L Lewandowski

P Reiter

B Birkenbach

B Bruyneel

E Clement

J Eberth

H Hess

C Michelagnoli

R M Perez-Vidal

M Zielinska

Pulse-Shape Analysis and position resolution in highly segmented HPGe AGATA detectors

Keywords: 29, 30, Kv X-and γ-ray spectroscopy -29, 40, Gx Tracking and position-sensitive detectors -29, 40, Wk Solid-state detectors -07, 05, Tp Computer modeling and simulation

The performance of the Pulse-Shape Analysis (PSA) in AGATA HPGe detectors was investigated and improved employing a γ-ray source measurement based on e -e + annihilation radiation after decays of 22 Na by β + decay. The first interaction positions of the two 511 keV γ rays were determined and the connecting line of these two positions was compared to the known source position as a measure for the PSA performance. The position resolution and its dependence on the PSA parameters were investigated by varying most relevant input quantities: the charge carrier mobility of the holes, the response of the employed measuring electronics especially the preamplifier rise time. The relative statistical weight of charge signals and transient signals was scrutinized. The optimal distance metric of the grid-search algorithm and its impact on the position resolution was determined.

Introduction

AGATA in its final configuration will be a highly efficient 4π γ-ray spectrometer consisting of 180 highly segmented HPGe detectors [1]. It will provide utmost detection efficiency and performance employing the γ-ray tracking (GRT) technique which is based on the individual interaction positions of all γ rays within the segmented HPGe detector volumes of the array [2]. The interaction positions of the γ rays within the individual segments of the HPGe detectors are obtained by means of Pulse-Shape Analysis (PSA) using the adaptive grid search [3]. The main idea of PSA with highly segmented HPGe detectors is a detailed comparison between the measured detector pulses and a set of reference pulse shapes which are generated for a narrow three dimensional grid in order to identify the most probable interaction position within the active detector material. These reference pulse shapes comprise information created by moving charge carriers and influenced signals. The pulse shapes unambiguously depend on the three dimensional location of the interaction point where electron hole pairs are created after the interaction of the incoming γ ray. The position information of the grid point together with the pulse shape information of the core and the segment signals are bundled in lookup libraries. To achieve the necessary position resolution of a few millimeters the calculated signal pulses from different interaction points have to be equal or very similar to the measured pulses.

The theoretical signal pulses are extracted from simulations which request very accurate information on the properties of each individual detector. The procedure how these position dependent detector pulses are generated for the AGATA detectors is given by the AGATA Detector Library (ADL) described in Ref. [4]. Each individual HPGe detector is characterized by its geometry, space-charge distribution, crystal-axis orientation, crosstalk properties and the response function of the electronics. The interaction positions allocated by the PSA depend also on the grid-search algorithm that is employed to perform the comparison of the measured and simulated pulses. Moreover, the results of this comparison depend on the time alignment between measured and calculated pulses, the distance metric and the weighting of the individual segment pulses.

Recent studies demonstrated that the AGATA PSA can resolve the interaction position of γ rays in segmented AGATA detectors with an energy dependent position resolution of a few millimeters [5,6]. The AGATA tracking efficiency is also within the specifications. However, an inspection of the interaction position yielded for example an unexpected clustering of position points as a result of the PSA, which are not in agreement with the expected distribution of hits within the Ge detector volume. Especially, measurements with a 60 Co source show deviations from isotropic distributions and a clustering of hits in specific parts of the detector volume or even a clear surplus of hits at single grid points (see chapter 3) is observed. The clustering of positions and accumulation of grid points in the final results of the AGATA PSA inspired a refined investigation of the AGATA PSA.

In the first part of this paper a reminder to the principles of the AGATA PSA will be given and results from latest source measurements will be discussed. The second part covers the coincident detection of 511 keV γ rays at 180 • and the reconstructed emission point. Its comparison to the known source position enables a detailed assessment of the PSA performance. In the third part of the paper input parameters are varied and optimized by maximizing the position resolution of the PSA.

PSA principles

The Pulse-Shape Analysis combines the measured digitized traces of AGATA detector pulses from individual HPGe detectors and the AGATA data library (ADL) employing the adaptive grid search [3,4] algorithm. The measured detector signals from individual γ-ray events are taken from the 36 segment signals and the core signal from the analogue preamplifiers after 100 MHz digitization or 10 ns time steps with the AGATA electronics (see also Ref. [1]). The ADL contains simulated pulses of every segment and the central electrode (core) for every possible interaction position in the detector. The detector is divided into a grid with a set of simulated signals associated with every grid point. All the pulses from the position dependent simulation are compared to the measured detector pulses. The set of pulses with the best matching of measured and simulated pulses is used to assign the interaction position of the γ ray.

The adaptive grid search yields first an interaction position which is the result of a three dimensional search in a coarse grid with a length of 6 millimeters per grid point. Then a refined second search is conducted around the interaction position found in the coarse search. The fine grid has a length of 2 millimeters per grid point. The comparison of measured and simulated signals is done by minimizing the following figure of merit:

Figure of Merit = j,ti |A j,m (t i ) -A j,s (t i )| p (1)
A j,m (t i ) and A j,s (t i ) are the pulse heights at the time t i of segment j of the measured (m) or simulated (s) pulse. Only the hit segment, its nearest neighbors and the core signal are considered. Typically, a time range of the first 400 ns or 40 time steps after the determined T 0 are used to determine the figure of merit. The exponent p is a real number greater zero that has to be chosen. The adaptive grid search provides only one interaction per detector segment. The black sector of a circle indicates the positions with the same distance to the source. Sets of grid points at a constant detector depth z have approximately the same distance to the source. This is indicated for the front part at z = 0 -2 mm (red marked area). A maximum deviation to the sphere is about 2.5 mm compared to the smallest radial distance of 226 mm.

Results of the Pulse-Shape Analysis

A measurement with a 60 Co source was performed at the Grand Accélérateur National d'Ions Lourds (GANIL) with 29 HPGe detectors comprised in nine triple-cluster and one double-cluster detectors. The source was located in the center of the sphere at a distance of 226 mm. The data set was analyzed using the adaptive grid search and the AGATA data library introduced in section 2. For a given distance z along the main axis of the HPGe detector each grid point has approximately the same distance to the source. The distance deviates by approximately 1% for different points of that plane located at the fringes of the detector volume (see Fig. 2). Therefore, the expected number of hits for a detector plane with constant z-value is estimated to be equal within statistical errors for a source measurement. However, the interaction positions allocated by the PSA results show deviations from these expectations which cannot be explained by the statistical nature of the mea- surement. The six fold structure of the segments is visible although the interaction probability should not be impacted by the segmentation (for more details on the segment structure refer to [1]). Some grid points at the outer edges of the detector or in the middle of segments show more hits than expected (see Fig. 3). The deviations from the smooth distribution indicate that some of the allocated interaction positions systematically disagree with the physical interaction points (see also Fig. 4).

The pulses that are created by the AGATA Data Library depend on a variety of detector properties and the acquisition electronics. For the calculation several input parameters for each individual HPGe detector are needed to generate the most reliable set of pulse shapes for the ADL. Most of these input parameters are measured routinely, e.g. the cross talk properties of the detectors of its electronics network. However, some quantities, like the space charge distribution inside the Ge crystal volume, are hard to access and are only partially available from the crystal manufacturer.

In order to understand the clustering of interaction points and to optimize the PSA performance a method was developed that scrutinizes the PSA input parameters within meaningful ranges especially in case the parameters are not measurable or known with uncertainties. It will be introduced in the following section 4.

Coincidence measurement of γ rays after e + e -annihilation

The position sensitivity of several AGATA detectors was determined by analyzing the data of a 22 Na source measurement. In the decay process of 22 Na a positron is emitted. This positron annihilates with an electron very close to the point of emission. It is assumed that the source is point like and that the positron annihilates at the point of emission. The positron emitted by 22 Na has a maximum energy of 540 keV and a mean energy of 180 keV. The mean range of the positron in polyethylen is about 0.24 mm [START_REF] Dryzek | Detection of Positron Implantation Profile in different Materials[END_REF]. The assumption that two 511 keV γ rays are emitted with an angle of 180 • is very well justified. The coincident detection of these γ rays allows to assess the quality of the PSA results. For this purpose, the shortest distance of a straight connecting line between the two found interaction points to the known source position is determined (see Fig. 5 for details). A similar approach was also used in the past to compare different simulated signal bases by [START_REF] Klupp | [END_REF].

For the measurement the 22 Na source was positioned within one of the pentagonal shaped holes. It was placed 3.2 cm inside the spherical shell formed by the canisters of the triple cluster detectors close to the five surrounding individual HPGe detectors (see Fig. 6). Coincident events with an energy of 511±3 keV were selected allowing only for two interactions in the complete array in two individ-ual crystals. The background of random coincidences was reduced by gating on angles larger than 150 • between two γ rays positions and the source (see Fig. 9). Assuming a constant background of 250 counts/ • in the range of 150-180 • only 1.53% of all events in that region are estimated to be from background. Very large deviations from 180 • cannot be caused by the position uncertainty of the PSA. The distance from the connecting line of the measured interaction positions to the source position (see Fig. 5) was required to be shorter than 14 mm. Events with larger distances were discarded since they are most likely caused by random coincidences.

The trigger rate for the five inner crystals was approximately 1 kHz and 200 Hz for all other detectors. For the event building a timing window of ± 2.5 µs was chosen. Fig. 7 shows the time stamp differences of the individual detectors before and after applying the aforementioned data analysis conditions. With these gates, a data set of about 468 k events was selected for the final analysis. The mean distance from the distribution of minimum distances between the source position and the connecting line (see Fig. 10), accumulated for all coincidences, was used as a measure for the quality of the PSA. In order to investigate and improve the PSA performance the input parameters were varied and optimized in a variation process.

The 22 Na-coincidence method is used to verify properties of the crystal and the electronics as well as parameters of the algorithm. The position uncertainty of both interactions of different detectors contribute to the final performance measurement. Therefore, crystal specific properties, such as the impurity concentration of the individual crystals, are not subject of the following procedure.

Improving PSA performance

The results of the PSA rely on the detailed comparison of calculated detector signals which are generated by the AGATA data library. These signals depend on the individual HPGe crystal properties and the signal processing by analog and digital electronics. Moreover, the results depend on the adaptive grid search algorithm that compares measured and simulated traces. The 22 Na-coincidence method introduced in section 4 is employed to optimize the characterization of the detector and electronics parameters as well as the grid search algorithm. A variation procedure is employed to re-evaluate and to improve the individual PSA parameters. For this purpose the input parameter of interest (e.g. a crystal property) was varied in a certain range around the previously used value. For each individual variation step the same 22 Na-coincidence data set was analyzed again, leading to new interaction positions found by the PSA. This set of new interaction positions was then searched for γγ coincidences as described in section 4. As a result of the variation procedure the local minimum of the mean distance of the lines to the source (see Fig. 8 and 10) was determined.

The reanalysis of the data set is CPU time consuming which is mainly caused by the grid search algorithm of the PSA. In addition the analyzed data set was chosen to be sufficiently large to ensure a high significance. The used data set consisted of 6.24 • 10 8 individual interactions leading to typical computing times of around a day for a single variation step using a 32 CPU core high performance server. Therefore, variations were performed for single parameters because a multidimensional variation was not feasible due to time constraints. However, clearly correlated quantities were studied simultaneously like e.g. the hole velocity parameter and the preamplifier rise time.

Transfer function

The measured pulses are processed and shaped by the preamplifier and the digitizer electronics [START_REF] Pascovici | [END_REF]10]. In order to compare the measured pulses with calculated signals the transfer function of the electronics is needed for the convolution of the the simulated signals with this transfer function. The properties of the transfer function is described in a simplified approach by a single rise time τ of the preamplifier. Consequently, this rise time was varied and the different results were evaluated with the method presented in section 4. The optimal value for the position resolution given by the mean value is found to be around 45 ns, (see results of Fig. 11), a value which is about 10 ns larger than expected from measurements of the preamplifier rise times [11]. This deviation is expected to be caused by an additional contribution to the measured signal rise time from the digitizers electronics. Another explanation for a slower measured rise time may also be given by the drift velocities of the charge carriers emplyed in the simulation. Therefore, the mobility of charge carriers and its interplay with the transfer function was investigated separately (see section 5.2).

Hole mobility

The drift velocity of the charge carriers is essential for calculating precise pulses for the different interaction positions. For the hole drift velocity the empirical approach of [12,13,14] is employed:

v D = µE (1 + ( E E0 ) β ) 1 β (2)
Where v D is the drift velocity of the holes, µ the mobility of the holes and E the electrical field strength. E 0 and β are empirically determined parameters. The used values are given in table 1 which were derived by Bruyneel et al. in the same way as described in [11]. The hole mobility µ was varied in the range between 40 and 70 cm 2 mVs . Due to the time consuming manner of the variation procedure the mobility was assumed to be the same in every crystallographic direction. This is a valid approximation as the mobility in the 1 0 0 and 1 1 1 directions only differ by less than one percent. The results in Fig. 12 show the best PSA performance for a hole mobility of µ = 55 cm 2 mVs . The variation was performed with a rise time of τ = 35 ns. The results in section 5.1 show that a slower preamplifier response yields improved results. Using a larger preamplifier rise time has a very similar consequence on the simulated traces like using a reduced mobility. Therefore, both of these input parameters were investigated in combination by varying both in an iterative procedure. For this either the hole mobility µ or the rise time τ were fixed and the other parameter was varied.

Best results (in terms of a minimal mean distance d) were obtained for a rise time of τ = 40 ns and µ = 55 cm 2 mVs . Note that the drift velocity also depends on several other factors as for example the impurity profile, the empirical parameters E 0 and β as well as the electrical field strength.

Weighting of transient signals

The signals of the hit segment and the core are very sensitive to a change in the radius of the interaction position but change only slightly for different angles φ or different z values (see Fig. 15, the terms radius, z and φ are used as in a cylindrical coordinate system of the HPGe detector volume). The transient signals are essential for extracting correct φ angles and z values as they provide a very characteristic change of signal shapes as a function of these coordinates (see Fig. 13 and14). Therefore, the transient signals need to have a considerable impact on the final figure of merit although they are typically relatively small compared to the signals of hit segment and core. The figure of merit landscape for a single event is depicted in Fig. 15 illustrating the higher sensitivity to radial changes than to angular changes in the interaction position.

The contribution of the transient signals to the final figure of merit is in average about 35%, depending on the choice of p in eq. 1 and on the interaction position. A weighting was introduced to explore the contribution of the transient signals to the final figure of merit value and to increase the angular position resolution. The modified weighted figure of merit is given by:

Figure of Merit = j w j ti |A j,m (t i ) -A j,s (t i )| p (3)
Here w j is a weighting coefficient that is one for the hit segment and the core. The weighting factor was determined for the nearest neighbors. Segments that are neither hit nor adjacent to a hit segment are not considered. The weighting coefficient was varied assessing the PSA performance for each variation step. The results are shown in Fig. 16 and an optimal value of about w j = 2.75 is obtained. The PSA yields better results by increasing the weighting coefficient

Distance metric

The calculation of the figure of merit (see eq. 1) depends as well on the choice of the exponent p, that determines the distance metric (e.g. p=2 corresponds to an Euclidean metric which is used for standard χ 2 minimization). The value of p has considerable effect on the comparison between measured and simulated pulses and on the result which minimizes the figure of merit best. Large values of the exponent p correspond to a stronger weighting of large deviations of measured and simulated pulses. In this way the impact of noise is reduced as these deviations are typically small. On the other hand, the transient signals are most relevant for the correct position determination by the PSA. Depending on the interaction position, these can be very small compared to the signals of the hit segment or of the core. Therefore, p cannot be not too large.

To investigate this behaviour PSA was performed for different values of the exponent p. Each time the PSA result was assessed with the 22 Na-coincidence method introduced in section 4. The results are depicted in Fig. 17. The found minimum corresponds to a value of p = 0.4 which shows that smaller values than the Euclidean metric is favored in this case. The result is consistent with previous findings where an optimal value of p = 0.3 was found using the quality of the Doppler correction from in-beam data as performance criterium [15]. This also implies that the differences between simulation and measurement are not Gaussian like (see also Fig. 18).

An ideal scenario would be free of systematic errors; the deviations between measurement and simulation only arise from the noise of the signal and the difference of both is expected to be Gaussian. However, the observed differences of measurement and simulation cannot be solely explained by noise. Additional deviations are presumably caused by the non perfect data base of simulated signals and simplifying assumptions that are used in the grid-search algorithm which leads to systematic deviations between measurement and simulation, for example the simplifying assumption of only one interaction per segment.

As shown in section 5.3 a weighting of the transient signals is favorable. This implies that a different distance Two different exponents q and p were evaluated separately in an iterative procedure by leaving one constant and varying the other. The weighting factor for the transient signals was set to 1 for this investigation. Results of the two last iterative steps are shown in Fig. 19. The best PSA result is obtained for p = 0.4 and q = 0.7. Note that in the last iteration with a constant q of 0.7 the PSA-performance parameter changes only very slightly in the range from 0.1 to 0.5. The main improvement is caused by the condition p < q. This is most likely caused by the reduced signal to noise ratio in the small transient signals.

Conclusion and outlook

The results of the AGATA Pulse-Shape Analysis were investigated and improved by exploiting the 180 • γγ coincidences of annihilated positrons emitted from a 22 Na source. An enhanced position resolution was achieved with an adjusted electronic response which is described by a rise time τ that is 5 ns larger than previously measured. Simultaneously, the mobility of the holes µ was reduced by about 10% to 55 cm 2 mVs . A smaller mobility and a slower electronic response have a similar impact on the simulated pulse shapes. Therefore, these two parameters were determined in an iterative procedure. The final mobility is in Table 2. A summary for the initial and final values of the distance metric parameters p and q, the weighting coefficient wj, the hole mobility µ and the preamplifier rise time τ is shown. The small difference of the mobility µ in the 1 0 0 and 1 1 1 directions could not be resolved. good agreement with the previously measured value [11].

Remaining differences might originate from the transfer function as well as from the impurity profile of the crystal.

In addition the results of the grid-search algorithm were improved by implementing a weighting factor for the transient signals of the nearest neighbors of the hit segment. A weighting factor of 2.75 yielded the best results. For future investigations it might be worthwhile to introduce an energy dependent weighting factor which increases for higher energies. For low energy depositions the signal to noise ratio is small, especially for the transient signals. In these cases a stronger weighting of these signals might not be favorable. However, a different approach has to be applied in order to determine the optimal energy dependence of weighting factors. Unfortunately, the 22 Na-coincidence method is restricted to energy depositions of 511 keV.

It was shown that an Euclidean metric, corresponding to an exponent value of two in the figure of merit calculation, does not give best PSA results. Improvements were achieved by splitting the figure of merit calculation into two parts for the hit segment and core, as well as for the neighboring segments. The best PSA performance was observed by choosing the exponent to 0.4 for the hit segment and core and to 0.7 for the neighboring segments.

The final improved PSA results are shown in the distribution of interaction positions in Fig. 21 and22. A clear reduction of the non-physical allocation of hits is obtained: The visibility of the segment structure is diminished. The number of hits at large radii is no longer smaller than expected. The amount of interactions in single high statistics grid points is decreased to a more reasonable level. A comparison of the distances of the coincidence lines to the source is shown in Fig. 20. The standard configuration yields a mean distance of 2.73 mm and a standard deviation of 1.85 mm whereas in the optimized configuration a mean distance of 2.54 mm and a standard deviation of 1.80 mm is obtained.

The presented procedure was able to improve the PSA performance, although remaining limitations have to be accounted for. The choice of the parameters and its variation is motivated by an empiric approach. The starting values were taken from measurements and then varied in the final configuration of the spectrometer and its PSA grid algorithm. The resulting improved parameters, for example for the transfer function, are the ones that yield the best position resolution, but do not necessarily have to coincide with the physical ones. This is caused by the fact that the PSA performance could only be determined for a subset of input parameters.

In future, a full scan of the N-dimensional performance landscape should be performed and the long processing time of each variation step (where N is the number of input parameters) has to be coped with. In this way the variation will provide the global optimal configuration in the N dimensional space. Also the γ-ray energy should not be restricted to 511 keV. Previous investigations showed that PSA performance depends on the energy deposition at the interaction point due to the better signal to noise ratio at higher energies [16]. The restriction to a single energy may produce biased results and a continued effort is needed to resolve the energy dependence of the Pulse-Shape Analysis.

The 22 Na-coincidence method was established as a fast and reliable tool to study the results of the PSA, improve the simulated signal basis and the position resolution. The electronic response, the hole mobility and features of the grid search algorithm were investigated and adjusted. Despite the improvements made some unexpected distribution of hits, e.g. a clustering of hits, in the detector is still observed. Therefore, a continued investigation is needed to obtain better reference pulses and an improved search algorithm. In particular two interactions inside a single segment should be considered. The current algorithm assumes only single interactions inside a segment. As a result two hits inside a segment are allocated at the energy weighted bary center of the interaction. The disentanglement of one hit and two hit interactions inside a single segment is very challenging and requires a high fidelity of the reference basis. Several scanning tables were developed to obtain reference pulses at known interaction positions [17,18,[START_REF] Hernndez-Prieto | High spatial resolution -camera devoted to characterization of electrical response in HPGe segmented detectors[END_REF]. In future a detailed comparison with scanning table results should be utilized to simulate an improved signal basis. This research was supported by the German BMBF under Grants 06K-167, 06KY205I, 05P12PKFNE, 05P15PKFN9, 05P18PKFN9. AGATA was supported by the European funding bodies and the EU Contract RII3-CT-2004-506065.

Figure 1 .

 1 Figure 1. Picture of the AGATA array at GANIL which comprised 29 individual HPGe detectors at this time. The crystals have a tapered hexagonal geometry with an asymmetric shape to fit into a 4π configuration with a 180 detector geometry. A beam pipe and an open target chamber is located in the central position of the setup. A 60 Co source was placed in the middle of the vacuum chamber for calibration.1

Figure 2 .

 2 Figure 2. Schematic side view of the AGATA HPGe detector.The black sector of a circle indicates the positions with the same distance to the source. Sets of grid points at a constant detector depth z have approximately the same distance to the source. This is indicated for the front part at z = 0 -2 mm (red marked area). A maximum deviation to the sphere is about 2.5 mm compared to the smallest radial distance of 226 mm.

Figure 3 .

 3 Figure 3. The distribution of hits allocated by the PSA is shown for crystal A001 for two different depth values of a) z = 6 -8 mm and b) z = 28 -30 mm. The first picture a)shows a reduced number of hits for large radii. In addition the six fold structure of the segments is visible. The second picture b) illustrates that the PSA assigns a much higher amount of hits in single grid points at the upper right and lower left corners marked with circles. For both pictures a homogeneous distribution of hits with statistical fluctuation is expected.

Figure 4 .

 4 Figure 4. The radii of the interaction positions are plotted versus the detector depth z for crystal A001. The black line marks the mean radius for the given depth z. The source is placed in front of the crystal at a distance of 226 mm. A clustering of hits is observed at specific depth values z which is not expected illustrating a systematic deviation between the PSA results and the physical interaction position.

Figure 5 .

 5 Figure 5. Two 511 keV γ rays are emitted after e + e -annihilation and both are detected in coincidence in opposite Ge crystals. The connecting line of the two interaction positions found by the PSA may not cross the known source position. This is mainly caused by the finite position resolution of the PSA and to a minor part by the range of the positron. The minimum distance of the connecting line to the source position provides a measure for the performance of the PSA.

Figure 6 .

 6 Figure 6. The source was placed 3.2 cm inside the pentagonal shaped hole which is typically used for the beam line. This geometry allows the coincident detection of the 180 • correlation of the two γ rays for several crystals and ensures that data is also available for the segments located at the back side of the crystals.

Figure 7 .

 7 Figure 7. The differences of the time stamps T Si,j for all individual detector combinations i and j are plotted in a). In addition to correlated interactions close to zero a constant background is observed. The same difference is plotted in b) after applying gates to multiplicity, energy, angle and distance reducing the background nearly to zero (for details see text).

Figure 8 .

 8 Figure 8. A visualization of the measured interaction positions of all 511 keV γγ coincidence events is shown (colored distribution). A line is drawn between the two interaction positions. These lines pass by in close distance to the source position which is marked with a red dot. For clarity only lines from 100 coincident events are plotted.

Figure 9 .

 9 Figure 9. The distribution of angles between the two 511 keV interactions is shown. For background reduction only events with an angle larger than 150 • considered which corresponds to 96.56% of all events.

Figure 10 .

 10 Figure 10. The distance of the connecting lines between found coincidences and the source position is shown. The mean of this distribution is used as a measure for the PSA performance. The standard AGATA PSA was used and a mean distance of 2.73 mm and a standard deviation of 1.85 mm was achieved.

Figure 11 .

 11 Figure 11. The rise time of the transfer function was varied in a wide range of 15-65 ns around the expected best value which is obtained at τ = 45 ns.

Table 1 .Figure 12 .

 112 Figure 12. Variation of the hole mobility in germanium. To reduce the needed computational time the mobility for the 1 0 0 and 1 1 1 directions were chosen identically. The PSA performs best for a mobility of µ = 55 cm 2 mVs .

Figure 13 .

 13 Figure 13. Simulated traces are shown for different radii at an fixed angle φ = 15 • and a depth z = 40 mm for a) the hit segment and b) for the left neighbor. The pulse shapes of the hit segment are very sensitive to a change in the radius of the interaction. Even more, the transient signals in the neighboring segment also change the signal shape in a characteristic way for different radii. Here the polarity of the signal changes going from inner to outer radii and the induced signals nearly vanish for intermediate radii in the middle of the segment.

Figure 14 .

 14 Figure 14. Simulated traces for different angles φ are shown for a fixed radius of r = 35 mm and a depth of z = 40 mm for a) the hit segment and b) for the left neighbor. The change in the pulse shape of the hit segment is relatively small whereas the transient signals provide a pronounced change in amplitude for different φ angles.

Figure 15 .

 15 Figure 15. The figure of merit associated with every possible interaction position with a fixed z is shown for a single event. The minimum, corresponding to the allocated interaction position, is marked with a red circle. The change of the figure of merit is much more pronounced in radial direction than for different angles φ.

Figure 16 .

 16 Figure 16. Variation of the weighting coefficient in the figure of merit calculation, see eq. 3. Better PSA performance was achieved with a weighting coefficient of about 2.75 compared to the standard non weighted comparison of measurement and simulation.

Figure 17 .

 17 Figure 17. Variation of the exponent p in the calculation of the figure of merit. The minimum at p = 0.4 corresponds to the best PSA results.

Figure 18 .

 18 Figure 18. The difference of measured Am and simulated As pulses using the first 400 ns (corresponding to 40 samples) after the trigger time T0 for the a) signals from hit segment and core and b) the transient signals is shown for a multitude of events. Only events with one interaction per crystal were used and the pulses Am and As were normalized to the energy of the interaction E. The resulting distributions are not Gaussian like but have left and right tails.

Figure 19 .

 19 Figure 19. Variation of the new distance metric parameters p and q from eq. 4. An improved PSA performance is achieved by treating the the transient signals individually in the comparison of measurement and simulation.

Figure 20 .

 20 Figure 20. The distribution of distances of the lines connecting the coincident interaction positions to the source position is shown for the initial and optimized set of PSA input parameters.

Figure 21 .

 21 Figure 21. The optimized results of the PSA are shown for the same slices as in Fig. 3. Picture a) shows the slice for z=6-8 mm and b) for z=28-30 mm. The same color code was chosen like in Fig. 3.

Figure 22 .

 22 Figure 22. A comparison between initial (left) and optimized (right) PSA results is shown for a) z=4-6 mm and b) z=14-16 mm. A distinct improvement of the allocated interaction positions is observed. The same color code was chosen for a) and a') and b) and b').

Picture by P. Stroppa, CEA


Clément Huguet

Joshua Opeyemi

Eleonore Olatunji

Phila Real

Aurelie A Raharivelomanana

Urbain
email: urbain@unistra.fr

Joshua Olatunji

Eleonore Real

Phila Raharivelomanana

Aurélie Urbain

Glucagon-like peptide-1 secretagogues from the leaves of Oparanthus teikiteetinii

Keywords: Oparanthus teikiteetinii, benzofuran derivatives, coumaran, sesquiterpene lactones, glucagon-like peptide-1 secretion

Introduction

Oparanthus teikiteetinii (J.Florence & Stuessy) R.K.Shannon & W.L.Wagner is a tree endemic to the Marquesas Islands, French Polynesia, which belongs to the Asteraceae family and to the subtribe Coreopsidinae. Firstly described under the genus Bidens [START_REF] Florence | Sertum polynesicum 3 : un Bidens (Asteraceae) nouveau de l'île de Nukuhiva (Marquises, Polynésie Française)[END_REF], it has been recently revised to the genus Oparanthus [START_REF] Ryding | Phylogeny, distribution, and classification of the Coreopsideae (Asteraceae)[END_REF][START_REF] Shannon | Oparanthus (Asteraceae, subtribe Coreopsidinae) revisited[END_REF].

No traditional uses have been reported for this species that has been found only in the restricted area of the Toovi plateau on the island of Nuku Hiva, and no phytochemical nor pharmacological studies have been conducted until now. According to phylogenetic trees, O. teikiteetinii is rather closely-related to Fitchia speciosa, endemic to the Cook Islands [START_REF] Kimball | Phylogeny of Coreopsideae (Asteraceae) using ITS sequences suggests lability in reproductive characters[END_REF]. This latter species was found to contain mainly sesquiterpene lactones, as most of the Asteraceae, and dihydrobenzofuran derivatives [START_REF] Bohlmann | New sesquiterpene lactones and other constituents from Fitchia speciosa[END_REF]. As part of our continuous efforts to identify natural GLP-1 secretagogues, we decided to evaluate the potential of O. teikiteetinii to stimulate the secretion of glucagon-like peptide-1 (GLP-1) in vitro in a mouse intestinal cell culture model. GLP-1 is an intestinally secreted peptide involved in blood glycemia regulation [START_REF] Nicolaus | Endogenous GLP-1 regulates postprandial glycemia in Humans: relative contributions of insulin, glucagon, and gastric emptying[END_REF]. As its secretion is impaired in patients with type 2 diabetes, compounds improving GLP-1 secretion levels could therefore represent a novel therapeutic strategy [START_REF] Nauck | Normalization of fasting hyperglycaemia by exogenous glucagon-like peptide 1 (7-36 amide) in type 2 (non-insulindependent) diabetic patients[END_REF][START_REF] Nauck | Glucagon-like peptide 1 (GLP-1) as a new therapeutic approach for Type 2-diabetes[END_REF][START_REF] Vilsbøll | Reduced postprandial concentrations of intact biologically active glucagon-like peptide 1 in type 2 diabetic patients[END_REF]. In our in vitro assay, the EtOAc extract from the leaves of O. teikiteetinii increased significantly the extracellular secretion of GLP-1 in STC-1 cells. The phytochemical investigation of this extract led to the isolation of 15 compounds, including mainly benzofuran and benzopyran derivatives (1-11), together with 4 sesquiterpenoids (12-15). All compounds were evaluated for GLP-1 secretagogue activity and some of them (7, 11, and 12) were found to be significantly active. This is the first report on phytochemical composition and biological activity of Oparanthus teikiteetinii.

Results and discussion

The EtOAc extract from the leaves of O. teikiteetinii increased the extracellular secretion of GLP-1 in STC-1 cells by 249 ± 2% when tested at a concentration of 30 μg/mL (Supplem. S1). This extract was subjected to successive chromatographic separations leading to fifteen compounds (1-15) (Figure 1 and Supplem. S2), including two new dihydrobenzofuranes identified as

(2R)-2-(prop-1-en-2-yl)-2,3- dihydro-1-benzofuran-5-ol (1) and (2S,3R)-2-(prop-1-en-2-yl)-2,3-dihydro-1-benzofuran-3,5-diol (2).
Eight other coumaran derivatives were isolated and identified by careful comparison of their spectral data with those reported in the literature as tremetone (3) [START_REF] Banskota | Chemical constituents of Brazilian propolis and their cytotoxic activities[END_REF][START_REF] Bonner | Toxic constituents of white snakeroot[END_REF][START_REF] Lee | Tremetone and structurally related compounds in white snakeroot (Ageratina altissima): A plant associated with trembles and milk sickness[END_REF], 3-hydroxytremetone (4), 6-hydroxytremetone (5), 3,6-diydroxytremetone (6), 12-hydroxytremetone (7), 3-hydroxytremetol (8), megapodiol (9), and 6-hydroxy-megapodiol (10) [START_REF] Jarvis | Non-trichothecenes from Baccharis megapotamica[END_REF][START_REF] Spring | Sesquiterpene lactones and benzofurans in glandular trichomes of three Pappobolus species[END_REF]. According to optical rotation and comparison with data in literature, all the isolated 2-substituted dihydrobenzofuran derivatives were found to have a 2R absolute configuration and the 3-OH, 2-substituted derivatives a 2S,3R configuration, which is in accordance with previous literature reports [START_REF] Wang | New sesquiterpenes and benzofuran derivatives from the aerial parts of Asterothamnus centrali-asiaticus[END_REF]. A bibenzopyran derivative, jasopyran (11), was also obtained [START_REF] Ahmed | Two germacrane sesquiterpenes and a bibenzopyran from Jasonia montana[END_REF], together with four sesquiterpene lactones: costunolide (12) [START_REF] Rao | Costunolide, a new sesquiterpene lactone from costus root oil[END_REF][START_REF] Rao | Terpenoids. XXI. Structure of costunolide, a new sesquiterpene lactone from Costus root oil[END_REF], dihydrocostunolide (13), asperilin (14) [START_REF] Herz | Determination of the absolute configuration of hydroxylated sesquiterpene lactones by Horeau's method of asymmetric esterification[END_REF]Herz and Viswanathan, 1964), and grandulin (15). The structure of the latter was wrongly reported as a constituent of Inula grandis [START_REF] Nikonova | Grandulin and grandicin -new sesquiterpene lactones from Inula grandis[END_REF][START_REF] Nikonova | Structure of grandicin and grandulin[END_REF]. Therefore, the natural occurrence of grandulin is herein reported for the first time.

Compound 1 was isolated as colorless oil. Its UV spectrum presented maxima of absorption at 228 and 300 nm. Its molecular formula was determined to be C 11 H 12 O 2 on the basis of the observed peak at m/z 177.0909 [M+H] + , indicating six degrees of unsaturation. The 13 C and 1 H NMR data displayed typical signals of a 2,3-dihydrobenzofuran ring, confirmed by HMBC correlations. The proton and carbon signals are shown in Table 1. The aromatic ring was shown to be hydroxylated in para (δC 150.9), and the furan ring substituted in C-2 (δC 85.4) by a methylethenyl group composed of one olefinic quaternary carbon (δC 144.7), one olefinic methine (δC 110.4), and one methyl (δC 15.9). The methylene signal observed in DEPT and 1 H spectrum was assigned to position 3 (δC 34.8;δH 3.25,dd,J = 15.7,8.3 Hz;2.96,dd,J = 15.7,8.3 Hz). Thus compound 1 was identified as 2,3-dihydro-2-(1methylethenyl)-5-benzofuranol. This compound has been previously reported as a synthetic intermediate in the synthesis of coumaranol derivatives [START_REF] Schädel | Synthesis of coumaran-5-ols -new model compounds for bioantioxidants[END_REF], but is now reported for the first time as a natural product and named oparanol.

Compound 2 was obtained as slightly orange oil and presented a UV spectrum similar to the one of compound 1. Its molecular formula was deduced to be C 11 H 12 O 3 (m/z 191.0707, [M-H] -). The 1 H and 13 C NMR spectra of compound 2 were remarkably similar to those of compound 1, confirming a 5 hydroxyl-benzofuran skeleton substituted by a methylethenyl group in C-2 (see Table 1 for 13 C and 1 H data). The downfield values for C-3 (δC 76.6) indicated the presence of an hydroxyl group in position 3, confirmed by the absence of a methylene signal in the 1 H spectrum but the presence of an oxygenated methine signal at 5.02 (d, J = 4.3 Hz). The relative configuration was assumed to be trans on the basis of the small coupling constant observed between H-2 and H-3 (4.3 Hz), according to the Karplus equation and to literature [START_REF] Zalkow | On the absolute configuration of toxol at C-3. Vicinal H-H coupling constants in 2-alkyl-3-hydroxydihydrobenzofurans[END_REF]. Therefore, 2 was elucidated as 2,3-dihydro-2-(1-methylethenyl)-benzofuran-3,5-diol and named 3-hydroxy-oparanol.

All compounds were evaluated for their capacity to stimulate GLP-1 secretion in an in vitro model of STC-1 cells in culture (Table 2). The dihydrobenzofuran derivative 12-hydroxy-tremetone (7) was found to be the most active resulting in a 3-fold increase in GLP-1 secretion in STC-1 cells (305 ± 26%). Jasopyran (11) and costunolide (12) were also found to contribute to the bioactivity of the extract, as they stimulated GLP-1 secretion by 218 ± 41% and 156 ± 39% respectively. Furthermore, none of the isolated compounds affected STC-1 cells viability at the tested concentration, reinforcing their therapeutic potential. However, in order to confirm the potential of O. teikiteetinii compounds as antidiabetic agents, further in vitro and in vivo assays are required to identify mechanisms of actions and to assess lack of toxicity. As O. teikiteetinii is considered as a threatened species due to its extremely narrow distribution, alternative sourcing is mandatory. Chemical synthesis could be considered thank to the rather simple structure of active constituents [START_REF] Sangeeta | Synthetic routes and biological activities of benzofuran and its derivatives: a review[END_REF].

Also, alternative natural sources could be used. Compound 7, the most active constituent in our bioassay, has been previously reported from various Helichrysum species, already used for essential oil production [START_REF] Valverde Lopez | Benzofuran derivative isolated from Helichrysum stoechas[END_REF][START_REF] Zapesochnaya | Phenolic compounds of Helichrysum italicum[END_REF].

Supercritical CO 2 extraction from Helichrysum italicum flowers can be performed to yield an extract containing up to 23% of compound 7 [START_REF] Jerković | Optimization of supercritical CO 2 extraction of dried Helichrysum italicum flowers by response surface methodology: GC-MS profiles of the extracts and essential oil[END_REF], providing therefore another source to study in depth its therapeutic potential. This would enable the preservation of biodiversity while contributing to the development of potential new drugs for the management of type 2 diabetes.

Experimental

General experimental procedures

Optical rotations were measured on a Jasco P-2000 polarimeter (λ 589 nm, path length 1.0 cm). NMR experiments were conducted on a Bruker 500 MHz Avance II spectrometer equipped with a 13 C/ 1 H cryoprobe. HRESIMS analyses were carried out on an Agilent 6520 Q-ToF mass spectrometer.

Preparative HPLC was conducted on a Gilson 322 LC system using an Axia Phenomenex C18 column (100 mm × 21.2 mm). Mobile phase consisted of a mixture of acetonitrile (ACN) and water (H 2 O) containing 0.05% v/v formic acid at a flow rate of 25 mL/min. Vacuum liquid chromatography (VLC) was performed using 40-60 µm silica gel (Sigma Aldrich). Sephadex LH-20 (Sigma Aldrich) was used for gel filtration chromatography. Thin-layer chromatography (TLC) was performed on pre-coated silica gel plates (silica gel 60 F254, Merck). HPLC grade solvents were purchased from Sigma Aldrich.

Plant material

Extraction and isolation

The air dried leaves of Oparanthus teikiteetinii (153.9 g) were crushed and extracted by maceration with EtOAc (100 mL/50 g, 3 x 3 hours). The filtrates were combined and evaporated to obtain a crude EtOAc extract (28.15 g). The EtOAc extract was fractionated using vacuum liquid chromatography (150 g of silica 60 Å, 40-63 µm, 230-400 mesh), each fraction obtained by eluting with 300 mL of the following solvents: cyclohexane (c-Hex), c-Hex-CHCl 3 (1:1), CHCl 3 , CHCl 3 -EtOAc (75:25), CHCl 3 -EtOAc (1:1), EtOAc and CH 3 OH (500 mL) to afford seven fractions; F1 (0.71 g), F2 (0.66 g), F3 (6.86 g), F4 (14.18 g), F5 (2.47 g), F6 (1.09 g), and F7 (2.08 g). F3 (750 mg) was fractionated on Sephadex-LH 20 eluting with CHCl 3 /CH 3 OH (1:1) to obtain twelve sub fractions (F3S1-F3S12). Sub fraction S-10 (62.1 mg) was purified by preparative HPLC (20% ACN during 3 min, to 65% ACN in 12 min) to yield compound 12 (41.2 mg). Sub fraction S-11 (201.2 mg) was purified in the same conditions to afford compounds 3 (108.3 mg), 5 (24.9 mg), and 12 (40.9 mg). F4 (800 mg) was fractionated using Sephadex LH-20 CHCl 3 /CH 3 OH (1:1) to afford ten main sub fractions (F4S1-F4S10). F4-S9 (402.4 mg) was purified by preparative HPLC (20% ACN during 3 min, to 65% ACN in 12 min) to yield compounds 3 (91.9 mg), 12 (109.9 mg), and 13 (6.0 mg). Using the same conditions, compounds 1 (2.5 mg), 3 (64.8 mg), 5 (8.9 mg), 8 (2.5 mg), and 12 (10.7 mg) were obtained from sub fraction F4-S10 (133.9 mg). F5 (700 mg) was also chromatographed on Sephadex-LH 20 column eluting with CHCl 3 /CH 3 OH (30:70) to obtain eight sub fractions (F5S1-F5S8). Sub fraction F5-S7 (129.6 mg) was further purified by preparative HPLC (20% ACN during 3 min, to 65% ACN in 12 min) to afford compounds 7 (1.0 mg), 8 (5.5 mg), 11 (1.6 mg), 6 (1.0 mg), and 14 (2.6 mg). Sub fraction F5-S8 (38.7 mg) was also purified on Axia column (20% ACN during 3 min, 20 to 65% in 19 min, then 65 to 80% in 4 min) to yield 15 (17.0 mg), 9 (4.1 mg), and 8 (2.5 mg). F6 (800 mg) was first fractionated on Sephadex LH-20 eluting with CHCl 3 /CH 3 OH (30:70) to obtain seven sub fractions (F6S1-F6S7). Sub fraction F6S6 (120 mg) was subjected to preparative HPLC (35 to 65% ACN in 13 min, then to 100% ACN in 5 min, flow rate 15 mL/min) to afford compounds 2 (1.0 mg), 4 (8.0 mg), and 9 (23.1 mg). Sub fraction F6S7 (20.2 mg) led to compound 10 (1.1 mg) after preparative HPLC.

(2R)-2-(prop-1-en-2-yl)-2,3-dihydro-1-benzofuran-5-ol (1)

Colorless oil; [α] -102.5 (c 0.18, MeOH); UV (nm): 203, 228, 300; HRESIMS m/z 177.0909 [M+H] + , (calcd error -0.6 ppm); 1 H NMR and 13 C NMR data: see Table 1. , (calcd error -3.5 ppm); 1 H NMR and 13 C NMR data: see Table 1. 3.3.3. (3aR,8R,8aR,9aR)-8-hydroxy-5,8a-dimethyl-3-methylene-3a,4,4a,7,8,8a,9,9a-octahydronaphtho[2,3-b]furan-2(3H)-one (15) White amorphous powder; [α] +45.9 (c 0.10, MeOH); UV (nm): 215; HRESIMS m/z 249.1480

3.3.2. (2S,3R)-2-(prop-1-en-2-yl)-2,3-dihydro-1-benzofuran-3,5-diol (2)
[M+H] + , (calcd error -2.1 ppm); 1 H NMR and 13 C NMR data: see Table 1.

Bioassays

GLP-1 secretion assay

GLP-1 secretion assays were carried out as described previously [START_REF] Tsoukalas | Pregnane glycosides from Cynanchum marnierianum stimulate GLP-1 secretion in STC-1 cells[END_REF]. Briefly, STC-1 cells were cultured at 37°C under a 5% CO 2 atmosphere in high glucose (4.5 g/L) complete Dulbecco's modified Eagle's medium (DMEM, supplemented with 10% non-heat-inactivated fetal bovine serum (FBS), penicillin 100 IU mL -1 , and streptomycin 100 IU mL -1 ). Cells were passed at subconfluency every 3-4 days. Twenty-four hours prior induction, 10 4 cells per well in 100 μL complete low glucose (1 g/L) DMEM were seeded in a 96-well plate. Prior to treatment, cells were washed two times with HBSS (Thermo Fischer 14025092), after addition of 100 μL HBSS they were returned back in the incubator for 30 min. The samples DMSO stock solutions were diluted at 100 μM in HBSS containing a constant DMSO concentration of 1 %. Finally, 125 μL of the diluted solutions were applied to the cells for 2 hours at 37°C in a 5% CO 2 atmosphere. The GLP-1 content of 100 μL of the cell supernatant was then quantified in an ELISA assay (Merck Millipore, EGLP-35K) according to the manufacturer instructions. For each molecule the assay was performed in quadruplicate and results expressed as the percentage of the GLP-1 content secreted by cells treated with HBSS containing only 1% DMSO. For the statistical analysis, a one-way ANOVA with post hoc Dunnett test was performed (* p adj  0.05; *** p adj  0.001).

Cytotoxicity assay

For the image cytometry assay, the STC-1 cells after treatment with the molecules were incubated during 20 minutes with DMEM complete medium containing propidium iodide, Hoechst reagent, and calcein (1 µM each). After incubation, pictures of each well were taken with a Celigo Imaging Cytometer (Nexcelom Bioscience) at visible and fluorescent light and processed with the Celigo program in order to quantify the percentage of viable cells.

  Fresh leaves (1.75 kg) of Oparanthus teikiteetinii were collected in January 2015 in Nuku-Hiva, Marquesas Islands, French Polynesia, and identified by Dr Jean-François Butaud, botanist. The harvesting permit n°0923 was delivered from DIREN in November 2014. A voucher specimen (No CM1717_OT 01-2015) is deposited at the herbarium of the University of French Polynesia.

  Slightly orange oil; [α] -97.2 (c 0.18, MeOH); UV (nm): 203, 228, 302; HRESIMS m/z 191.0707 [M-H] -

Table 1 .

 1 1 H and 13 C NMR data for compounds 1, 2, and 15 (methanol-d 4 , δ in ppm, J in H , 500 MH for 1 H NMR, 125 MH for 13 C NMR).

			1		2		15
	position	δ C	δ H	δ C	δ H	δ C	δ H
	1					121.4	5.38 (m)
	2	85.4	5.07 (t, 8.3)	93.4	4.75 (d, 4.3)	32.1	2.38 (m) 2.33 (m)
	3	34.8	3.25 (dd, 15.7, 8.3) 2.96 (dd, 15.7, 8.3)	76.6	5.02 (d, 4.3)	77.4	3.64 (m)
	4	113.6	6.63 (d, 1.5)	111.6	6.81 (m)	40.7	-
	5	150.9	-	151.2	-	34.2	2.05 (m) 1.35 (m)
	6	111.6	6.52 (m)	116.6	6.69 (m)	82.1	4.04 (t)
	7	108.3	6.51 (m)	109.4	6.66 (m)	51.0	2.59 (br s)
	8	152.9	-	153.2	-	20.7	2.12 (m) 1.68 (m)
	9	127.3	-	128.7	-	50.8	2.42 (br d, 11.9)
	10	144.7	-	142.7	-	133.2	-
	11	110.4	5.04 (s) 4.86 (s)	110.7	5.06 (m) 4.89 (overlap)	139.6	-
	12	15.9	1.74 (s)	16.2	1.79 (s)	171.4	-
	13					115.6	6.03 (s) 5.52 (s)
	14					22.2	1.84 (s)
	15					9.96	0.89 (s)

Table 2 .

 2 Glucagon-like peptide-1 secretagogue activity and cell viability induced by compounds 1-15 (tested at 100 µM on STC-1 cells).Data are expressed as the mean ± SD of four independent experiments.

	Compound	GLP-1 secretion stimulation (%)	Cell viability (%)
	control	100 ± 10	96 ± 1
	7 a	305 ± 26	96 ± 1
	11 a	218 ± 41	97 ± 1
	12 b	156 ± 39	94 ± 1
	15	145 ± 15	96 ± 1
	1	121 ± 9	97 ± 1
	10	109 ± 11	95 ± 1
	14	104 ± 6	97 ± 1
	4	104 ± 16	91 ± 2
	13	102 ± 20	95 ± 2
	3	101 ± 20	96 ± 1
	9	100 ± 16	95 ± 1
	6	94 ± 9	96 ± 1
	2	93 ± 25	91 ± 2
	8	91 ± 13	96 ± 1
	5	81 ± 9	97 ± 1

a *** p adj < 0.001 b * p adj < 0.05 Figure 1. Structures of compounds 1-2 and 15.

Present address : Faculty of Traditional Thai Medicine, Prince of Songkla University, Hat Yai 90112, Thailand

Acknowledgments

This work was financially supported by the Foundation for the Development of the Chemistry of Natural Products and their Applications (CNRS/Academy of Sciences in Paris). We gratefully acknowledge Nicolas Humbert, Dr Christian D. Muller, and Dr Julien Godet for assistance with microplate reading, image cytometry, and statistical analysis, respectively.


Christophe Duplais

Nicolas Papon

Vincent Courdavault
email: vincent.courdavault@univ-tours.fr

Keywords:

HAL is

revealing the molecular mechanisms for the loss and re-emergence of iridoid biosynthesis in 1 the Nepeta lineage.

3

Iridoids are produced by several plant families and act as chemical defense against 4 herbivores and plant pathogens. In the Lamiaceae (approximatively 200 genera, 7,000 5 species) which is composed of seven major subfamilies, iridoids are widely distributed across 6 all the subfamilies but are absent in a single subfamily, i.e. the Nepetoideae (Fig. 1A).

7

Importantly, since iridoids are also present in the sister family of the Lamiaceae, i.e. the 8 Verbenaceae, it is likely that genes involved in iridoid biosynthesis have evolved from a 9 common ancestor while the capacity of producing iridoids has been lost in the clade of 10 Nepetoideae. However, there is a noteworthy exception because iridoids are found in the 11 Nepetoideae genus Nepeta. These plants are known as catmint or catnip, due to the euphoria-12 inducing effect of the nepetalactone iridoids on the behavior of felines. The intriguing 13 presence of iridoids in Nepetoideae thus raises the question are the same enzymes utilized or 14 are novel enzymes involved that have led to the re-emergence of the biosynthesis of these 15 metabolites (Fig. 1A).

16

The structural core of iridoids is a cisor trans-fused cyclopentanopyran. Unlike the 17 biosynthesis of terpenoids generally resulting from the cyclization of a linear terpene 18 carbocation by terpene synthase (TS), the key step to form the iridoid bicycle is a reductive 19 cyclization of 8-oxogeranial by iridoid synthase (ISY) followed by either a Diels-Adler reaction 20 or a Michael addition (6). ISY was originally discovered in the Madagascar periwinkle 21 (Catharanthus roseus, Apocynaceae), a major source of anticancer drugs derived from iridoid 22 monoterpene indole alkaloids (6,7). Interestingly, the sequence and crystal structure reported 23 from the Madagascar periwinkle showed that ISY is not similar to TS but more closely related of the re-emergence of ISY supports the hypothesis that an ancestral enzyme with a minor 2 side ISY activity gradually evolved into a novel iridoid biosynthetic enzyme with high ISY 3 activity, following gene duplication and selection (Fig. 1C).

4

The tour de force was to compare the evolution and diversification of NEPS with the 5 emergence of ISY activity and to elucidate the chronology events leading to the assembly of 6 the nepetalactone gene cluster. By comparing PRISE and NEP chronograms, the authors 7 predicted that the gain of the most recent common ancestor of the NEPS gene was 8 concomitant to the second gene duplication of the ISY ancestor (around 25 Ma ago), while the 9 NEPS family expansion occurred at the same time that ISY relative activity dramatically 10 increased and P5R is was lost (20 to 9 Ma ago). Ultimately, a dispersed duplication 11 event allowed ISY to integrate into the NEPS locus while the original copy at the PRISE locus 12 was pseudogenized since redundant (9 Ma ago, today). These discoveries strongly suggest 13 that ISY and NEPS catalytic activities have coevolved with strong interplay between 14 corresponding genomic regions (Fig. 1C).

16

The evolution of iridoids in Lamiaceae thus stands out as a fascinating example for tracing

24

  to the PRISE (progesterone 5-reductase/ISY) enzyme family, a short-chain NADPH-25 dependent dehydrogenase (8,9).26To understand the evolution of ISY in aromatic plants, a novel chemical-genomic-27 phylogenetic approach using the transcriptomes of 48 Lamiaceae species was recently 28 published (10). Unexpectedly, the functional validation of these candidates revealed that 29 although ISY activates 8-oxogeranial to give an enolic intermediate it does not catalyze the 30 consecutive cyclization into nepetalactone (11). Instead, the newly identified class of 31 nepetalactol-related short-chain dehydrogenase enzymes (NEPS) achieves the cyclization of 32 the reactive intermediate and controls the stereoselectivity of the outcome products (12). 33 Furthermore, biosynthetic gene clusters composed of ISY and NEPS were identified by mining 34 Nepeta genomes (5). Interestingly, these clusters also include major latex protein-like genes 35 (MLPL) whose biochemical characterization showed they react in a similar manner to NEPS to 36 form cis-trans nepetalactone stereoisomer (Fig. 1B). 37 38 With these key pieces in hand, Lichman et al. investigated the loss and re-emergence of 39 iridoid biosynthesis during the evolution of Nepetoideae. Firstly, genome resources of Nepeta 40 cataria and Nepeta mussinii were compared with those of the iridoid non-producer Hyssopus 41 officinalis (indicated by a red asterisk in Fig. 1A) and confirmed that the absence of iridoids 42 results from the loss of the ISY gene in this last species as in other Nepetoidae for which 43 omics data are available. Surprisingly, it also directly correlates nepelactone biosynthesis to a 44 regain of ISY in N. cataria and N. mussinii. Furthermore, phylogenetic analyses clearly indicate 45 that ISY genes of Nepeta form a distinct clade in the Lamioideae subfamily, which strongly screening of in vitro activities of extant and predicted ancestral PRISE. Overall, the key finding 1

  17the origins of plant metabolites and the re-emergence of their biosynthesis in Nepeta. The 18 present study provides unprecedented insights suggesting that this phenomenon relies on 19 repeated and innovative evolution further widening our knowledge of the production of 20 nepetalactones compared to iridoids in the rest of the mint family. The proposed chronology of 21 enzyme selection and diversification also suggests that the formation of gene clusters may not 22 drive metabolic innovation but rather organize following enzyme evolution under strong initial 23 pressure. Now highlighted, this twisted evolutionary story raises new puzzling questions 24 ranging from the role of protein-protein interactions between ISY with NEPS/MLPL during the 25 stereoselective formation of nepetalactones, to the biotic and abiotic factors responsible for 26 the co-evolution of ISY and NEPS. Additionally, since iridoids also occur across different insect 27 taxa, the comparison of plant and insect iridoid synthases is a crucial entry point to address 28 the evolutionary convergence for producing these common compounds (13). The evolution of 29 iridoids represents a remarkable model to further study enzyme and metabolite evolution 30 across the tree of life.

  Evolutionary Genomics Consortium (2018). Phylogenomic mining of the mints reveals multiple mechanisms 1 contributing to the evolution of chemical diversity in Lamiaceae. Mol. Plant. 11, 1084-1096.

2 11 .

 11 Sherden NH, Lichman B, Caputi L, Zhao D, Kamileen MO, Buell CR, O'Connor SE. (2018) Identification of iridoid 3 synthases from Nepeta species: Iridoid cyclization does not determine nepetalactone stereochemistry. Phytochemistry 4 145, 48-56. 5 12. Lichman BR, Kamileen MO, Titchiner GR, Saalbach G, Stevenson CEM, Lawson DM, O'Connor SE. (2019) Uncoupled 6 activation and cyclization in catmint reductive terpenoid biosynthesis, Nat. Chem. Bio. 15, 71-79.

7 13 .

 13 Beran F, Köllner T, Gershenzon J, Tholl D. (2019) The chemical convergence between plants and insects: biosynthetic 8 origins and functions of common secondary metabolites. New Phytologist 223, 52-67.

9 14 .Figure 1 .

 141 Figure caption 15Figure 1. Investigating the molecular basis of the re-emergence of iridoid biosynthesis in the Nepeta lineage.

cyclization of the reactive intermediate and controls the stereoselectivity of the outcome products. (C) Proposed chronology

suggests a distinct and parallel evolution of ISYs. Secondly, the evolution of ISY in Nepeta was 47 assessed using ancestral sequence reconstruction to infer the PRISE phylogeny. This of events that have likely occurred in Nepeta for the re-emergence of nepetalactone biosynthesis.


Ajaya K Nayak

Vivek Kumar

Peter Werner

Eckhard Pippel

Roshnee Sahoo

Franҫoise Damay

Ulrich K Rößler

Claudia Felser

Stuart S P Parkin

Discovery of Magnetic Antiskyrmions Beyond Room Temperature in Tetragonal Heusler Materials

Skyrmions, topologically stable spin textures, are of great interest for new generations of spintronic devices 1-3 . In general, the stabilization of skyrmions has been achieved in systems with broken inversion symmetry, where the asymmetric Dzyaloshinskii-Moriya interaction (DMI) modifies the uniform magnetic state to a swirling state 4,5 . Depending on the crystal symmetries, two distinct types of skyrmions, Bloch 5,6 and Néel types 7 , have been observed experimentally. Here, we present, the experimental manifestation of a special type of spinswirling, namely antiskyrmions, in a family of acentric tetragonal Heusler compounds with D 2d crystal symmetry. A spiral magnetic ground-state, which propagates in the tetragonal basal plane, is transformed into a skyrmion lattice-state under magnetic fields applied along the tetragonal axis over a wide temperature interval. Direct imaging by Lorentz Transmission Electron Microscopy (LTEM) shows field stabilized antiskyrmion lattices and isolated antiskyrmions between 100 K and 400 K, and zero-field metastable antiskyrmions at low temperatures.

Skyrmions are topologically stable vortex-like objects with a swirling spin configuration which

have recently been observed in several bulk materials and thin films [START_REF] Rossler | Spontaneous skyrmion ground states in magnetic metals[END_REF][START_REF] Muhlbauer | Skyrmion Lattice in a Chiral Magnet[END_REF][START_REF] Yu | Real-space observation of a two-dimensional skyrmion crystal[END_REF][START_REF] Kézsmárki | Neel-type skyrmion lattice with confined orientation in the polar magnetic semiconductor GaV4S8[END_REF][START_REF] Yu | Near room-temperature formation of a skyrmion crystal in thin-films of the helimagnet FeGe[END_REF][START_REF] Seki | Observation of Skyrmions in a Multiferroic Material[END_REF][START_REF] Tokunaga | A new class of chiral materials hosting magnetic skyrmions beyond room temperature[END_REF][START_REF] Milde | Unwinding of a Skyrmion Lattice by Magnetic Monopoles[END_REF][START_REF] Heinze | Spontaneous atomic-scale magnetic skyrmion lattice in two dimensions[END_REF][START_REF] Moreau-Luchaire | Additive interfacial chiral interaction in multilayers for stabilization of small individual skyrmions at room temperature[END_REF] . Skyrmions are potential candidates for new generation spintronic devices [START_REF] Jonietz | Spin Transfer Torques in MnSi at Ultralow Current Densities[END_REF][START_REF] Schulz | Emergent electrodynamics of skyrmions in a chiral magnet[END_REF][START_REF] Fert | Skyrmions on the track[END_REF] , such as a novel high density, solid-state storage memory device -Racetrack Memory [START_REF] Parkin | Magnetic domain-wall racetrack memory[END_REF][START_REF] Yang | Domain-wall velocities of up to 750 ms -1 driven by exchange-coupling torque in synthetic antiferromagnets[END_REF][START_REF] Parkin | Memory on the Racetrack[END_REF] . The existence of skyrmions was predicted in extended magnets with acentric crystalline structures more than 25 years ago [START_REF] Bogdanov | Thermodynamically Stable Vortexes in Magnetically Ordered Crystals -Mixed State of Magnetics[END_REF][START_REF] Bogdanov | Thermodynamically stable magnetic vortex states in magnetic crystals[END_REF] . In these magnets, the magnetization, twisted by relativistic spin-orbit couplings in the magnetic ground-state state, is usually a one-dimensional helix, a Dzyaloshinskii spiral. Evidence of the existence of skyrmions emerged from experiments on a small class of chiral magnetic systems with broken inversion symmetry, where they were discovered only in a very narrow temperature-magnetic field region near the magnetic ordering temperature of their magnetic phase diagram [START_REF] Muhlbauer | Skyrmion Lattice in a Chiral Magnet[END_REF] . Later the skyrmionic phase was stabilized over a wider temperature range in thin plates of certain chiral magnets [START_REF] Yu | Real-space observation of a two-dimensional skyrmion crystal[END_REF][START_REF] Yu | Near room-temperature formation of a skyrmion crystal in thin-films of the helimagnet FeGe[END_REF] .

Depending upon the spin rotation and by analogy to the two fundamental types of Bloch and Néel domain walls, two distinct types of Bloch and Néel skyrmions have been observed experimentally to date. Here we report, using Lorentz transmission electron microscopy, the observation of a new type of skyrmion, an antiskyrmion, in a Mn-Pt-Sn inverse Heusler compound. The antiskyrmions exist over a wide temperature range even above room temperature both as isolated objects and in ordered lattices. anti-skyrmion, and (c) Néel skyrmion. The corresponding helimagnetic structures from which these various skyrmionic states emerge are shown schematically in Fig. 1d,e, andf. Chiral magnets with the cubic B20 crystal structures with T symmetry display Bloch type skyrmions 5,6,8- 10 . Bloch skyrmions imaged using Lorentz transmission electron microscopy (LTEM) should show a ring type pattern (Fig. 1g). Unlike Bloch skyrmions, a cross-sectional view of the spin arrangement in a Néel skyrmion represents a cycloid, where spins rotate in a plane along the propagation direction (Fig. 1f). In the case of Néel skyrmions viewed in LTEM, the deflected electrons make a closed loop, hence, no intensity modulation is expected (Fig. 1I). Polar magnets with C nv symmetry [START_REF] Kézsmárki | Neel-type skyrmion lattice with confined orientation in the polar magnetic semiconductor GaV4S8[END_REF] and multilayer films with interfacial DMI host Néel skyrmions [START_REF] Heinze | Spontaneous atomic-scale magnetic skyrmion lattice in two dimensions[END_REF][START_REF] Moreau-Luchaire | Additive interfacial chiral interaction in multilayers for stabilization of small individual skyrmions at room temperature[END_REF][START_REF] Boulle | Room-temperature chiral magnetic skyrmions in ultrathin magnetic nanostructures[END_REF][START_REF] Woo | Observation of room-temperature magnetic skyrmions and their currentdriven dynamics in ultrathin metallic ferromagnets[END_REF][START_REF] Jiang | Blowing magnetic skyrmion bubbles[END_REF] .

Antiskyrmions were theoretically predicted in certain tetragonal materials with acentric crystal structures and D 2d symmetry [START_REF] Bogdanov | Thermodynamically Stable Vortexes in Magnetically Ordered Crystals -Mixed State of Magnetics[END_REF][START_REF] Bogdanov | Thermodynamically stable magnetic vortex states in magnetic crystals[END_REF][START_REF] Bogdanov | Magnetic structures and reorientation transitions in noncentrosymmetric uniaxial antiferromagnets[END_REF][START_REF] Koshibae | Theory of antiskyrmions in magnets[END_REF] . Cross-sectional schematics along four different directions reveal both helicoid and cycloid spin propagations (see Fig. 1b ande) in an antiskyrmion. This unique rotation of the spin is expected to result in a distinct LTEM pattern as shown in Fig. 1h. In the present spin notation, the transmitted electron beam will converge towards the skyrmion center in the vertical direction, whereas, it will diverge in the horizontal direction.

Therefore, a single antiskyrmion is expected to form two bright and two dark spots in a LTEM. Although antiskyrmions were predicted some time ago 17,18,22,23 they have not yet been found as isolated objects nor as arrays. Reports in ref. [START_REF] Zhang | Creation of artificial skyrmions and antiskyrmions by anisotropy engineering[END_REF] in engineered Co/Pt multilayers of anti-skyrmions are rather simply achiral spin textures without skyrmionic properties. Similarly, complex spin textures in the B20 compound MnGe are simulated to be arrays of skyrmions and anti-skyrmions [START_REF] Tanigaki | Real-Space Observation of Short-Period Cubic Lattice of Skyrmions in MnGe[END_REF] but the symmetry of this compound only allows for simple skyrmions.

As the antiskyrmions break the cylindrical symmetry and carry a quadrupolar moment of the magnetostatic charges, their properties may differ from those of Bloch and Néel skyrmions. Thus, they are very interesting objects in their own right. In a broader perspective, acentric magnetic carrying skyrmions are of foremost interest for spintronic applications. A key requirement is a material with above room temperature magnetic ordering, the stability of skyrmion states over an extended temperature range, and the ability to integrate films of such materials into magnetic devices. Of especial importance is to control the magnetic anisotropy, which can influence the competition between a spiral magnetic state, skyrmionic textures, and collinear spin-structures. In this regard, magnetic materials with tetragonal crystal structures possess an advantage of unidirectional anisotropy for which they are widely used in spintronics. Indeed, Heusler compounds, X 2 YZ (where X, Y are transition metals and Z is a main-group element), are well known for their potential applications in spintronics, especially in tunneling based devices [START_REF] Jeong | Termination layer compensated tunnelling magnetoresistance in ferrimagnetic Heusler compounds with high perpendicular magnetic anisotropy[END_REF] . Mn 2 YZ based magnetic Heuslers provide a perfect platform for the design of anisotropic and acentric room-temperature magnets with flexible magnetic configurations [START_REF] Nayak | Design of compensated ferrimagnetic Heusler alloys for giant tunable exchange bias[END_REF][START_REF] Sahoo | Compensated Ferrimagnetic Tetragonal Heusler Thin Films for Antiferromagnetic Spintronics[END_REF][START_REF] Nayak | Large Zero-Field Cooled Exchange-Bias in Bulk Mn 2 PtGa[END_REF] .

The atomic order that forms an 'inverse Heusler' structure in these crystals type is acentric, whereas the higher symmetry in the cubic form does not favor a chiral inhomogeneous DMI. In many of these crystals a cubic to tetragonal transformation "switches on" the chiral inhomogeneous DMI. Recently, a non-collinear spin configuration was observed in the acentric tetragonal Heusler compound Mn 2 RhSn [START_REF] Meshcheriakova | Large non-collinearity and spin-reorientation in the novel Mn 2 RhSn Heusler magnet[END_REF] . Here we have focused on inverse tetragonal Heusler compounds in which we have replaced Rh by the heavier Pt to increase spin-orbit coupling and thereby DMI. However, we find that Mn 2 PtSn can not be stabilized without the introduction of significant Mn vacancies. A single phase material Mn 1.4 PtSn that crystallizes in an acentric tetragonal structure with space group 𝐼42𝑚 could be obtained with uniformly distributed Mn vacancies exclusively in the Mn-Pt NaCl type substructure [START_REF] Jamijansuren | Mn 3 Pt 2 Sn 2 -A vacancy stabilized Heusler-like compound[END_REF] . Additionally, we found that a small amount of Pd that was substituted in place of Pt enhances the tetragonality (supplementary information). Therefore, we focus on Mn 1.4 Pt 0.9 Pd 0.1 Sn in the present study. In this compound the direct magnetic exchange sets the basic magnetic configuration of the Mn moments in the unit cell: there are two distinct Mn sub-lattices, each with their own magnetic moment. The DMI twists this basic magnetic moment configuration along one given propagation direction, thereby giving rise to a long-range helimagnetic structure. However, the crystal structure of this material belongs to a crystal class with D 2d point symmetry. This ensures that the twisting of the ferromagnetic order takes place only in the tetragonal basal plane. For this symmetry, theoretical considerations have shown that a field applied close to the tetragonal c-axis always stabilizes a skyrmion-lattice state, as there is no competing conical spiral state in the magnetic phase-diagram [START_REF] Meshcheriakova | Large non-collinearity and spin-reorientation in the novel Mn 2 RhSn Heusler magnet[END_REF] . This is completely different from the acentric cubic helimagnets, where the applied field always favors conical helices propagating along the field direction [START_REF] Muhlbauer | Skyrmion Lattice in a Chiral Magnet[END_REF][START_REF] Yu | Real-space observation of a two-dimensional skyrmion crystal[END_REF] . Here we find from LTEM that indeed there exists a long range helimagnetic structure with a period that is larger than ~100 nm. The skyrmions appear as a lattice with application of magnetic field for temperatures up to 400 K, which is the magnetic ordering temperature for the present material.

The magnetization versus temperature M (T) data measured in a field of 0.1 T for Mn 1.4 PtSn and Mn 1.4 Pt 0.9 Pd 0.1 Sn are plotted in Fig. 2a. The parent compound Mn 1.4 PtSn exhibits a Curie temperature, T C , of about 400 K followed by a second transition to a state with a higher magnetization below ~160 K. This suggests that the change in temperature leads to a reorientation of the moments of the Mn sub-lattices. Neutron diffraction studies on powdered samples of Mn 1. 4 PtSn clearly demonstrate a considerable change in the intensity of the (101) and (004) ferromagnetic peaks around the spin-reorientation transition, signifying a change in the magnetic structure at this temperature (inset of Fig. 2a). As expected, Mn 1.4 Pt 0.9 Pd 0.1 Sn displays a similar T C as that of Mn 1.4 PtSn due to the equal number of valence electrons [START_REF] Graf | Simple rules for the understanding of Heusler compounds[END_REF] . However, the low temperature spin-reorientation transition is decreased to approximately 125 K in the Pd-doped sample.

The bright field transmission electron microscopy (TEM) study shows a martensitic-like twinned microstructure for Mn 1.4 Pt 0.9 Pd 0.1 Sn as shown in Fig. 2b Application of magnetic field along [001] modifies the helimagnetic stripes into skyrmions (Fig. 2f). It can be mentioned that in the following at all temperatures (except at 100 K) the skyrmion phase was better nucleated when the field was applied at some angle to [001] (see Fig. 4f). Once the skyrmion phase was stabilized with an oblique field the sample was rotated to apply field along

[001] direction and a stable antiskyrmion lattice was formed. It is also important to mention that as the temperature approaches T C , the skyrmion phase can be better nucleated even with applying field directly along [001].

For a close look at the nature of the skyrmions in the present system, an underfocused LTEM image of a single skyrmion taken at 0.29 T field applied parallel to [001] direction is displayed in Fig. 3a. Most interestingly, it exhibits two bright spots and two dark spots along [010] and [100] directions, respectively. The modulation of the contrast can be discerned from the appearance of two peaks with a dip in the middle for the line profile taken along [010] (lower inset) and two dips with a peak in the middle for the line profile taken along [100] (upper inset). This picture clearly matches to the expected LTEM pattern as shown in Fig. 1h, signifying the presence of antiskyrmions. A complete reversal of the contrast is observed in the overfocused LTEM image as shown in Fig. 3b. The formation of a hexagonal lattice of antiskyrmions is depicted in Fig. 3c.

A slight distortion of the lattice along [010] direction might be related to the presence of a small amount of in-plane field due to a slight misorientation (≈ ±3°) of the sample away from the exact [001] direction. This is because in our thin crystalline foils so-called bend-contours occur when observing the TEM samples at the pole position, which disturbs the visibility of skyrmions. At low in-plane fields the distortion is mostly in the [010] direction as the helix propagates along [100].

Due to the tetragonal D 2d symmetry it is expected that the skyrmions lattices undergo characteristic distortions in oblique fields, as their axis remains locked to the tetragonal axis, and the skyrmions are consequently distorted with the perfectly radial core shifting from the centers of the honeycomb lattice cells of the hexagonal densely packed skyrmion lattitce.

A theoretical simulation of the antiskyrmion lattice in a small oblique field is shown in Fig. 3d. It illustrates the skewed appearance of the skyrmions, which leads to a slight rectangular modification of the honeycomb lattice. The effect is easily understood, as the in-plane component of the magnetic field favors and broadens the quarter of the circulating magnetization along the field, at the cost of the opposite part. A complete distortion of the lattice can be seen in Fig 3e ,   when the magnetic field was applied at an angle of about 20° to [001] by rotating the sample along the [110] direction. In this case the skyrmions appear as elliptical shapes in the Lorentz micrographs with the center of the skyrmions strongly off-centered. The appearance of doubleskyrmion-like features results from the projection of the skyrmion tubes that are oriented obliquely to the illuminating electron-beam and to the applied field. As the rotation angle decreases from Fig. 3e to Fig. 3g, the original quadrupole-like internal magnetization distribution of the antiskyrmions is revealed. For rotation angle θ=8° strong bright spots can be seen at the upper half of the antiskyrmions (Fig. 3g), whereas, the strong bright spots appear at the lower half for θ= -5° (Fig. 3h). In both cases the antiskyrmion lattice exhibits a large distortion along [010]. Nearly symmetrical bright and dark spots with an almost hexagonal nature of the lattice can be seen in Fig. 3i, for an applied field of 0.29 T along [001] (within ±3° limit). A small increase in the field to 0.33 T perturbs the regular arrangement of the skyrmions in the lattice (Fig. 3j). This field corresponds to the stability limit of the equilibrium lattice phase. Owing to their topological stability, a great number of antiskyrmions remain as metastable excitations in the homogeneous field-polarized collinear state. For further higher fields antiskyrmions disappear from the relatively thinner region of the sample and only stabilize at the thicker region (Fig. 3k). Finally, the antiskymion lattice evolves into an array of single antiskyrmions, which disappear for fields above 0.49 T at room temperature (Fig. 3l).

Since Mn 1.4 Pt 0.9 Pd 0.1 Sn exhibits a T C of about 400 K, it is expected that the material should display skyrmions up to 400 K. Underfocused LTEM pattern taken at 350 K in presence of a field of 0.22 T shows a lattice of antiskyrmions (Fig. 4a). At higher temperatures the skyrmion phase can be stabilized at lower fields. Figure 4b shows antiskyrmions at 100 K for a field of 0.33 T applied along [001]. At temperatures below the spin-reorientation transition of the present material, it was difficult to nucleate an antiskyrmion lattice by applying magnetic field even at an angle to [001] after cooling down the sample in zero field. Therefore, the sample was field cooled to 100 K at 0.24 T and subsequently the field was increased or decreased to observe antiskyrmions at different fields (see supplementary information). The antiskyrmion lattice obtained after reducing the field to zero is depicted in Fig. 4c. At zero field each antiskyrmion can be seen with two symmetrical bright spots along [010], whereas, the black spots were smeared out with the background. The zero-field stabilized lattice of antiskyrmions likely is a metastable state, underlining the topological stabilization against the decay into the simple spiral 1D-modulated ground-state.

It is often found that the transition from the helical phase to skyrmions and subsequently to the field polarized state is visualized with the presence of a kink in the M (H) measurements [START_REF] Kézsmárki | Neel-type skyrmion lattice with confined orientation in the polar magnetic semiconductor GaV4S8[END_REF][START_REF] Li | Robust Formation of Skyrmions and Topological Hall Effect Anomaly in Epitaxial Thin Films of MnSi[END_REF] .

M (H) isotherms measured at different temperatures for the bulk polycrystalline Mn 1.4 Pt 0.9 Pd 0.1 Sn are shown in Fig. 4d. It can be clearly seen that M (H) curves for temperatures down to 150 K exhibit a smeared out (due to polycrystalline nature) kink type of behavior (the region marked by arrows). For temperatures below 100 K an unsaturated magnetic behavior is obtained for fields up to 1 T. The regions marked inside the arrows indicate the phase transition between various magnetic states. H-T phase diagrams mainly derived from the LTEM measurements are shown in Fig. 4e. As can be seen the skyrmions can be found for the complete temperature region between 400 K and 100 K (the lowest measurement temperature in the present case). The strength of the magnetic field where the antiskyrmions appear increases with decreasing temperatures. Thus, a large region in the H-T phase diagram with stable and possible metastable antiskyrmion-lattice states is realized in the whole temperature region. For temperatures below 200 K the antiskyrmion lattice remains stable even when the field is completely removed. The observation of a stable antiskyrmion phase in zero magnetic field at low temperatures in the field decreasing path can be attributed to the anti-site disorder created by the substitution of Pd in place of Pt in Mn 1.4 Pt 0.9 Pd 0.1 Sn. The anti-site disorder act as pinning centers that helps the antiskyrmions to be quenched to a relatively low field when the field is reduced to zero. Since the sample does not exhibit a substantial magnetic hysteresis (see supplementary information) it can be readily considered that the observed effect does not arise from the materials intrinsic hysteretic behavior.

The role of disorder is also discussed in the achievement of stable zero field skyrmions in field cooling process in a cubic magnet [START_REF] Karube | Robust metastable skyrmions and their triangular-square lattice structural transition in a high-temperature chiral magnet[END_REF] . However, the stabilization of antiskyrmions at lower fields even without any field cooling/quenching [START_REF] Oike | Interplay between topological and thermodynamic stability in a metastable magnetic skyrmion lattice[END_REF] process makes the present system distinct from other skyrmion system. Nevertheless, it is also for the very first time that skyrmions can be stabilized for a temperature up to 400 K in a bulk system.

While we have demonstrated the formation of antiskyrmions in Mn 1.4 PtSn and Mn 1.4 Pt 0.9 Pd 0.1 Sn, we anticipate that the tunable nature of the Heusler family will allow for the formation of a wide range of skyrmionic structures, by varying the number of valence electrons, the spin-orbit coupling, the symmetry of the crystal structure, and thereby the magnetocrystalline anisotropy and the magnetization. In particular, the presence of more than one magnetic sublattice in the family of Heusler materials makes it possible to tune the total magnetic moment to zero [START_REF] Nayak | Design of compensated ferrimagnetic Heusler alloys for giant tunable exchange bias[END_REF][START_REF] Sahoo | Compensated Ferrimagnetic Tetragonal Heusler Thin Films for Antiferromagnetic Spintronics[END_REF] , so as to potentially give rise to antiferromagnetic skyrmions. Such antiferromagnetic skyrmions are predicted to move in a straight line when moved by current in contrast to the curved motion of conventional skyrmions [START_REF] Barker | Static and Dynamical Properties of Antiferromagnetic Skyrmions in the Presence of Applied Current and Temperature[END_REF] . Interestingly, the quadrupole moment of the magnetostatic charge distribution in the antiskyrmions of acentric magnets allows the control of their motion by applying inhomogeneous magnetic fields.

Figure 1a ,

 1a Figure 1a,b and c illustrate the spin textures of three classes of skyrmions: (a) Bloch skyrmion, (b)

  Fig. 2c, additional contrast modulation is observed on the platelet with an [001] orientation in case

  skyrmion. E, For an antiskyrmion the magnetization rotates both as a transverse helix (within the dashed rectangles 1and 3 in b) and as a cycloid (within the dashed rectangles 2 and 4 in b). g, Schematic in-plane arrangement of the moments in a Bloch skyrmion (green arrows) and the Lorentz deflections of the transmitted electrons (shown in red arrows) to give a ring type LTEM pattern of the deflected electrons (lower part of the figure). h, In-plane spin arrangement of an antiskyrmion. In this case the spins deflect towards the center in the vertical direction, whereas the deflection is outward in the horizontal direction. The corresponding LTEM pattern with two bright spots and two dark spots is exhibited in the lower part of h. i, In-plane spin arrangement of a Néel skyrmion and the corresponding LTEM pattern.

Figure 2 .

 2 Figure 2. Structural and magnetic properties. a, Temperature dependence of low-field (0.1 T)

Figure 3 .

 3 Figure 3. Room temperature skyrmions in Mn 1.4 Pt 0.9 Pd 0.1 Sn. a, Under-focused LTEM image of a single antiskyrmion with field applied along [001] direction. The lower and upper insets show the intensity profiles of the in-plane magnetization along [010] and [100] directions. b, Overfocused LTEM image of the single antiskyrmion shown in a. c, Under-focused LTEM showing a hexagonal lattice of antiskyrmions. d, Theoretical calculation of an antiskyrmion lattice in an oblique field. e-h, Under-focused LTEM images of antiskyrmions taken in 0.24 T with different rotation angle (q) as shown by schematic diagram in the inset of e. i-l, Under-focused LTEM images of antiskyrmions taken at different fields applied along [001].

Figure 4 .

 4 Figure 4. Temperature dependence of antiskyrmions and phase diagram. Under-focused

Acknowledgements:

We thank Horst Blumtritt and Norbert Schammelt for their help in preparing TEM lamella for the present study. This work was financially supported by the ERC Advanced Grant No. (291472) "Idea Heusler".

Author Contributions:

A.K.N., C.F. and S.S.P.P. conceived the original idea for the project. A.K.N. performed Lorentz TEM investigations with help of E.P. and P.W. The bulk materials were synthesized by V.K and A.K.N.. F.D., R.S. carried out the powder neutron diffraction study. A.K.N. and R.S. performed magnetic measurements. A.K.N. and S.S.P.P. wrote the manuscript with substantial contributions from all authors.

Additional information:

Supplementary information is available in the online version of the paper. Reprints and permissions information is available online. Correspondence and requests for materials should be addressed to S.S.P.P. (stuart.parkin@mpi-halle.mpg.de).

Competing financial interests:

The authors declare no competing financial interests.