21.ii.2019, S. Hugel leg. (2019MADASH 186, ♀ Allotype), MNHN (MNHN-EO-ENSIF10803). same as holotype, (2019MADASH 011 ♀, 042 ♂), coll. SH. same as allotype, (2019MADASH 187 ♀), CAS (CASENT0872623). Madagascar, province Toliara, région Atsimo-Andrefana, Tulear II, Belalanda, Ifaty, 23.1413°S, 43.6152°E, 18.ii.2019, S. Hugel leg. (2019MADASH 068 ♂, 069 ♀), coll. SH. Madagascar, province Toliara, région Atsimo-Andrefana, Tulear II, Belalanda, Tsivonoe, 23.1502°S, 43.6268°E, 18.ii.2019, S. Hugel leg. (2019MADASH 145 ♀), coll. SH.

  . 1.Toliaridectes meridionalis n. gen. n. sp., here described. Madagascar, province Toliara, région Anôsy, Réserve de Berenty; 25.0096°S, 46.3079°E, 26.iii.2005, N. Cliquennois leg., MNHN (MNHN-EO-ENSIF 10799). : since the female has been collected in a relatively distinct area (ca. 90 km apart), in a more humid and coastal environment, I prefer to exclude it from the type series since further specimens may prove it to belong to another species.Other females possibly belonging to that species but from more northern localities:[Madagascar, province Toliara, region Atsimo-Andrefana,] Sakaraha, Lambomakandro, III-56 A.R.,MNHN; Lohela, Mahala, 11.VI.1964; Madagascar Sud, D. Wintrebert Rec., MNHN; LoHena, Mahala, 11.VI.1964 Madagascar Sud, D. Wintrebert Rec., MNHN.

	Holotype male. Male paratype. [Madagascar, province Toliara, region Androy, district Beloha], Beloha; iii.56.
	AR.
	Female (not a type). Madagascar, province Toliara, région Anôsy, Réserve de Sainte-Luce;
	24.8028°S, 47.1636°E, 15.iii.2005, N. Cliquennois leg., coll. SH.

Remark

. Toliaridectes n. gen. male head and pronotum

  together with Meconematini Burmeister, 1838, Arytropteridini[START_REF] Caudell | Orthoptera Fam. Locustidae Subfam. Decticinae[END_REF] and -more surprisingly-Hetrodinae Brunner von Wattenwyl, 1878 would probably have to be defined. . 1-3: head and pronotum in dorsal view, before removing the forewings for examination. 4-6: head and pronotum in left side view. 7-9: face in frontal view. 1, 4, 7: Toliaridectes meridionalis n. gen. n. sp. 2, 5, 8:

	FIGURES 1-9Toliaridectes wendenbaumi n. gen. n. sp. 3, 6, 9: Toliaridectes antsycurvis n. gen. n. sp. All
	pictured specimens are holotypes.
	FIGURES 10-24. Toliaridectes n. gen. legs. 10-12: left fore tibia inner view. 13-15: left fore
	tibia outer view. 16-18: left mid tibia anterior view. 19-21: left mid tibia posterior view. 22-
	24: left hind femur outer view. 10, 13, 16, 19, 22: Toliaridectes meridionalis n. gen. n. sp. 11,
	14, 17, 20, 23: Toliaridectes wendenbaumi n. gen. n. sp. 12, 15, 18, 21, 24: Toliaridectes
	antsycurvis n. gen. n. sp. All pictured specimens are holotypes.
	FIGURES 25-

33. Toliaridectes n. gen. male forewings. 25

  , 28, 31: left forewing in dorsal view (removed from specimen). 26, 29, 32: right forewing in dorsal view (removed from specimen). 27, 30, 33: file in ventral view. 25-27: Toliaridectes meridionalis n. gen. n. sp. 28-30:

	Toliaridectes wendenbaumi n. gen. n. sp. 31-33: Toliaridectes antsycurvis n. gen. n. sp. All
	pictured specimens are holotypes.
	FIGURES 34-54. Toliaridectes n. gen. male terminalia. 34, 41, 48: subgenital plate (ventral
	view). 35, 42, 49: left paraproct (removed from specimen). 36, 43, 50: male suranal plate
	(dorsal view, the subgenital plate is lightened for better visualization). 37, 44, 51: left cercus
	(dorsal view). 38, 45, 52: left cercus (inner view). 39, 46, 53: left cercus (ventral view). 40, 47,
	54: left cercus (outer view). 34-40:

Toliaridectes meridionalis n. gen. n. sp. 41-47:

  Toliaridectes antsycurvis n. gen. n. sp. All pictured specimens are holotypes.

	Toliaridectes wendenbaumi n. gen. n. sp. 48-54: Toliaridectes antsycurvis n. gen. n. sp. All
	pictured specimens are holotypes.
	FIGURES 55-57. Toliaridectes n. gen. male genitalia (dorsal view, posterior side on top).
	55: Toliaridectes meridionalis n. gen. n. sp. 56: Toliaridectes wendenbaumi n. gen. n. sp.
	57:

FIGURES 58-63. Toliaridectes n. gen. female terminalia

  . 58, 60, 62: subgenital plate (ventral view, posterior side on top). 59, 61, 63: ovipositor (left side view). 58-59: Toliaridectes meridionalis n. gen. n. sp. 60-61: Toliaridectes wendenbaumi n. gen. n. sp. 62-63: Toliaridectes antsycurvis n.

gen. n. sp. FIGURES 64-67. Toliaridectes antsycurvis n. gen. n. sp. male call. Specimen

  2019MADASH042recorded in natura in Andrevo Bas 30°C 15-16.ii.2019 at 0h30, few seconds before taking the picture from figure 70.

FIGURES 68-71. Toliaridectes n. gen. in natura

  . 68-69: Toliaridectes wendenbaumi n. gen. n. sp., male in resting position (68); female eating a caterpillar (69). 70-71: Toliaridectes antsycurvis n. gen. n. sp., male calling (70; specimen 2019MADASH042, see figures 64-67); female in oviposition (71).

	FIGURE 72.

Distribution of Toliaridectes n. gen. TablesTABLE 1 .

 1 Measurements of Toliaridectes meridionalis n. gen. n. sp. (in mm).

		Body	Pronotum Pronotum	Fore femur	Mid femur	Hind femur	Hind femur	Ovipositor
		length	length	width	length	length	length	width	length
	♂ Holotype ♀	17.5 21.4	6.4 7.1	4.7 6.8	5.7 7.0	5.3 6.2	16.1 20.4	3.4 3.8	-14.5

TABLE 2 .

 2 Measurements of Toliaridectes wendenbaumi n. gen. n. sp. (in mm).

		Body	Pronotum Pronotum	Fore femur	Mid femur	Hind femur	Hind femur	Ovipositor
		length	length	width	length	length	Length	width	length
	♂ Holotype Paratype 428	17.6 16.0	6.1 6.1	4.3 4.8	4.9 5.5	4.8 5.5	15.5 16.3	3.2 3.1	--
	♀ Allotype min	21.9 21.3	6.4 6.2	5.4 5.4	6.2 6.2	6.3 6.1	19.7 18.6	3.5 3.5	12.4 11.2
	max	23.0	6.4	5.6	7.0	6.7	19.7	3.6	13.9

TABLE 3 .

 3 Measurements

		Body Pronotum Pronotum	Fore femur	Mid femur	Hind femur	Hind femur	Ovipositor
		length	length	width	length length Length width	length
	♂ Holotype 16.0 min 12.9	6.1 5.6	5.0 4.1	5.2 4.5	5.2 4.5	15.8 14.4	3.4 3.0	--
	max	16.0	6.1	5.0	5.2	5.2	15.8	3.4	-
	♀ Female min	19.7 17.7	6.8 6.8	5.7 5.7	6.2 6.2	6.0 6.0	19.0 19.0	3.9 3.6	8.8 8.8
	max	19.7	6.9	6.2	6.7	6.7	20.1	4.2	11.9

of Toliaridectes antsycurvis n. gen. n. sp. (in mm).

Acknowledgments

. The fieldwork on which this study is based could not have been completed without the gracious support of the Malagasy people, Brian Fisher (California Academy of Science) and all the arthropod team of the Madagascar Biodiversity Center for their help and advices, particularly Balsama Rajemison, Jean-Claude Rakotonirina, Jean-Jacques Rafanomezantsoa, Chrislain Ranaivo, Hanitriniana Rasoazanamavo, Nicole Rasoamanana, Clavier Randrianandrasana, Dimby Raharinjanahary, Njaka Ravelomanana, Manoa Ramamonjisoa. I thank Naturevolution team (https://www.naturevolution.org/) and particularly Evrard Wendenbaum for having organized multiple surveys in Makay region. I thank Nicolas Cliquennois for his continuous and fruitful efforts to collect Orthoptera in South Western Indian Ocean islands. Thanks to Darren Goldin and Aran Hinton for their help in the field in Tulear. I thank Steve Goodman for his precious advices. This work was conducted with support from Insects and People of the Southwest Indian Ocean (IPSIO, https://www.ipsio.org), a network funded by the Critical Ecosystem Partnership Fund (CEPF). Permits to research, collect and export arthropods were obtained from the Ministry of Environment and Forest as part of an ongoing collaboration between the Madagascar Biodiversity Center, and the Ministry of Environment and Forest, Madagascar National Parks and Parc Botanique et Zoologique de Tsimbazaza.