Akiko Sasaki

Yu Tsutsumi

Jean-Paul Amoureux

Accelerating high-resolution NMR of half-integer quadrupolar nuclei in solids: SPAM-MQMAS and SPAM-STMAS

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

Introduction

Quadrupolar nuclei (I > 1/2) represent more than 70% of NMRactive nuclei. Nevertheless, their use in NMR is limited owing to the presence of the quadrupole interaction which can cause a significant loss of sensitivity and resolution. In solid-state NMR of these nuclei, magic angle spinning (MAS) 1 is often employed to eliminate the first-order quadrupolar broadening and partly reduce the second-order one. For half-integer spin quadrupolar nuclei (I = 3/2, 5/2, 7/2 and 9/2), high-resolution NMR methods such as double rotation (DOR) 2 and dynamic angle spinning (DAS) 3 have been successfully implemented to obtain isotropic spectra. However, these methods require special probes with limited spinning frequencies. Multiplequantum MAS (MQMAS) 4 and satellite-transition MAS (STMAS) 5 experiments are two-dimensional (2D) correlation experiments that allow to obtain isotropic spectra under MAS conditions. In MQMAS, a symmetrical multiple-quantum (MQ) coherence is evolved in t 1 and correlated with the centraltransition (CT) coherence observed in t 2 , while in STMAS, a single-quantum satellite transition (ST) is evolved in t 1 and correlated with CT in t 2 . Both MQMAS and STMAS methods exploit the refocusing of the second-order quadrupolar broadening at t 2 = Rt 1 , where the constant R depends on the spin value and the order of coherences used. 6,7 A 2D Fourier transformation (FT) of such signals leads to a 2D spectrum where each species appears as a ridge lying along the slope of R. A projection onto an axis perpendicular to R yields an isotropic spectrum free of anisotropic quadrupole interaction while a cross section along the ridge retains the anisotropically broadened MAS line-shape. Alternatively, a shearing transformation produces a 2D spectrum in which the MAS spectrum lies horizontally along the direct dimension (F 2 ). The isotropic spectrum can then be obtained from a projection onto the indirect dimension (F iso ). Although MQMAS is widely used as a routine method owing to the ease in experimental set up, its intrinsically low sensitivity due to the use of the usually 'forbidden' multiple-quantum coherences is a limiting factor in many practical applications. On the other hand, despite its high sensitivity, STMAS is more challenging to perform than MQMAS because of its technical requirements, such as stable spinning frequency and accurate magic angle setting, mandatory to eliminate the first-order quadrupole broadening. 8 MQMAS and STMAS are in fact complementary as their comparison allows to detect the presence of motions close to the various observed species. 9 For the ease of a direct comparison of all MQMAS and STMAS spectra, a unified representation has been proposed, in which an identical ppm scaling is used for F iso . Please do not adjust margins

ARTICLE

In MQMAS and STMAS experiments, most often used pulse sequences are composed of three pulses: two hard pulses followed by a soft one. The initial hard pulse excites the coherence of interest (MQ or ST) and, after t 1 evolution, the second hard and the final soft pulses perform an effective conversion to observable CT coherences. Pulse sequences proposed for MQMAS and STMAS experiments to achieve absorptive line-shapes are of two categories: amplitudemodulated, which contains a z-filter, 11,12 or phasemodulated, 13,14 which is based on a full-echo acquisition. In the amplitude-modulated z-filter approach, a pair of symmetrical coherences are initially excited, evolved in t 1 and then converted to CT via a z-filtered p = 0 state selected by phase cycling. Frequency sign discrimination in t 1 is ensured by a suitable data treatment (States or TPPI). In the phasemodulated full-echo approach, on the contrary, only a single coherence pathway is selected by phase cycling. By the insertion of an appropriate interval () after the second hard pulse, the echo signal is shifted in the time domain so that whole echo signals are acquired for all t 1 increments. A dependent first-order phase correction gives rise to purely absorptive line-shapes. As a rule of thumb, the full-echo acquisition is often preferred for samples with long T 2  (e.g. crystalline compounds) to avoid the losses occurring during 2, whereas the z-filtered approach is often used for samples with short T 2  (e.g. amorphous or disordered materials). The problems of distorted line-shapes observed when using a single pathway have been discussed in detail in the case MQMAS experiments performed on a well-crystallized sample of RbNO 3 . 15 In the following we are concerned with the amplitude-modulated approach, which is by far the most used method.

Soft-pulse-added-mixing (SPAM)

A large variety of sensitivity enhancement techniques (RIACT, 16 FAM, 17,18 DFS, 19 FASTER, 20 HS, 21 RAPT, 22 and QCPMG-MQMAS

23

) have been proposed and proved useful in MQMAS and/or STMAS contexts, although their optimization process may sometimes be found time-consuming owing to the presence of multiple parameters that need to be controlled. In 2004 and 2005, Gan et al. 24 and Amoureux et al. O NMR. [25][26][27] To illustrate the SPAM method, pulse sequences and coherence transfer pathways of triple-quantum MQMAS and DQF (doublequantum filtered) STMAS 28 experiments are shown in Fig. 1 for z-filter and SPAM approaches. All the pulse sequences mainly consist of three pulses, one for excitation (p1) and two for conversion (p2 and p3), except that the DQF-STMAS includes an additional CT-selective -pulse (p4) immediately before the second hard pulse (p2) to remove unwanted autocorrelation peaks to simplify the spectral analysis without significant loss of sensitivity. 28 In conventional z-filter approaches (Fig. 1a,b), signals from a pair of symmetrical coherence transfer pathways, echo (E) and antiecho (AE), are simultaneously selected, evolved during t 1 and converted via the z-filtered p = 0 state so that the observed signal is a combination of E and AE signals with equal amplitudes, which consequently yield a 2D absorptive line-shape. For example, p = { 3  0  -1} is selected in z-filter MQMAS in Fig. 1a and p = { 1  2  0  -1} is selected in z-filter STMAS in Fig. 1b. In SPAM methods, on the other hand, symmetrical coherence transfer pathways are treated separately, and signals are acquired sequentially in an alternating manner. By properly setting the phase of the CTselective p3 pulse, a constructive addition of signals from all p = (+1, 0, -1) pathways can be achieved, resulting in an effective signal accumulation. For example, as shown in Fig. 1c-f, p = {3  (1, 0, -1)  -1} and p = {-3  (1, 0, -1)  -1} are selected in SPAM-MQMAS, and p = {1  2  (1, 0, -1)  -1} and p = {-1  -2  (1, 0, -1)  -1} are selected in SPAM-STMAS, respectively. Upon consideration of Wigner rotation matrix elements, Gan indicated a possible 70% gain in signal intensity by using these (1, 0, -1) pathways compared to that of the single p = 0 pathway. 24 Numerical simulations performed by Amoureux and co-workers revealed that the SPAM signal can be further enhanced, depending on the C Q value, and that a maximum enhancement of ca. 2 was obtained at C Q = 0.5-1 MHz for I = 3/2 and C Q = 1-4 MHz for I = 5/2. 25 It should be noted that, although SPAM requires separate acquisition of E and AE signals, the total number of t 1 increments to achieve the same isotropic resolution is identical in both z-filter and SPAM experiments. This is because, in the hyper-complex (States) data treatment used in z-filter experiments, each t 1 increment is in fact recorded twice, one with sine and one with cosine modulation, to ensure frequency discrimination in the indirect dimension. Therefore, for a given experimental time, SPAM can yield a signal enhancement of up to 2 owing to the effective accumulation of signals from p = (1, 0, -1) pathways.

It should be reminded that in the conventional methods, there is a z-filter delay of a few tens of s in between the second hard pulse (p2) and the last soft pulse (p3) to 'purge' the nondesired coherences not situated on the p = 0 state, and thus these two pulses are phase-cycled individually. With SPAM, on the contrary, these two pulses can be considered as a single composite pulse, 29 without any delay in between them, and they are thus phase-cycled globally. Since the main difference between z-filter and SPAM methods is the phase cycling that derives from the desired coherence pathways, the optimum pulse lengths are expected to be similar. This was demonstrated theoretically by Gan for p3 upon consideration of Wigner rotation matrices, and experimentally on 27 Al (I = 5/2) MQMAS signals of berlinite for p2 or p3. 24,26 In addition to the implementation of SPAM into MQMAS and STMAS experiments, Amoureux et al. also introduced an additional experimental trick, namely the "truncated SPAM" for the 2D acquisition of MQMAS and STMAS signals. 25 In the truncated SPAM, either only a part or no AE signals are acquired, which we denote, "partly" and "fully"-truncated SPAM, respectively. To illustrate this concept, a time-domain representation of E and AE signals experimentally recorded on 27 Al (I = 5/2) of berlinite and 23 Na (I = 3/2) of Na 2 SO 4 is shown in Fig. 2. It should be reminded that, by definition, the signal that moves forward in the t 2 acquisition window comes from the echo pathway, whereas the signal that moves backward and disappears from the t 2 acquisition window derives from the antiecho pathway. Therefore, for MQMAS the echo pathway during t 1 is p = -3 or +3 for I = 3/2 or  5/2, respectively. The coefficients relevant to MQMAS and STMAS experiments are summarized in Table S1 of the Supplementary Information. In a 2D time-domain representation, similar to that shown in Fig. 2, E and AE signals move along the opposite slopes of 1/R. For example, taking 27 Al (I = 5/2) MQMAS in Fig. 2a, |R| = 19/12 and the E and AE move along the slopes of 12/19 and -12/19, respectively. As the number of t 1 increments increases, eventually, there will be a point in t 1 at which AE signals are no longer present in the t 2 acquisition window and only noise is acquired. By skipping the AE acquisition beyond this point, the total experimental time required for 2D acquisition can be largely reduced. This is schematically shown in Fig. 3 where a series of FIDs is compared between z-filter (E + AE), SPAM (E = AE), partlytruncated SPAM (0 < AE < E) and fully-truncated SPAM (AE = 0), where we designate E and AE as the number of echoes and antiechoes, respectively. In z-filter (E + AE), E and AE signals are acquired simultaneously, whereas in SPAM (E = AE), they are acquired in an alternate manner. In partly-truncated SPAM (0 < AE < E), the AE acquisition is skipped beyond an arbitrarily point and a blank dataset is inserted in place of AE signals, while in fully-truncated SPAM (AE = 0), no AE signals are acquired. With truncated SPAM approaches, a sensitivity enhancement can be simply achieved by increasing the number of scans (NS) for echo signals proportionally to the number of antiecho signals being skipped. For example, for a fixed experimental time, if no antiecho signals are recorded at all (AE = 0), then NS can be simply doubled. If the number of antiechoes is set to a quarter of the number of echoes (E:AE = 4:1), then NS can be increased by 1.6, and so on. More generally, the multiplication factor for NS is given by 2E/(E+AE). It should be noted, however, that some dispersive parts may become visible in the 2D SPAM spectra when an insufficient number of AE are recorded. This may obscure the resonances and hinder an accurate spectral analysis, especially for well-crystalized samples presenting close narrow resonances and species with small C Q . 25 30 in which only an echo pathway is selected prior to population transfer via dipolar interaction (MQ-D-HETCOR) or J coupling (MQ-J-HETCOR). These high-resolution SPAM methods have been applied to 27 Al-31 P and 27 Al-29 Si through-space and through-bond HETCOR spectra and they provided a considerable sensitivity gain.

Despite its robustness and simplicity, the SPAM concept has not been widely applied since its introduction and only a few number of publications exist since then. 18,29,[31][32][33] This limited application of SPAM methods, is mainly due to three reasons: (i) solid-state NMR spectroscopists rarely use the echoantiecho method, (ii) the data treatment of the truncated SPAM methods was not easy to do before, and (iii) no simple rule for the best E:AE ratio was proposed. However, (i) this complexity of the data treatment is now completely overcome with the new NMR consoles, (ii) we provide in this article a simple rule for the E:AE ratio, and (iii) very simple and robust SPAM sequences are presently available (see Supplementary Information). Here, we further exploit the SPAM approach to accelerate the acquisition of MQMAS and STMAS spectra with respect to sensitivity. Its effectiveness and ease in experimental setup are demonstrated using both simulations and experiments performed on I = 3/2 ( Rb) and 9/2 ( 93 Nb) nuclei with a wide range of C Q values. It will be shown that sensitivity enhancements of up to 4 can be attained by SPAM-MQMAS and SPAM-STMAS without additional timeconsuming pulse optimization. Recent developments of highfield (B 0 ) spectrometers and fast MAS probes have potential to facilitate the solid-state NMR investigations of half-integer quadrupolar nuclei. In the context of SPAM, we also present a comparison of several experimental approaches with respect to repetition delay (RD) and spinning frequency ( R ), discussing the precautions upon making a judicious choice of highresolution NMR methods (MQMAS versus STMAS) for halfinteger quadrupolar nuclei in solids.

Methods

All experiments were performed using a Bruker Avance NEO spectrometer with a B 0 = 14. ) were packed as purchased or as synthesized. All MQMAS experiments were performed using the triple-quantum versions described in Fig. 1a,c and all STMAS ones with the DQF (double-quantum filtered) versions described in Fig. 1b,d. The coefficients of MQMAS and STMAS experiments and quadrupolar parameters of selected compounds are provided in Tables S1 andS2, respectively. Prior to any 2D signal acquisition, for each sample all pulse lengths were experimentally optimized using a 1D version that corresponds to either the first row or the first echo of the 2D acquisition, for z-filter or SPAM experiments, respectively. Chemical shift

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scales, shown in ppm, were referenced using the sample of interest itself as a secondary reference, and MQMAS and STMAS spectra were referenced after shearing transformation according to the unified representation. 10 Prior to any STMAS investigations, accurate adjustment of the spinning axis to the magic angle was performed using a DQF version of the split-t 1 shifted-echo pulse sequence on the sample of interest itself. 34 Further experimental details are given in the figure captions. We used the TS4.0.6 version of Topspin software. It must be noted that with previous versions, in the case of for I = 3/2 nuclei the minimum number of AE that can be acquired with SPAM is not 0, but 1.

All simulations were performed using the SIMPSON density matrix simulation program. 35 The variable input parameters were the nucleus of interest, spinning frequency ( R ), rf field strength ( 1 ), and the quadrupolar coupling constant (C Q ). The quadrupole interaction, with  Q = 0, was taken into account up to the second-order, without scalar or dipolar coupling. Input pulse programs and coherence selections were chosen to emulate the real MQMAS and STMAS sequences shown in Fig. 1 without any delay, except for the rotor-synchronized STMAS experiments (t 1min = T R -p1/2 -p4 -p2/2). All pulses were applied on the gravity center of the line-shape using  1 = 90-160 kHz for hard pulses and  1 = 5-20 kHz for CT-selective soft pulses. Input parameters intrinsic to powder averaging under MAS conditions (crystal file, number of -angles and maximum time step (t) over which the Hamiltonian is considered time independent) were tested for convergence, and a combination of ZCW20  10 -angles with t = 0.1 s was sufficient to produce a contour plot of signal intensity versus pulse lengths for the given range of C Q used in the following investigations. Since the imaginary part of the signal was found to be minor, only the real part of the signal is plotted as the signal intensity. Further computational details are given in the figure captions.

Results and discussion

4-1 Pulse length optimization

As previously discussed by Gan 24 and Amoureux, 26 the optimum pulse lengths for SPAM are expected to be close to those of z-filter. This has been verified using simulations. Na (I = 3/2) nuclei. Only the signal intensity of the echo pathway has been considered since it gives rise to the overall signal intensity, while the antiecho signal mainly contributes to cancel the residual dispersive part of the echo signals.

Upon comparison of contour plots between z-filter and SPAM, it can be observed that the optimum pulse lengths are identical in both MQMAS and STMAS approaches. For example, for 27 Al with C Q = 4 MHz, the optimum MQMAS pulse lengths for z-filter and SPAM are {p1, p2, p3} = {4.0, 1.4, 16.7} s, and similarly {p1, p2, p3, p4} = {1.8, 1.1, 16.7, 33.4} s for both z-filter and SPAM-STMAS approaches.

Since the optimum pulse lengths are identical with z-filter and SPAM, no additional experimental time is required for further optimization upon implementation of SPAM experiments. This makes SPAM a simple, efficient and userfriendly approach compared to other sensitivity enhancement techniques that might involve complicated and timeconsuming parameter optimization procedure.

4-2 Sensitivity enhancement and reduction in experimental time

Despite their robustness and simplicity, SPAM methods have not been widely used since their introduction and not many examples are available in the literature to demonstrate the effectiveness of SPAM as a sensitivity enhancement technique for MQMAS and STMAS acquisition. In the following subsections, we present a selected series of comparison of MQMAS and STMAS spectra of inorganic crystalline powder samples with various C Q values for I = 3/2 ( 23 Rb). We show that, for a given experimental time, a signal enhancement of 2 to 4 is easily achieved by SPAM compared to z-filter. In addition, although SPAM was originally developed for sensitivity gains of MQMAS and STMAS signals, it can readily be used for reduction of the experimental time to obtain an identical isotropic spectrum. In the following, three sets of MQMAS or STMAS experiments were compared: zfilter, SPAM (E = AE), and truncated SPAM (0  AE < E) in which the AE acquisition is partly or fully skipped. An identical combination of optimum pulse lengths was used in z-filter and SPAM, as anticipated from the simulations and confirmed experimentally. Rb (Rb 2 SO 4 ) STMAS spectra recorded using z-filter and SPAM approaches. The experimental time (T exp ), repetition delay (RD), and total number of t 1 increments (Nt1) were fixed, while the number of scans (NS) was the same in z-filter and SPAM (E = AE) but doubled in fully truncated SPAM (AE = 0 or 1) to amount to the same T exp . Compared to zfilter, the signal intensity of SPAM (E = AE) is twice as large, as anticipated by the previous numerical simulations 25 and our simulated contour plots in Fig. 4. This signal enhancement originates from the constructive addition of (+1, 0, -1) coherences intrinsic to the SPAM concept. With the fully truncated SPAM (AE = 0), a signal enhancement of 4 can be observed with respect to z-filter. This gain can be decomposed as 2  2, where the first factor of 2 comes from the constructive addition of (+1, 0, -1) coherences intrinsic to SPAM and the second is related to the doubling of NS to compensate for the time saved by skipping the AE acquisition. For example, in the case of 27 Al MQMAS of AlPO 4 -14 in Fig. 5a, given T exp , a signal enhancement of 2 is attainable by SPAM (E = AE) and of 4 by fully truncated SPAM (AE = 0).

4-2-1 Sensitivity enhancement

4-2-2 Reduction in experimental time

Although SPAM was originally developed to increase the intensity of MQMAS and STMAS signals, it can also be used to reduce T exp to obtain an identical isotropic spectrum. Figs. 5c,d show the isotropic projections of 27 Al MQMAS of berlinite or 23 Na STMAS of NaH 2 PO 4 , and the experimental times to achieve such identical isotropic resolutions. With respect to zfilter, SPAM (E = AE) and fully truncated SPAM (AE = 0) can be twice and four times faster, respectively. For example, in the 27 Al MQMAS case of berlinite (Fig. 5c), to obtain the same isotropic projections, recording the z-filter took 4 hrs (24 scans  200 t 1 increments), SPAM (E = AE) 2 hrs (12 scans  (100 E  100 AE)), and truncated SPAM 1.2 hrs (12 scans  (100 E  20 AE)). This considerable time-saving advantage of SPAM, as well as the associated sensitivity gain, are particularly useful when the isotropic signal acquisition via MQMAS or STMAS is combined with other experiments such as hetero-nuclear correlation experiments.

4-3 Analysis of SPAM 2D spectra

So far, only the isotropic 1D projections were used to demonstrate the advantages of SPAM over z-filter. However, as the integrated surface of the dispersion is always zero, these 1D isotropic projections are immune to any dispersive signal. This is not the case of the SPAM 2D spectra. The E signal is directly related to the intensities of the 2D line-shapes, while the AE signal cancels the dispersive parts of the resonances to achieve absorptive line-shapes. 25 This implies that, when an insufficient number of AE signals is acquired, some dispersive parts may become apparent in the 2D SPAM spectra, possibly hindering an accurate spectral analysis. However, it has been shown experimentally that only a small number of AE is required to minimize the dispersion of distributed samples such as glasses, 26 while a significant number of AE may be necessary for crystalline materials. 25,29 This presence of dispersion is systematically illustrated in Fig. 6 using 85 Rb MQMAS and STMAS spectra of RbNO 3 . In z-filter STMAS (Fig. 6a), three Rb sites, with Rb1 and Rb3 overlapping, are clearly present, whereas in z-filter MQMAS (Fig. 6d), the Rb2 site with the largest C Q is barely visible. In the isotropic projection of these spectra, the signal intensity of STMAS is approximately 5 times higher than that of MQMAS. In SPAM (E = AE) spectra (Fig. 6b,e), the signal intensity is doubled with respect to that of z-filter, as expected. In fully truncated SPAM spectra (Fig. 6c,f), the signal intensity is scaled up by a factor of 4 as NS was doubled. In the fully truncated SPAM-STMAS spectrum (Fig. 6c), however, the dispersive tails are significantly visible above the noise due to the lack of cancellation by AE signal. This is not the case with the fully truncated SPAM-MQMAS spectrum (Fig. 6f), in which the dispersive tails are just above the noise level. This is related to the fact the dispersion is proportional to the signal intensity and that STMAS exhibits a ca. five times higher signal intensity than MQMAS for this compound.

A question that arises here is, "is it possible to find a ratio of the number of echo and antiecho (E:AE), in between 1:1 and 1:0, which simultaneously minimizes the dispersion and maximizes the intensity even for species with small C Q in well-crystallized samples?" In order to answer this question, a set of SPAM (E = AE) spectra were recorded for wellcrystallized samples containing nuclei with I = 3/2 ( 23 Rb) with various C Q values. Starting from the experimental SPAM (E = AE) dataset, a series of truncated SPAM ensembles with arbitrary E:AE ratios were then created by zero-filling the data points that correspond to the skipped AE signals. This processing is schematically illustrated in Fig. S1, using an experimental SPAM dataset with E = AE = 50 as an example. The series of time-domain SPAM datasets with arbitrary E:AE ratios were then Fourier-transformed and the resulting 2D spectra were analyzed qualitatively (Fig. 7), by the examination of the spectral appearance, and quantitatively (Fig. 8), by calculating the ratio between negative and positive integrals over an arbitrary area of 2D spectra. Fig. 7 shows two examples of qualitative examination of the spectral appearance of a series of 2D SPAM spectra of wellcrystallized samples with arbitrary E:AE ratios. In 27 Al SPAM-MQMAS spectra of AlPO 4 -14, the large C Q sites (Al2 and Al3) are already absorptive even with AE = 0, while the small and moderate C Q sites (Al1 and Al4) give rise to dispersive tails.

With AE = 16 (E:AE = 8:1), the moderate C Q site (Al4) becomes absorptive and eventually all sites are absorptive with AE = 32 (E:AE = 4:1). Similarly, in 87 Rb SPAM-STMAS spectra of Rb 2 SO 4 , the larger C Q site (Rb2) is already absorptive even with AE = 1, and as the number of AE increases, both sites become absorptive with AE = 19 (E:AE = 4:1). From this spectral appearance of well-crystallized samples, one deduces that fully-truncated SPAM can be used for large C Q sites. On the contrary, for small C Q sites, truncated SPAM with E:AE  4:1 is better to minimize the dispersion and maximize the signal intensity simultaneously.

As for the quantitative analysis, Fig. 8 summarizes the ratio of negative to positive integrals (/) of the resonances plotted versus the ratio of the number of AE to that of E (AE/E) for various C Q values. The integrals were calculated over an arbitrary area of 2D spectra, and they are assumed proportional to dispersion and signal intensity, respectively. The convergence was tested for I = 5/2 ( 27 Al and 85 Rb) and I = 3/2 ( 87 Rb and 23 Na). It can be seen that the ratio of negative to positive integrals decreases as the ratio AE/E increases from zero and it is minimum at E:AE = 4:1. There is no change in this ratio (/) when AE > E/4, i.e. no further minimization of dispersion. This is in good agreement with the qualitative analysis made on the examination of spectral appearance of 2D SPAM spectra (Fig. 7).

Based on the qualitative and quantitative analyses made in this section, the use of truncated SPAM with E:AE = 4:1 is recommended for small C Q sites with well-crystallized samples, whereas that of fully truncated SPAM seems to be possible for large C Q sites. This potential use of AE = 0 may look surprising

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This journal is © The Royal Society of Chemistry 20xx because of the dispersive components that exist in the 2D lineshapes. However, it must be first reminded that for each crystallite, the dispersive component corresponds to a 2D signal with positive and negative parts. This means that in the case of a powder sample, these dispersive components cancel each other's in the central part of broad 2D resonances. This cancellation process cannot fully occur in the case of narrow resonances or on the external parts of broad resonances. However, it must be remembered that skipping all AE allows doubling the signal with respect to the case "E = AE". Most of the time the spectrometer time is limited, and it may be interesting to divide by 2 a long acquisition time, even if this introduces a small amount of dispersion. It is important to remind that complete MQMAS and STMAS 2D line-shape simulations are very difficult to perform. Indeed, to be accurate these simulations should have to take care of numerous parameters such as: (i) distribution of surroundings, (ii) rf-field inhomogeneity, (iii) pulse transients, (iv) Q of the probe, and (v) fluctuations of spinning-speed and magicangle in the case of STMAS! To demonstrate the small distortions afforded with a limited number of AE in the case of a moderately distributed sample, we have recorded the 27 Al SPAM-MQMAS 2D spectrum of -alumina, with AE = E = 25, and we have treated the data a posteriori with a decreasing number of AE. The results, shown in Fig. 9, demonstrate that no spectral distortion is observed when E:AE  6:1.

As a conclusion, we recommend using the ratio E:AE = 4:1, which simultaneously minimizes the dispersion and maximizes the intensity in all types of samples (well-crystalized or not) and C Q values (small or large).

4-4 Detection of minor signals:

23 Na (I = 3/2: NaVO 3 )

In the previous section, we demonstrated the sensitivity advantage of truncated SPAM (E:AE = 4:1). We should note, however, that this advantage is only effective when a sufficient number of echoes is recorded, i.e. the echo signals decayed completely in t 1 , as for all other MQMAS and STMAS methods.

A question may arise: what is the best sequence for the detection of minor quadrupolar signals under high resolution? This problem is analyzed in Fig. 10 showing 23 Na spectra of commercial NaVO 3 . This sample is known to consist of -and -NaVO 3 polymorphs, 36 and from the MAS line-shape shown in -g. It can be seen that the isotropic signal intensities of minor signals increase from z-filter to SPAM (E:AE = 4:1) by a factor of ca. 4, as expected. However, a significant baseline distortion is introduced in the isotropic projection. This distortion, is intrinsic to STMAS due to the rotor-synchronized non-zero evolution time: t 1min = T R -p1/2 -p4 -p2/2. It is also at the origin of the negative green parts in the 2D spectra of Fig. 10c andd and being proportional to the signal is hence doubled in Fig. 10d compared to Fig. 10c. These dispersive parts may be cancelled afterward by using a linear prediction. However, one also observes that the detection of these minor species remains easy on the isotropic projection (Fig. 10g). We should note that the use of MQMAS to detect these minor signals is very inefficient as its signal intensity was found four times lower than that of STMAS for this sample.

4-5 Comparison of experimental approaches

The recent development of high-field spectrometers and fast MAS techniques has made solid-state NMR investigations of half-integer quadrupolar nuclei more and more accessible, owing largely to the intrinsic increase in sensitivity at high B 0 fields and a possible resolution enhancement by the narrowing effect of MAS line-width under fast MAS conditions. When a given sample of interest is suspected to possess overlapping multiple sites with possibly distributions of chemical shifts or quadrupolar couplings, it has become routine for solid-state NMR users to record high-resolution spectra.

For a finite spectrometer time, NMR spectroscopists are often required to make a strategic decision on hardware, methods and parameters to be used. While STMAS requires an accurate orientation for the rotor axis and a stable spinning speed to cancel the 1 st -order quadrupole interaction, the intrinsic insensitivity of MQMAS may demand a large number of scans (NS) and hence a long experimental time (T exp ). Fast MAS conditions may narrow the CT MAS linewidth of nuclei subject to large quadrupolar interactions and hence may increase the resolution between overlapping sites. However, the sample volume is then inevitably decreased, and the rotorsynchronized MQMAS and STMAS acquisitions may require a larger number Nt 1 of increments and hence a longer T exp . Furthermore, the choice of the repetition delay (RD) may affect the relative intensities if multiple sites with different T 1 are present. For a given spectrometer time, therefore, an interplay between the choice of (i) method, (ii) spinning frequency, (iii) repetition delay, (iv) number of scans and (v) number of t 1 increments, controls the quality of highresolution spectra of half-integer quadrupolar nuclei.

In the following, a comparison of experimental approaches is made in the context of 23 Na and 87 Rb (I = 3/2) SPAM-MQMAS and SPAM-STMAS experiments with respect to repetition delay and spinning frequency, aiming to discuss the precautions upon making a judicious choice of high-resolution NMR methods of half-integer quadrupolar nuclei. Also, 93 Nb (I = 9/2) NMR of previously studied compounds is revisited to demonstrate the applicability of MQMAS and STMAS experiments to this nucleus in the context of SPAM approaches.

4-5-1 Repetition delay:

23 Na (I = 3/2: sodium citrate dihydrate)

So far, only the absolute signal intensity of the indirect isotropic projection was considered to demonstrate the SPAM enhancement, on assumption that the t 1 noise is insignificant compared to the signal intensity. In the direct dimension, however, the thermal noise makes a significant contribution. A comparison of the theoretically expected maximum of S/N ratios of z-filter and SPAM approaches is summarized in Table Na MQMAS and STMAS investigations. In Fig. S2, we show a signal recovery profile of 23 Na MAS spectra of sodium citrate dehydrate versus the repetition delay (RD). From this profile T 1  1.5 s, and we expect that 50 and 97 % recoveries are ensured with RD = 0.5 and 5 s, respectively. For a given spectrometer time, we have set up 23 Na MQMAS and STMAS experiments with RD = 0.5 and 5 s, as summarized in Table 2. It should be remembered that the number of t 1 increments (Nt 1 ) to achieve the same isotropic resolution is twice in STMAS that in MQMAS (Table S1).

10

Consequently, the numbers Nt 1 of increments required to achieve the same isotropic resolution are also in the ratio of 2.

In the case of sodium citrate dehydrate, Nt 1 = 96 and 192 were chosen, and the number of scans was then adjusted in the unit of minimum phase cycling (24 for MQMAS, 12 for SPAM-MQMAS, 32 for STMAS and 16 for SPAM-STMAS) so that T exp  9 hours. Fig. 11 compares the isotropic MQMAS and STMAS projections of 23 Na z-filter and SPAM (E:AE = 4:1) spectra recorded according to the experimental set up in Table 2. We should note that the relative intensities of isotropic peaks are modified when RD changes (compare Figs. 11b andd or f and h), and it is highly likely that T 1 (Na2) < T 1 (Na1, Na3). It should be reminded that MQMAS is 3-5 times less sensitive than STMAS. This results in an apparently low intensity of Na1 in MQMAS compared to STMAS, most apparent when comparing Figs. 11b andf. Nonetheless, the signal gain by SPAM over zfilter was observed to be in the range of 2.8-3.3, which is in good agreement to the theoretically expected value of ca. 3.2 for SPAM (E:AE = 4:1) in Table 1. Similarly, the S/N gain by SPAM lies in the range of 1.6-2.7 (calculated from the last three lines of Table 2), which corresponds to the theoretically expected value of ca. 2.3 in Table 1. For a given experimental time, SPAM-STMAS (E:AE = 4:1) is likely to be the method of choice to achieve an optimum sensitivity, i.e. the highest S/N in Table 2. Moreover, upon comparison of the integrated intensities of the isotropic projections of Figs. 11b,d,f,h,a long relaxation value (RD = 5 s  3T 1 here) looks to provide betterquantified isotropic spectra with integrated ratios close to 1:1:1.

4-5-2 Spinning frequency:

87 Rb (I = 3/2: Rb 2 SO 4 )

Recent developments of fast MAS techniques have undoubtedly broadened the scope of solid-state NMR. In the context of MQMAS and STMAS, increasing the spinning speed enhances the resolution by zeroing the CSA and dipolar interactions, and by narrowing the MAS line-width in case of very large C Q values, which facilitate the signal acquisition. It also increases the isotropic spectral widths of rotorsynchronized experiments, SW iso , which are proportional to this spinning speed, thus limiting the folding of the resonances, which greatly eases the subsequent spectral analysis. These effects are demonstrated in Fig. 12 using 87 Rb z-filter MQMAS and STMAS spectra of Rb 2 SO 4 recorded with  R = 20 and 62.5 kHz using a 1.3 mm rotor. It can be seen that SW iso is doubled in STMAS with respect to MQMAS (as mentioned in the previous section) and that it increases proportionally to  R . It should be remembered, however, that the number Nt 1 of increments to achieve the same isotropic resolution increases proportionally to SW iso , hence leading to longer experimental times. For example, for MQMAS in Fig. 12a,c, Nt 1 = 50 and 160 increments were used at  R = 20 and 62.5 kHz, respectively. Nevertheless, the larger SW iso reduces the risk of signal aliasing or spectral crowding and alleviates the subsequent spectral analysis. Another advantage of fast MAS techniques is the fact that they are associated to small rotor diameters that can deliver very high rf-fields, which are highly recommended for the p1 and p3 hard pulses, and are difficult to access for low- nuclei. 37 Consequently, fast MAS conditions offer a possibility of widening the range of C Q that can be investigated by highresolution NMR methods. Fig. 13 shows a series of 87 Rb signal intensities simulated with  R = 20 and 62.5 kHz for a range of C Q . It has been known that MQMAS efficiency decreases when  R increases, 37 as shown in our simulations (Fig. 13a,b) and experiments (Fig. 12a,c). On the other hand, STMAS requires rotor-synchronized delays to refocus the large first-order broadening. The minimum evolution time is not zero but t 1min = T R -p1/2 -p4 -p2/2. The 2D signal intensities are determined by the signal at t 1min and they are thus attenuated by the losses that occur during this minimum delay due to irreversible processes and second-order quadrupolar dephasings that are not refocused by MAS. Under fast MAS conditions, however, these losses are minimized and hence the STMAS efficiency increases, especially for large C Q values. This is confirmed by our simulations (Fig. 13c,d) and experiments (Fig. 12b,d) in which the intensities of isotropic peaks are 3-4 times larger at  R = 62.5 compared to 20 kHz. Fig. 14 shows a series of isotropic projections of 87 Rb MQMAS and STMAS spectra of Rb 2 SO 4 recorded with various SPAM approaches. As expected from simulations (Fig. 13), the optimum pulse lengths are identical with z-filter and SPAM, irrespective of  R . With STMAS, the expected signal enhancements of 2 ( 1.9) and 4 ( 3.8) were observed for SPAM (E = AE) and SPAM (E:AE = 4:1), respectively. With MQMAS, slightly larger enhancements of 2.7-2.8 and 5.5-5.6 were observed, but these gains are difficult to accurately determine due to the poor S/N of z-filter signals. Considering that the STMAS sensitivity is approximately 5 times larger than that of MQMAS for this sample, the use of SPAM-STMAS under fast MAS conditions is a promising approach to expand the range of C Q that can be investigated by high-resolution NMR.

4-5-3 Application to

93 Nb (I = 9/2)

We demonstrated in Fig. 2, that the slopes of 1/R along which the E and AE signals move in time depend on the spin 31 Fig. 16 shows a series of isotropic slices of MQMAS and STMAS spectra of Nb 3 (NbO) 2 (PO 4 ) 7 with  R = 20 or 62.5 kHz. Under these conditions, the STMAS intensity is roughly 3.5 times higher than MQMAS. Although we observed the expected SPAM enhancement,  R = 20 kHz was not fast enough and a partial overlap of the center-band and the first spinning sidebands of the CT was observed in the MAS spectra. Also, the isotropic MQMAS spectra at  R = 20 kHz (Fig. 16a) are severely aliased, demonstrating the superiority of faster spinning with larger spectral width. Although the previous study suggested C Q = 30 MHz, 31 we suspect the presence of peaks with much smaller C Q in our sample, which are probably the cause of sinc wiggles visible in the MQMAS spectra at 62.5 kHz spinning (Fig. 16b). The signal truncation is less pronounced in the STMAS spectra (Fig. 16d) as the signal intensity of the main peak with large C Q is much higher than that of MQMAS. Fig. S3 shows a similar series of isotropic slices of MQMAS and STMAS spectra of Cs 4 Nb 11 O 30 recorded with various SPAM approaches. Under these conditions, the STMAS intensity was found approximately 6.5 times higher than that of MQMAS for this sample with C Q = 16 MHz. 31 Overall, for 93 Nb isotopes we recommend the use of STMAS at  R = 62.5 kHz for optimum sensitivity and resolution.

Conclusions

The main purpose of this publication is to describe the successful implementation of SPAM high-resolution experiments for half-integer quadrupolar nuclei. This has been done for spin-3/2, 5/2 and 9/2 nuclei in inorganic compounds with known NMR parameters. We have shown that a signal gain of ca. 4 and a gain in S/N of ca. 2.3 can be expected with the same simple setting than with z-filter. We have shown that SPAM-STMAS provides a much larger signal than the mostly used z-filter MQMAS. It has been shown recently that with an adapted Bruker MAS iProbe and a modern speed controller, the technical difficulties usually related to STMAS are fully controlled, which means than now high-resolution of halfinteger quadrupolar nuclei can be obtained, even with insensitive compounds. The sensitivity advantage of SPAM will be particularly helpful for signal acquisition of nuclei or samples with intrinsically low S/N such as low-, low natural abundance and/or large C Q . We expect that SPAM-MQMAS and SPAM-STMAS experiments will be a promising approach for further applications, in which signal acquisitions using conventional pulse sequences have been unsuccessful, or for more sophisticated experiments such as efficient isotropic signal acquisition in high-resolution hetero-nuclear correlation experiments. We believe that these results will be presented elsewhere in the foreseeable future.

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If you are experiencing difficulty using our template, please consult our 'Using the Template' Guide, found here. ) and corresponding signal-to-noise ratio (S/N) of each Na site obtained from F2 slice using the "sino" command on TopSpin. ig.11. Isotropic projection of 23 Na (I = 3/2) MQMAS and STMAS spectra of sodium citrate ihydrate recorded using various experimental approaches (as shown in Table 2) with fixed T exp (8-0 hrs) at B 0 = 14.1 T,  R = 20 kHz,  1 ( 23 Na): {p1, p2, p3, p4} = {95, 95, 9.5, 9.5} kHz, MQMAS: {p1, 2, p3} = {5.0, 2.5, 15} s, STMAS: {p1, p2, p3, p4} = {1.5, 1.5, 12.5, 25} s. P Q : {Na1, Na2, Na3} = 2.0, 1.9, 1.7} MHz. In (b,d,f,h), the ratios of integrated signal intensities with respect to the ighest peak of each spectrum is shown in curly brackets.

Journal Name 2 |

 2 J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx

  Fig.4 shows a series of plots of calculated MQMAS and STMAS signal intensities with respect to each pulse length for a large range of C Q values for 27 Al (I = 5/2) and 23

Figs

  Figs.5a,b show a comparison of isotropic projections of 27 Al (AlPO 4 -14) MQMAS and 87

30

 30

Fig. 10a ,

 10a Fig.10a, our sample seems to contain mostly -NaVO 3 and a tiny amount of -NaVO 3 , with {C Q (MHz),  Q ,  CS (ppm)} = {1.42, 0.27, -10.3} for -NaVO 3 and {Na1: 1.50, 0.58, -15.6} plus {Na2: 0.765, 0.06, -4.8} for -NaVO 3 . Figs.10b-d show the 23 Na zfilter, SPAM (E = AE = 96) and truncated SPAM (E:AE = 96:24 = 4:1) STMAS 2D spectra of NaVO 3 , with the corresponding isotropic projections below, expanded for minor resonances in Figs.10e-g. It can be seen that the isotropic signal intensities of minor signals increase from z-filter to SPAM (E:AE = 4:1) by a factor of ca. 4, as expected. However, a significant baseline distortion is introduced in the isotropic projection. This distortion, is intrinsic to STMAS due to the rotor-synchronized non-zero evolution time: t 1min = T R -p1/2 -p4 -p2/2. It is also at the origin of the negative green parts in the 2D spectra of Fig.10c and dand being proportional to the signal is hence doubled in Fig.10d compared to Fig.10c. These dispersive parts

1

  Citations should appear here in the format A. Name, B. Name and C. Name, Journal Title, 2000, 35, 3523; A. Name, B. Name and C. Name, Journal Title, 2000, 35, 3523. 2 …

Fig. 4 .

 4 Fig.4. Simulated signal intensity of (a,b) z-filter and SPAM-MQMAS, (c,d) z-filter and SPAM-STMAS with respect to each pulse length (p1, p2, p3) for a range of quadrupolar coupling constants (C Q ) of (a,c) 27 Al (I = 5/2) and (b,d) 23 Na (I = 3/2) nuclei at B 0 = 14.1 T,  R = 20 kHz,  1 ( 27 Al): {p1, p2, p3, p4} = {90, 90, 5, 5} kHz,  1 ( 23 Na): {p1, p2, p3, p4} = {95, 95, 9.5, 9.5} kHz. Pulse lengths used within each plot: (a) 27 Al MQMAS: {p1, p2, p3} = {4.0, 1.4, 16.7} s, (c) 27 Al STMAS: {p1, p2, p3, p4} = {1.8, 1.1, 16.7, 33.3} s, (b) 23 Na MQMAS: {p1, p2, p3} = {6.5, 2.2, 13.0} s, (d) 23 Na STMAS: {p1, p2, p3, p4} = {1.8, 1.6, 13.0, 26.0} s.

Fig. 6 .

 6 Fig.6. 85 Rb (I = 5/2) MQMAS and STMAS spectra of RbNO 3 at B 0 = 14.1 T,  R = 20 kHz, RD = 0.5 s, T exp = 80 min each,  1 ( 85 Rb): {p1, p2, p3, p4} = {35, 35, 9, 9} kHz, MQMAS: {p1, p2, p3} = {8.0, 3.0, 7.5} s, STMAS: {p1, p2, p3, p4} = {3.0, 3.0, 7.5, 15} s. (a) E + AE = 100, NS = 96, (b) E = AE = 50, NS = 96, (c) E = 50, AE = 1, NS = 192, (d) E + AE = 50, NS = 192, (e) E = AE = 25, NS = 192, (f) E = 25, AE = 1, NS = 384. P Q : {Rb1, Rb2, Rb3} = {3.7, 4.9, 3.4} MHz.

Fig. 8 .

 8 Fig.8. Ratio of negative to positive integral (/) versus the ratio of the number of antiecho to echo (AE/E). (a) I = 5/2: AlPO 4 -14 (green, C Q = 1.8, 4.1, 5.6, 2.6 MHz), berlinite (yellow, C Q = 4.1 MHz), RbNO 3 (blue, P Q = 3.4-4.9 MHz). (b) I = 3/2: Na 2 SO 4 (orange, C Q = 2.6 MHz), NaH 2 PO 4 (yellow C Q = 1.6-2.4 MHz), Na citrate (green, P Q = 1.7-2.0 MHz), Rb 2 SO 4 (blue, C Q = 2.5, 5.3 MHz). An estimated convergence line is indicated at E:AE = 4:1.

Fig. 12 .

 12 Fig.12. 87 Rb (I = 3/2) z-filter (a,c) MQMAS and (b,d) STMAS spectra of Rb 2 SO 4 and corresponding isotropic projection at B 0 = 14.1 T, RD = 0.5 s with (a,b)  R = 20 kHz or (c,d)  R = 62.5 kHz using a 1.3 mm rotor.  1 ( 87 Rb): {p1, p2, p3, p4} = {160, 160, 20, 20} kHz, MQMAS: {p1, p2, p3} = {3.0, 1.5, 6} s, STMAS: {p1, p2, p3, p4} = {1.4, 1.0, 6.0, 10} s. (a,b) T exp = 0.5 hrs with (a) NS = 72, E + AE = 50, (b) NS = 32, E + AE = 100, (c,d) T exp = 1.5 hrs with (c) NS = 72, E + AE = 160, (d) NS = 32, E + AE = 320. C Q : {Rb1, Rb2} = {2.5, 5.3} MHz.

Fig. 13 .

 13 Fig.13. Simulated z-filter and SPAM signal intensity of (a,b) MQMAS and (c,d) STMAS with respect to each pulse length (p1, p2, p3) and C Q at B 0 = 14.1 T with (a,c)  R = 20 kHz or (b,d)  R = 62.5 kHz.  1 ( 87 Rb): {p1, p2, p3, p4} = {160, 160, 20, 20} kHz. For each plot, the two other pulse lengths were fixed at their optima. Pulse lengths used within each plot: (a,b) {p1, p2, p3} = {3.5, 1.5, 5.0} s, (c,d) {p1, p2, p3, p4} = {1.2, 1.0, 6.0, 12.0} s.

Fig. 14 .

 14 Fig.14. Isotropic projection of 87 Rb (I = 3/2) (a,b) MQMAS and (c,d) STMAS spectra of Rb 2 SO 4 recorded with various SPAM approaches at B 0 = 14.1 T with (a,c)  R = 20 kHz or (b,d)  R = 62.5 kHz using a 1.3 mm rotor.  1 ( 87 Rb): {p1, p2, p3, p4} = {160, 160, 20, 20} kHz. C Q : {Rb1, Rb2} = {2.7, 5.3} MHz.

Fig. 15 .

 15 Fig.15. Simulated z-filter and SPAM signal intensity of (a,b) MQMAS and (c,d) STMAS with respect to each pulse length (p1, p2, p3) and C Q at B 0 = 14.1 T with (a,c)  R = 20 kHz or (b,d)  R = 62.5 kHz.  1 ( 93 Nb): {p1, p2, p3, p4} = {128, 128, 8, 8} kHz. For each plot, the two other pulse lengths were fixed at their optima. Pulse lengths used within each plot: (a,b) {p1, p2, p3} = {2.0, 0.6, 6.0} s, (c,d) {p1, p2, p3, p4} = {1.0, 0.4, 6.0, 12.0} s.

Fig. 16 .

 16 Fig.16. Isotropic slice of 93 Nb (I = 9/2) (a,b) MQMAS and (c,d) STMAS spectra of Nb 3 (NbO) 2 (PO 4 ) 7 recorded with various SPAM approaches at B 0 = 14.1 T with (a,c)  R = 20 kHz or (b,d)  R = 62.5 kHz using a 1.3 mm rotor.  1 ( 93 Nb): {p1, p2, p3, p4} = {128, 128, 8, 8} kHz.

  ( 85 Rb) and 146.89 ( 93 Nb) MHz, equipped with HX MAS probes using either  = 3.2 mm rotors at  R = 20-22 kHz or  = 1.3 mm at  R = 20 and 62.5 kHz. For hard pulses, maximum rf field strengths of  1 = 120 ( Na 2 SO 4 , NaH 2 PO 4 , sodium citrate dihydrate, AlPO 4 -14, Berlinite, -Al 2 O 3 , RbNO 3 , Rb 2 SO 4 , NaVO 3 , CS 4 Nb 11 O 30 , and Nb 3 (NbO) 2 (PO 4 ) 7

	87 Rb), 95 (	23 Na), 90 (	27 Al), 35 (

1 T wide-bore magnet at a Larmor frequency of 158.75 ( 23 Na), 156.38 ( 27 Al), 196.37 ( 87 Rb), 57.94 85 Rb) kHz were attained with  = 3.2 mm, and  1 = 160 ( 87 Rb), 128 ( 93 Nb) kHz with  = 1.3 mm. A range of  1 = 5-20 kHz was used for CT-selective soft pulses. Powder samples (

  1. With SPAM (E:AE = 1:1), a maximum signal gain of 2 is expected while the noise increases by a factor of 2 as the E and AE signals are acquired separately and then combined upon processing. This gives rise to a gain of S/N of 1.

		4 with
	SPAM (E:AE = 1:1). With SPAM (E:AE = 4:1), NS is scaled up by a
	factor of 1.6 = 2E/(E+AE) and thus the maximum signal gain is 2
	 1.6 = 3.2, resulting in a gain of S/N of 2.3 = 3.2/2.
	These expected values have been experimentally verified in
	the following with	23

8 |

 8 J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx value and the nature of the coherences used (MQMAS or STMAS). Taking 27 Al (I = 5/2) in Fig.2a as an example, the MQMAS signals move along the slopes of 0.632 (12/19), while the STMAS slopes are equal to 3.429 (24/7).Consequently, the AE signal disappears from the t 2 acquisition window much faster in MQMAS than in STMAS. This effect is still more pronounced for MQMAS of I = 9/2 nuclei, for which the slopes are very small and equal to 0.396 (TableS1) and thus very few AE may be visible in t 2 acquisition window.

	93 Nb (I = 9/2) nuclei have 100 % natural abundance and a
	relatively high gyromagnetic ratio. However, they exhibit a
	large chemical shift range ( 4000 ppm) and their solid-state
	NMR spectra are often severely broadened due to large
	quadrupolar coupling. Both aspects inhibit the extraction of
	useful information concerning	93 Nb nuclei. Following the
	development of high-field spectrometers and fast MAS probes,
	93 Nb solid-state NMR spectra recorded at 21.1 T kHz have been reported, 39 and 93 Nb solid-state NMR has 38 and  R = 60
	indeed become more accessible as a reliable tool for material
	characterization.	39,40 Although numerous static and MAS
	studies of Nb-containing compounds exist, only a limited
	number of high-resolution MQMAS and STMAS spectra have
	been reported.	14,31,38,41-45	Combined with the recent
	acceleration in first-principles calculations of	93 Nb NMR
	parameters,	39,44-49 high-resolution NMR methods have great
	potential to facilitate the use of	93 Nb solid-state NMR in
	material investigations.
	Fig.15 shows a series of simulated	93 Nb signal intensity at
	14.1 T with  R = 20 and 62.5 kHz for a range of C Q . These
	results confirm that, as discussed in the former sections, (i) the
	optimum pulse lengths are identical between z-filter and SPAM,
	and (ii) that when the spinning speed increases, the MQMAS
	efficiency decreases while that of STMAS increases, especially
	for large C Q values. 93 Nb spectra of Nb 3 (NbO) 2 (PO 4 ) 7 and Cs 4 Nb 11 O 30 recorded at
	18.8 T and 20 kHz MAS have been reported.

Table 1 .

 1 Expected maximum gain over z-filter of signal-to-noise (S/N) ratio of SPAM approaches.

		z-filter	SPAM (1:1)	SPAM (2:1)	SPAM (4:1)	SPAM (1:0)
	Signal (S)	1	2	2.7	3.2	4
	Noise (N)	1	2	2	2	2
	S/N	1	1.4	1.9	2.3	2.8

Table 2

 2 Comparison of MQMAS and STMAS experimental approaches for sodium citrate dihydrate recorded with a comparable experimental time (T exp  9 hrs

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 3

scans were accumulated for each of

t 1 increments in zfilter, 24 scans for each of 128 E and 128 AE acquisition in SPAM (E = AE), and 48 scans for each of 128 E acquisition in fully-truncated SPAM (AE = 0). It can be concluded that, for a

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 7

Acknowledgements

The authors would like to thank Dr. Hideaki Kimura (Bruker Japan K.K.) and Dr. Sebastian Wegner (Bruker GmbH) for their help with implementation of SPAM experiments on TopSpin, and Dr. Julien Trébosc (University of Lille) for his kind advice on performing SIMPSON simulations.

Conflicts of interest

There are no conflicts to declare.


Armin Bischoff
email: armin.bischoff@univ-avignon.fr

Anna Pollier

Yann Tricault

Manuel Plantegenest

Bruno Chauvel

Pierre Franck

Antoine Gardarin

A multi-site experiment to test

Keywords: Conservation biological control, ecosystem services, field margin, natural enemies, natural regulation, semi-natural habitat

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

In many European regions, there is an ongoing loss of semi-natural habitats [START_REF] Billeter | Indicators for biodiversity in agricultural landscapes: a pan-European study[END_REF][START_REF] Ridding | Ongoing, but slowing, habitat loss in a rural landscape over 85 years[END_REF]) and this loss is among the major causes for the decline in biodiversity of many taxonomic groups [START_REF] Hallmann | More than 75 percent decline over 27 years in total flying insect biomass in protected areas[END_REF][START_REF] Ridding | Ongoing, but slowing, habitat loss in a rural landscape over 85 years[END_REF]. Biodiversity linked to seminatural habitats provides several important ecosystem services such as pollination, carbon sequestration, reduction in fertilizer and pesticide run off, as well as pest control [START_REF] Holland | Semi-natural habitats support biological control, pollination and soil conservation in Europe. A review[END_REF]. European Union member states have implemented agri-environment schemes to restore biodiversity in agricultural landscapes involving the creation or conservation of semi-natural habitats [START_REF] Haaland | Sown wildflower strips for insect conservation: a review[END_REF][START_REF] Holland | Semi-natural habitats support biological control, pollination and soil conservation in Europe. A review[END_REF]. One of the most widespread agri-environment schemes is the sowing of wildflower strips [START_REF] Haaland | Sown wildflower strips for insect conservation: a review[END_REF]. Wildflower strips are flowerrich mixtures of indigenous herbaceous species usually sown to the edges of arable fields.

Although initially developed in the late 1980ies to enhance biodiversity in general (neutral and functional), effects on ecosystem services such as biocontrol are increasingly discussed [START_REF] Tschumi | Perennial, species-rich wildflower strips enhance pest control and crop yield[END_REF][START_REF] Pollier | Sowing of margin strips rich in floral resources improves herbivore control in adjacent crop fields[END_REF]. In France, the research on this topic has been strongly revived since the governmental plan to reduce pesticide use by 50% between 2008 and 2018 failed ("Ecophyto 2018": [START_REF] Guichard | Le plan Ecophyto de réduction d'usage des pesticides en France : décryptage d'un échec et raisons d'espérer[END_REF] and the deadline was extended to 2025 in order to provide more time and resources for the development of alternative pest control measures.

Wildflower strips or equivalent semi-natural field margins provide shelter, overwintering habitats, and food resources such as nectar or alternative prey to different groups of natural enemies of pest insects [START_REF] Bischoff | Effects of spontaneous field margin vegetation and surrounding landscape on Brassica oleracea crop herbivory[END_REF][START_REF] Holland | Semi-natural habitats support biological control, pollination and soil conservation in Europe. A review[END_REF]. The provisioning of floral resources is crucial for several important natural enemies such as parasitic wasps, hoverflies and lacewings that change nutrition from carnivorous during larval stages to herbivorous (pollen, nectar) during the adult period [START_REF] Winkler | Nectar exploitation by herbivores and their parasitoids is a function of flower species and relative humidity[END_REF][START_REF] Araj | Comparing existing weeds and commonly used insectary plants as floral resources for a parasitoid[END_REF]. For this reason, quantity, quality and period of floral resource availability are key traits in designing wildflower strip mixtures [START_REF] Gardarin | Understanding plant-arthropod interactions in multitrophic communities to improve conservation biological control : useful traits and metrics[END_REF]. The positive effects of flower-rich semi-natural habitats including wildflower strips on abundance and diversity of natural enemies are well documented but a corresponding effect on pest abundance and/or crop damage has rarely been shown [START_REF] Chaplin-Kramer | A meta-analysis of crop pest and natural enemy response to landscape complexity[END_REF][START_REF] Haaland | Sown wildflower strips for insect conservation: a review[END_REF]. The reasons for this potential lack in the desired response of pest insect activity to an increased number of natural enemies in field margins are complex and not yet well understood [START_REF] Tscharntke | When natural habitat fails to enhance biological pest control -five hypotheses[END_REF]). One problem is the possible concomitant positive effect of such habitats on natural enemies and important pest insects [START_REF] Bischoff | Effects of spontaneous field margin vegetation and surrounding landscape on Brassica oleracea crop herbivory[END_REF][START_REF] Tscharntke | When natural habitat fails to enhance biological pest control -five hypotheses[END_REF]. Another important constraint may be energetic costs of movements combined with a relative attractiveness of semi-natural habitats resulting in a strong reduction of biocontrol efficiency [START_REF] Heimpel | Does Floral Nectar Improve Biological Control by Parasitoids[END_REF][START_REF] Wanner | Using the stable isotope marker 44 Ca to study dispersal and host-foraging activity in parasitoids: Stable isotope marking of parasitoids[END_REF]. Thus effects on crop herbivory need to be better studied and integrated when evaluating the biocontrol service of sown wildflower strips.

Climate has a large influence on plant diversity and community composition [START_REF] Thuiller | Climate change threats to plant diversity in Europe[END_REF]. Additionally, climate may change plant-insect interactions even on the same plant species [START_REF] Rasmann | Climate-driven change in plant-insect interactions along elevation gradients[END_REF]. So far, it is not well known at which scale such changes occur and whether plant mixtures for wildflower strips need to be adapted to climatic conditions in order to obtain a desired biocontrol effect. Currently, European species mixtures for wildflower strips are usually suggested at country scale although local origin is recommended [START_REF] Vander Mijnsbrugge | A question of origin: Where and how to collect seed for ecological restoration[END_REF][START_REF] Haaland | Sown wildflower strips for insect conservation: a review[END_REF]. Robust mixtures performing well under different climatic conditions may be a solution to simplify logistics of involved seed companies if regional seed sourcing is respected. They would also improve the resilience of established plant communities and associated insects to global climate change and the resulting increase in the probability of extreme weather events.

In our study, we tested the performance of a species-rich wildflower strip mixture in five French regions with different climate. We analysed the response of major natural enemy groups in the strips and the resulting effects on pest insects in adjacent winter wheat fields using a subset of four regions. To evaluate the biocontrol effects, we compared wildflower strips with both grass strips and spontaneously emerging vegetation. Sowing of grass strips to field margins is supported in French agri-environment schemes to reduce machinery impact on soil structure, soil erosion and water pollution by fertilizer and pesticide run-off close to water bodies [START_REF] Cordeau | Sown grass strips harbour high weed diversity but decrease weed richness in adjacent crops[END_REF]. Such commonly used, species-poor grass mixtures were thus the first baseline to which the biocontrol efficiency of wildflower strips was compared. Spontaneous vegetation was used as a second baseline because natural field margin succession is also common and thus more straightforward than using bare soil or cereal strips as controls [START_REF] Haaland | Sown wildflower strips for insect conservation: a review[END_REF]. Wildflower strips involving costs for seed material, soil preparation and sowing need to improve biocontrol performance compared to less costly practices representing our baselines. Both grass strips [START_REF] Collins | Influence of beetle banks on cereal aphid predation in winter wheat[END_REF] and spontaneous margins [START_REF] Bischoff | Effects of spontaneous field margin vegetation and surrounding landscape on Brassica oleracea crop herbivory[END_REF]) have been shown to provide resources and shelter to natural enemies of crop pests. We further used a correlative analysis to compare the influence of vegetation (independent of strip type) and climate on biological control.

In order to test the effect of sown wildflower strips on conservation biological control and the robustness of effects under different climatic conditions, we addressed the following research questions: (i) Does the selected wildflower strip mixture develop well under different French climates and increase floral resource provisioning in all study regions? (ii) Is this mixture in all regions successful in increasing natural enemy abundance and predation compared with grass strips and spontaneous vegetation? (iii) Does the potential increase in mixture-related resources translate in higher natural enemy densities in the field (spill-over) and in a better regulation of pest insects? (iv) How strong is the influence of vegetation characteristics compared to regional climate?

Material and methods

Study sites and design

The experimental design includes five study regions with three (Rennes) to five (Angers, Avignon, Grignon, Dijon) fields per region (Appendix A: Fig. A.1). Three study regions are located in North-Western France with temperate Atlantic climate characterised by mild winters and relatively low summer temperatures (Grignon, Rennes, Angers, Table 1). Among these three regions, the average annual temperature is higher in Angers than in the other two regions whereas winters are colder in Grignon. The Dijon region is located in central-eastern France. The climate is thus more continental, in particular with lower winter temperatures. The Avignon region is characterised by Mediterranean climate with high summer temperatures, mild winters and summer drought.

The experiment was set up in 2014 and analysed for two seasons. Both study years were warmer than the long-term average with a mild winter in 2014 and a warm summer in 2015 but differences between regions corresponded to the long-term climatic data (Appendix A: Table A.1).

In the regions of Rennes and Dijon, one field was lost in 2015 due to unexpected changes in crop and strip management. At the Grignon, Rennes, Angers and Dijon sites, the experiment was set up in oilseed rape field followed by wheat. In Avignon, the experiment was run in apple orchards. Thus, Avignon data were only included for measurements within margins. In Grignon, no data were available from the first year. In March 2014, three strip treatments were established in each of the 23 fields: (1) a wildflower strip (WS) with 32 flowering indigenous plant species (Appendix A: Table A.2). Cultivars or species non-native to the five study regions were excluded. The sowing density was 300 seeds/m² (0.8 g/m²) and density proportion of all species equal. The sown mixture comprised plant species of different height and flower morphology that produce high quantities of floral or extrafloral nectar at complementary flowering periods.

Annual, biennial and perennial plants were included to guarantee a sufficient cover in the first and subsequent years and to avoid expensive re-sowing. (2) A grass strip mixture (GS) with Lolium perenne (0.84 g/m² = 420 seeds/m²) and Festuca arundinacea (2.16 g/m² = 1540 seeds/m²). The two species are commonly used in France to obtain a fast vegetation cover outcompeting weed species and avoiding soil erosion. (3) A strip of spontaneously emerging vegetation (C). The soil of all treatments was ploughed and harrowed prior to sowing. The strip area was 30 m x 3 m and the three treatments were randomly assigned to a 90 m long field margin of each field. The strips were mown after the growing season in autumn 2014. The establishment of the three treatments in the same fields reduced bias resulting from differences in soil, microclimate and land use intensity but limited the analysis of long-distance spill-over since distance differences to different strip types were getting smaller in central parts of the field.

Measurements

Vegetation

The vegetation of margin strips was monitored in early June 2014 and in late June 2015. The presence of all vascular plant species was recorded in a survey area of 15 m x 3 m in the centre of the margin strips. Percentage cover of each plant species was visually estimated as the vertical projection of all aboveground organs. At the same time, the percentage of flowering plants was evaluated for each species. Individual flower cover was calculated by multiplying plant cover and the proportion of flowering plants in each species. The sum of individual flower cover was used as a proxy of total flower cover.

Natural enemies and predation in field margin strips

The insect surveys were conducted in the same weak as vegetation surveys corresponding to peak abundance of major pest insects. We selected hoverflies and ladybirds as natural enemy groups that occurred at all sites. In order to avoid disturbance, adult individuals were first counted from outside the strip in walking slowly along the edges of a 10 m x 3 m segment.

Afterwards hoverfly and ladybird larvae were recorded on the plants requiring a penetration of strips (observation time: 10 min in total x 2 observers). Hoverfly and ladybird counts for adults and larvae were pooled for analysis to avoid zero inflation. The observation period was between 10 AM and 4 PM under favourable weather conditions (sunny, not windy, no rain).

Aphid predation pressure was evaluated using bait aphids glued to sand paper (predation cards). We used the commercially available pea aphid Acyrthosiphon pisum as sentinel prey, assuming that predator identity and predation rates are similar for different aphid species (Östman 2004).

Predation cards were prepared by gluing (contact spray 'Cléopâtre ltd') three aphids on 5cm x 5cm black sand paper sheets. Aphid predation was evaluated after 24 h. An aphid was considered as predated when damaged or missing. We fixed one card on the ground in the centre of each margin strip in the Rennes, Angers, Grignon and Avignon region.

Crop herbivores, natural enemies and predation in adjacent fields

In June 2015, the effect of different strip types was also tested in adjacent winter wheat fields (n=17) at a distance of 5 m and 30 m from the margins. In addition to natural enemies and aphid predation, the abundance of cereal aphids and cereal leaf beetle larvae as major crop herbivores were included. For hoverflies and ladybirds, the observation method was similar to that used within margin strips: adult insects were first counted by observation along a segment of 3m x 10m parallel to the margin, from a distance and without touching the wheat tillers. In a second step, the number of larvae was recorded by close observation. Cereal leaf beetles (Oulema sp., larvae), cereal aphids (dominant species: Sitobion avenae and Metopolophium dirhodum) and aphid mummies were counted on 50 consecutive tillers along each segment. Aphid parasitism rate was estimated by dividing the total number of mummies by the sum of aphids and mummies. Aphid predation was measured as inside the margin strips by attaching a predation card to the ground of each segment corresponding to two cards per margin strip treatment. Aphid abundance, leaf beetle abundance and aphid parasitism were recorded in all experimental fields with winter wheat (Rennes, Angers, Grignon, Dijon) whereas hoverfly abundance, ladybird abundance and aphid predation were measured in a subset of three regions (Rennes, Angers, Grignon).

Analysis

Prior to statistical analysis, entomophilous flower cover was calculated based on total flower cover to obtain a more straightforward proxy of floral resource provisioning. The Biolflor database [START_REF] Klotz | Database on Biological and Ecological Traits on the Flora of Germany[END_REF] was used to identify entomophilous plants and to eliminate windpollinated species that do not produce nectar.

The strip treatment effect on vegetation characteristics, natural enemy abundance and aphid predation inside margin strips was analysed using generalized linear mixed models. Treatment, region and the region-by-treatment interaction were included as fixed, field within region as a random effect. Vegetation data and arcsin(sqrt(x))-transformed aphid predation were normally distributed and thus analysed using a Gaussian distribution and identity link. Count data were fitted using a Poisson distribution with log-link function. When overdispersion was detected, a negative binomial model with log-link function was applied. The glht function (multcomp package, Tukey) was used for multiple comparisons when treatment main effects were significant.

We used the same statistical model to analyse strip treatment effects within wheat fields but additionally included distance as a fixed effect. Thus the model comprised three fixed effects (region, treatment, distance), field within region as random effect and the two-way interactions region-by-treatment (fixed) and treatment-by-distance (fixed). For a higher parsimony of the model, we did not include the interaction between region and distance and the three-way interaction between region, treatment and distance. All GLMM analyses were run in R, version 3.3.1, using the glmer function of lme4 package (R Development Core Team 2015).

In order to compare the influence of climatic variables and vegetation characteristics beyond strip treatments on natural enemies and pest insects, a Redundancy Analysis (RDA) was applied to field margin data. Response variables were log-transformed to homogenise variances and to obtain similar scales. Independent of strip treatment, plant species number and entomophilous flower cover were included as explanatory variables to characterise vegetation. Summer temperature, the number of winter frost days and annual precipitation were used as major climatic variable potentially influencing response variables. Pseudo-F statistics using permutation tests with 499 unrestricted permutations was applied to evaluate the relative significance of explanatory variables in the full model. RDA including permutation tests was run in Canoco 5 (ter Braak & Smilauer 2012).

Results

Compared with the unsown strips with spontaneous vegetation, the wildflower strip treatment added on average eight plant species to the first year community and nine species in the second year (Fig. 1). Twenty-four sown species developed well in all regions, eight sown species were rare or absent (Table A2). In the first year, annual species from the soil seed bank dominated in all regions with sown species only representing on average 25% of soil cover and 40% of total vegetation cover in wildflower strips. All annuals such as Capsella bursa-pastoris, Cyanus segetum, Matricaria chamomilla, Papaver rhoeas and Sinapis arvensis but also some perennials such as Malva sylvestris flowered in the first year. In the second year, the cover of sown wildflower species increased to 50% with nearly all biennial and perennial species flowering.

The cover of Lolium perenne and Festuca arundinacea sown to grass strips reached on average 17% in the first and 40% in the second year. Several species sown to wildflower strips were part of the soil seed bank explaining their relatively high cover in unsown strips (Appendix A: Fig.

A.2).

The strip treatment had a significant effect on plant species richness, on the cover of sown wildflower strip species and on entomophilous flower cover, with the highest values for wildflower strips followed by spontaneous vegetation (Table 2, The strip treatment effect on flower cover did not significantly differ between regions. Strip treatment effects were stronger between wildflower strips and grass strips than between other treatments. In the first year, differences between wildflower strips and strips of spontaneous vegetation were for example not significant for flower cover (P=0.22) whereas differences between wildflower and grass strips were marginally significant (P=0.09). Differences between spontaneous vegetation and grass strips were never significant. Strip treatment effects on sown wildflower species and flower cover increased from the first to the second year.

Corresponding to the significant effect of strip treatment on flower cover, strip treatment also had a significant effect on hoverfly abundance with highest abundance in wildflower strips and lowest in grass strips (Table 2, Fig. 2). Interactions with region were not significant suggesting a similar strip treatment effect under different climatic conditions of the study regions. The significant strip treatment effect was mainly explained by a significant difference between wildflower strips and grass strips (year 1 marginally significant: P=0.092, year 2: P= 0.016) whereas the difference between wildflower strips and spontaneous vegetation was not or marginally significant (year 1: P=0.097, year 2: P=0.142). Neither strip treatment nor region had a significant effect on ladybird abundance. Aphid predation was significantly affected by strip treatment in the second year again being the highest in wildflower strips and the lowest in grass strips. In the first year, there was no treatment main effect on aphid predation but a significant interaction of treatment with regions. Predation was higher in wildflower than in grass strips in Angers and Rennes regions whereas in the Avignon region, the highest predation was measured in spontaneous vegetation.

In adjacent wheat fields, the significant effect on hoverfly abundance and aphid predation found within strips was confirmed (Table 3, Fig. 3). Again significant differences were observed between wildflower strips and grass strips (hoverflies: P<0.001, aphid predation: P=0.04) whereas the difference between wildflower strips and spontaneous vegetation was only significant for hoverflies (P<0.001). For hoverflies, a significant distance-by-strip type interaction occurred. The positive wildflower strip effect was limited to crop plants close to the margin (5 m, P<0.001) whereas the strip type effect on hoverflies was not significant at inner parts of the field (30 m). A marginally significant effect of strip type was detected for aphid parasitism with a higher parasitism of aphids at both distances of the wildflower strips.

Corresponding to the absence of significant differences within margins, the effect of strip type on ladybird abundance within wheat fields was not significant. The positive effects of wildflower strips on hoverflies, aphid predation and aphid parasitism did not result in a reduction of aphid abundance, nor leaf beetle abundance was affected by strip treatments.

A comparison of major climatic variables and characteristics of the field margin vegetation pooled for margin strip treatments showed a dominant influence of flower cover on natural enemies and aphid predation observed in field margins (Fig. 4). Flower cover was the only significant explanatory variable in the RDA on natural enemy abundance and predation in margin strips (PseudoF=14.4, P=0.004). It was strongly correlated with the first RDA axis explaining 30% of the total variation (second axis only 2%). At the same time, hoverfly abundance was correlated with flower cover whereas no correlation was observed for ladybirds.

Neither annual precipitation nor summer temperature nor winter harshness (number of frost days) were significantly correlated with natural enemy abundance and aphid predation.

Discussion

Treatment effects within strips and stability under different climate

The wildflower strip treatment had a consistent positive effect on plant species richness and flower cover in all study regions. The establishment rate of the sown species and the increase in plant species richness are in line with the results of a European scale experiment in orchards using country-specific wildflower strip mixtures [START_REF] Pfiffner | Design, implementation and management of perennial flower strips to promote functional agrobiodiversity in organic apple orchards: a pan-European study[END_REF]. Wildflower strip species spontaneously establishing from the soil seed bank had a higher cover in unsown than in grass strips. Thus, differences to sown wildflower strips were only significant to grass strips in the first year but not to unsown strips with spontaneous vegetation. Accordingly, first year flower cover was only different between wildflower strips and grass strips. Although treatment differences were not very strong between grass strips and spontaneous vegetation, these results suggest that sowing of competitive grasses may hamper the establishment of wildflowers known to improve pest biocontrol service.

Hoverfly abundance within margin strips followed the pattern of flower cover with a significantly higher density in margin strips of all study regions. The lack of significant interactions with region demonstrated the robustness of the positive wildflower strip effect under different climate from temperate oceanic to temperate subcontinental and Mediterranean. Again, the differences to grass strips were significant in both years whereas the differences to spontaneous vegetation were only significant in one year confirming the trend of floral suppression by grass sowing. Nectar accessibility is a key trait for positive interactions between flowering plants and hoverflies [START_REF] Van Rijn | Nectar accessibility determines fitness, flower choice and abundance of hoverflies that provide natural pest control[END_REF][START_REF] Gardarin | Understanding plant-arthropod interactions in multitrophic communities to improve conservation biological control : useful traits and metrics[END_REF]. Zoophagous hoverflies need flowers with a short corolla tube or with an open corolla to access nectar or even extra-floral nectar (Campbell et al., 2012;[START_REF] Van Rijn | Nectar accessibility determines fitness, flower choice and abundance of hoverflies that provide natural pest control[END_REF], which was considered in the choice of wildflower strip species. Correlative studies analysing the influence of unsown semi-natural habitats such as herbaceous field margins did not always show such a clear positive relationship between hoverfly abundance and floral cover [START_REF] Pollier | Effects of spontaneous field margin vegetation on the regulation of herbivores in two winter crops[END_REF][START_REF] Bartual | The potential of different semi-natural habitats to sustain pollinators and natural enemies in European agricultural landscapes[END_REF]. The stronger positive signal obtained for sown wildflower strips taking into account the floral requirements of hoverflies [START_REF] Tschumi | Perennial, species-rich wildflower strips enhance pest control and crop yield[END_REF][START_REF] Pollier | Sowing of margin strips rich in floral resources improves herbivore control in adjacent crop fields[END_REF]) demonstrated the potential for improving conservation biological control by optimising floral resource provisioning. The analysis of floral visits at two of the five sites demonstrated the high attractiveness of sown wildflower strip species compared with spontaneous vegetation [START_REF] Pollier | Sowing of margin strips rich in floral resources improves herbivore control in adjacent crop fields[END_REF].

Ladybirds did not show any significant response to the strip treatments and no interaction with study region was observed. Larvae and adults are predominantly carnivorous but gut content analyses showed that ladybirds also feed on floral resources and flowering plants may thus increase their lifespan (Walton & Isaac 2011). Accordingly, several studies have found a positive relationship between flower cover in semi-natural habitats and ladybird abundance [START_REF] Bischoff | Effects of spontaneous field margin vegetation and surrounding landscape on Brassica oleracea crop herbivory[END_REF][START_REF] Tschumi | High effectiveness of tailored flower strips in reducing pests and crop plant damage[END_REF][START_REF] Pollier | Effects of spontaneous field margin vegetation on the regulation of herbivores in two winter crops[END_REF][START_REF] Pollier | Sowing of margin strips rich in floral resources improves herbivore control in adjacent crop fields[END_REF]. In our study, ladybird abundances were both low and highly variable between fields within regions limiting the probability to detect significant differences.

Aphid predation was significantly affected by strip treatment in the second year when wildflower strip species were well established. Corresponding to the results on hoverfly abundance, highest predation was observed in wildflower strips and lowest in grass strips. Again, the result was robust under the different climates of our study regions resulting in a non-significant region-by-strip type interaction. The corresponding results of aphid predation and hoverfly abundance suggest a causal relationship since hoverfly larvae are major predators of aphids [START_REF] Laubertie | The contribution of potential beneficial insectary plant species to adult hoverfly (Diptera: Syrphidae) fitness[END_REF]. However, ground dwelling arthropods contribute to the predation of aphids on predation cards [START_REF] Blaise | The key role of inter-row vegetation and ants on predation in Mediterranean organic vineyards[END_REF]. Thus, the increase in predation may also be explained by a positive effect of wildflower strips on these arthropod groups [START_REF] Tschumi | High effectiveness of tailored flower strips in reducing pests and crop plant damage[END_REF].

The multivariate analysis comparing the influence of climatic factors and vegetation traits on predators and predation in the margin strips confirmed the particularly strong influence of floral cover on hoverflies (van Rijn & Wäckers 2016; [START_REF] Gardarin | Understanding plant-arthropod interactions in multitrophic communities to improve conservation biological control : useful traits and metrics[END_REF] and to a lesser degree on aphid predation. Floral cover explained much better the variation of response variables than climate. Climate may change plant-insect interactions as studies on latitudinal or altitudinal gradient showed [START_REF] Rasmann | Latitudinal patterns in plant defense: evolution of cardenolides, their toxicity and induction following herbivory[END_REF][START_REF] Rasmann | Climate-driven change in plant-insect interactions along elevation gradients[END_REF]. Often biotic interactions are getting stronger with warmer climate since the number of insects associated to plant species increases. However, in our study we sowed the same species everywhere limiting climate-related responses based on long-term adaptive processes.

Spill-over to adjacent wheat fields and control of pest insects

Insects depending on floral resources show limited movements from flower-rich margins into the field, and parasitism or predation rates may decrease far below maximum distances of movements [START_REF] Lavandero | Enhancing the effectiveness of the parasitoid Diadegma semiclausum (Helen): movement after use of nectar in the field[END_REF][START_REF] Wanner | Using the stable isotope marker 44 Ca to study dispersal and host-foraging activity in parasitoids: Stable isotope marking of parasitoids[END_REF]. Additionally, the higher availability of resources in semi-natural habitats may prevent natural enemies from moving into crop fields [START_REF] Heimpel | Does Floral Nectar Improve Biological Control by Parasitoids[END_REF].

In our study, positive wildflower strip effects on aphid parasitism and predation combined with the absence of a significant distance-by-strip type interaction indicated a spill-over of parasitoids and generalist predators to central parts of our fields. Although strip effects were weaker in the field than in field margins (predation: only wildflower vs grass strip significant, parasitism: only significant at P<0.1), the results clearly demonstrated the potential of wildflower strips in aphid control. Such an increase of aphid parasitism and/or predation in central parts of the field has not been found in previous studies (Hatt et al. 2017 at 15 m;[START_REF] Pollier | Effects of spontaneous field margin vegetation on the regulation of herbivores in two winter crops[END_REF] at 50 m) but markrecapture experiments showed that parasitoids can move over whole fields [START_REF] Lavandero | Enhancing the effectiveness of the parasitoid Diadegma semiclausum (Helen): movement after use of nectar in the field[END_REF][START_REF] Wanner | Using the stable isotope marker 44 Ca to study dispersal and host-foraging activity in parasitoids: Stable isotope marking of parasitoids[END_REF].

Hoverflies showed a higher abundance close to wildflower strips at 5m in the wheat fields but not at 30m suggesting dilution effects (treatment effects diminishing with distance from the margin) and limited spill-over. Such limited spill-over of hoverflies was also found in studies by Tschumi et al. (2016: potatoes, 1m) and Pollier et al. (2018: wheat, 5m) whereas [START_REF] Hatt | Pest regulation and support of natural enemies in agriculture: Experimental evidence of within field wildflower strips[END_REF] observed an increased hoverfly abundance in central parts of fields with wildflower strip margins. However, our study design may underestimate spill-over since inner parts of the fields may have been influenced by different strip treatments. This may have been avoided by establishing treatments in different fields but a one field -one treatment design increases the risk of confounding effects with other environmental factors such as field management, soil conditions, neighbouring habitats and microclimate.

The positive effect of wildflower strips on hoverflies, aphid predation and aphid parasitism in wheat fields did not result in lower aphid abundance close to the margin. The presence of wildflower strips had also no effect on cereal leaf beetles as the second major pest insect group.

Reviews by [START_REF] Chaplin-Kramer | A meta-analysis of crop pest and natural enemy response to landscape complexity[END_REF] and by [START_REF] Haaland | Sown wildflower strips for insect conservation: a review[END_REF] confirm that such a response of pest insects to an increase in natural enemy abundance is often lacking. In a study on cereal aphid regulation, the density of semi-natural habitats increased for example both natural enemy but also aphid abundance [START_REF] Thies | The landscape context of cereal aphidparasitoid interactions[END_REF]. A lower abundance of aphids and cereal leaf beetles in crop fields adjacent to wildflower strips was found by [START_REF] Tschumi | Perennial, species-rich wildflower strips enhance pest control and crop yield[END_REF] aphids in potato), Hatt et al. (2017: cereal aphids) and [START_REF] Tschumi | High effectiveness of tailored flower strips in reducing pests and crop plant damage[END_REF] cereal leaf beetles), corresponding to a higher natural enemy density. However, controls of these studies were cereal strips instead of spontaneous vegetation which increases the magnitude of wildflower strip effects on natural enemy abundance and thus the probability of detecting spill-over.

Conclusions and implications

The tested wildflower strip mixture established well in all study regions involving a large climatic gradient and increased plant diversity and flower cover compared with spontaneously emerging vegetation and sown grass strips. Such a robust plant species combination represents a cost-efficient and environmentally sound alternative to regionally differentiated species composition, in particular if local provenance can be provided in each region. Core mixtures of wide ecological amplitude may be complemented by several region-specific plant species if logistics is sufficiently developed [START_REF] Pfiffner | Design, implementation and management of perennial flower strips to promote functional agrobiodiversity in organic apple orchards: a pan-European study[END_REF]. Further tests are needed to adjust the optimum diversity level required for efficient biological control [START_REF] Gardarin | The hump-shaped effect of plant functional diversity on the biological control of a multi-species pest community[END_REF].

The increase in flower cover and diversity resulted in a consistent positive effect of wildflower strips on hoverfly abundance and aphid predation within the margin strips potentially improving the control of aphids as major herbivores in several arable crops [START_REF] Tschumi | Perennial, species-rich wildflower strips enhance pest control and crop yield[END_REF][START_REF] Pollier | Effects of spontaneous field margin vegetation on the regulation of herbivores in two winter crops[END_REF]. The widespread practice in France to sow grass mixtures in field margins showed rather negative effects on entomophilous flower cover and parameters of biocontrol since competitive grasses may hamper the development of herbaceous species.

We also demonstrated a spill-over effect of natural enemies into winter wheat fields but no reduction in major pest insect abundance was detected. Further multi-site experiments are needed to develop methods increasing the spill-over into crop fields and to obtain consistent and sufficiently strong effects on pest insect regulation. Differences between strip types are indicated using ">" (P<0.05), "(<)" (P<0.1) and "=" (not significant) with WS: wildflower strip, SV: spontaneous vegetation (unsown) and GS: grass strip. Region-by-strip type interactions are presented if significant.

  Fig. 1, Appendix A: Fig. A.2).

Fig. 1 .

 1 Fig. 1. Number of plant species (A, B), and flower cover (C, D) in different margin strip types

Fig. 2 .

 2 Fig. 2. Hoverfly abundance (A, B), ladybird abundance (C, D) and sentinel aphid predation (E,

Fig. 3 .

 3 Fig. 3. Hoverfly (A), ladybird (B), leaf beetle (C) and aphid abundance (D) measured on 50

Fig. 4 .

 4 Fig. 4. Ordination biplots of Redundancy Analysis (RDA) showing relations between

Table 2 .

 2 Chi-square values of Generalised Linear Mixed Models testing the effect of different strip treatments on vegetation, natural enemies and predation within field margins of different regions. Significant effects and interactions are indicated: ( * ) P<0.1, * P < 0.05, ** P < 0.01, ***

	P < 0.001.
	a) Year 1
		Df	Plant	Sown	Flower	Hoverfly	Ladybird	Aphid
			richness	cover	cover	number	number	predation
	Region	3	8.87*	1.71	1.12	15.92**	3.14	0.78
	Strip type	2	27.80*** 20.50*** 5.18 ( * )	6.50*	4.72	4.26
	Region x strip	6	10.11	4.80	3.02	9.18	4.02	11.27*
	b) Year 2
		Df	Plant	Sown	Flower	Hoverfly	Ladybird	Aphid
			richness	cover	cover	number	number	predation
	Region	4	8.85 ( * )	10.12*	19.41*** 10.68*	6.93	7.09*
	Strip type	2	49.64*** 22.66*** 14.57***	8.65*	1.43	4.99*
	Region x strip	8	18.92*	15.75*	11.03	5.50	4.14	0.72

Table 3 .

 3 Chi square values of Generalised Linear Mixed Models testing different strip treatments at 3 to 4 regions and two distances on pest insects, natural enemies and parasitism/predation within winter wheat fields (year 2). Significance levels: see Table2.

		Df	Hoverfly	Ladybird	Leaf beetle	Aphid	Aphid	Aphid
			number	number	number	number	predation	parasitism
	Region	2-3	6.88*	0.06	4.16	16.32*** 1.24	22.24***
	Strip type	2	33.74***	1.71	1.01	3.23	3.71*	2.46 ( * )
	Distance	1	4.53*	0.63	0.02	0.19	0.03	0.78
	Region x strip	4-6	19.64***	1.59	1.02	7.64	1.18	2.44*
	Distance x	2	28.39***	0.12	1.09	0.38	2.41	0.52
	strip

Acknowledgements

We thank M. Gosme, D. Le Floch, G. Grandeau, L.A.O. Dos Santos, T. Sollet, S. Le Barazer, E. Thenoux, C. Estellon, C. Thomas, M.B. Costa and A. Lubac for assistance in experimental set up, field work and data compilation. We are also grateful to farmers and land managers for hospitality and soil preparation, in particular to the Bergerie de Villarceaux and the experimental INRAE units of Avignon and Dijon. The study was funded by the French National Research Agency (project ANR-12-AGRO-0006 "PEERLESS").

Appendix A. Supplementary data

Differences between strip types are indicated using ">" (P<0.05). "(<)" (P<0.1) and "=" (not significant) with WS: wildflower strip. SV: spontaneous vegetation (unsown and GS: grass strip. Region-by-strip type interactions are presented if significant.