Ivan Castillo

Andrea P Torres-Flores

Diego F Abad-Aguilar

Armando Berlanga-Vázquez

Maylis Orio

Dr Diego Martínez-Otero

Prof Ivan Castillo

M. Sc P Andrea

Diego F Torres-Flores

M. Sc Abad-Aguilar

Armando Berlanga-Vázquez

Prof Maylis Orio

Cellulose Depolymerization with LPMO-inspired Cu Complexes

Keywords: biomass, catalysis, LPMO, biomimetic, copper

Bis(benzimidazole)amine-based copper complexes, with structural similarities to the active sites of Lytic Polysaccharide Monooxygenase enzymes (LPMOs), were tested for the oxidative degradation of cellulose. Spectroscopic characterization of the complexes, as well as structural authentication of one of them, confirm a tetragonal coordination environment with 3 nitrogen donors, as well as a thioether in axial positions. Aqueous oxidative degradation of cellulose was achieved with the Cu II complexes and H2O2 as oxidant through putative cupric-hydroperoxo intermediates. Conversion of cellulose was achieved in up to 67% yield of soluble oligosaccharide derivatives at ambient temperature and pressure. The products were analyzed in aqueous solution by HPLC-MS, confirming oxidative depolymerization of cellulose under ambient conditions, in an analogous fashion to LPMOs.

Copper monooxygenase enzymes have been the object of increasing attention due to their ability to selectively hydroxylate C-H bonds, including those of recalcitrant substrates such as methane. [1] Recently, the breadth of copper enzymes has extended to the family of Lytic Polysaccharide Monooxygenases (LPMOs), [2] which cleave the glycosidic bonds of recalcitrant saccharides such as cellulose and chitin by an oxidative mechanism that results in polymer degradation. [START_REF] Quinlan | Proc. Natl. Acad. Sci[END_REF][START_REF] Phillips | [END_REF] LPMOs are characterized by a monocopper site with three nitrogen donors, with a 'histidine brace' motif defined by an imidazole N-donor and a terminal amine, complemented by an additional histidinederived imidazole, as shown in Scheme 1. The development of catalytic systems akin to LPMO would have technological impact for the use of recalcitrant biomass as a source of renewable feedstocks, including D-glucose and its derivatives. [5] In addition, fundamental questions regarding the oxidizing co-substrate (dioxygen [2] or hydrogen peroxide [6] ), the active copper-oxygen species, [7] and the detailed mechanism of C-H activation remain to be answered.

A handful of inorganic model systems have been developed in attempts to gain insight on the mechanistic details of C-H activation that leads to polysaccharide degradation. Whether hydrogen peroxide acts as a shunt or as the true oxidant in biological systems, its use in conjunction with model copper complexes results in higher degradation activity in all cases. Two model systems containing a mixed imidazole/pyridylamine-based donor set, [8] and the bis(benzimidazole)amine-based 2BB [9] (Scheme 1), provide examples of confirmed oxidative degradation of model substrates. The more recently reported systems based on 2-pyridyl/diazepane, [10] bis(imidazole)amine, [11] and bis(2picolyl)amine [12] ligands degrade p-nitrophenolate-derivatized glucose and/or disaccharides, with the p-nitrophenolate leaving group quantified as the end product by UV-vis spectroscopy. In the first two cases, D-allose was also identified as product, instead of the expected gluconic acid upon hydrolysis of the initially formed gluconolactone; in the latter, the saccharide byproducts were not identified. Attempts to rationalize the catalytic activity of model complexes through analysis of physicochemical properties include correlations of the redox potential of the Cu II/I couple. Nonetheless, even the systems with relatively low half-wave potentials (E1/2) relative to those of LPMOs are active. [8] In this regard, only the (2BB)Cu II/I and bis(imidazole)amine-Cu II/I systems have redox potentials that are within the range of LPMOs [155-370 mV relative to the Standard Hydrogen Electrode (SHE)], [13] although (2BB)Cu II/I was incorrectly identified as having a low E1/2 = 76 mV vs SHE. [11] Scheme 1. Active site of fungal LPMO, the model system [(2BB)Cu II (H2O)2] +2 , and the benzimidazole-based ligands L 1 -L 4 employed in this work.

In light of the discrepancies mentioned above, and to provide more information of the types of systems that may oxidatively degrade the natural polysaccharide substrates to value-added chemicals such as oligo-, monosaccharides (D-glucose) and its dehydrated derivatives (5-hydroxymethylfurfural), we extended this chemistry to the previously reported bis(benzimidazole)amine-based ligands L 2 -L 4 in Scheme 1. [14] HN

N N HN N R N N N N N [(2BB)Cu II (H 2 O) 2 ] +2 L 1 , R = CH 2 SMe L 2 , R = CH 2 S(C 6 H 3 Me 2 ) L 3 , R = C 4 H 3 S L 4 , R = Ph NH N Cu N N NH N H 2 O Cu H Oxidized LPMO OH 2 OH 2 OH 2 +2 OH 2 +2
The novel derivative L 1 was prepared following the procedure reported previously for L 2 -L 4 , by introduction of the benzimidazole moieties to the nitrogen atom of 2-aminoethylmethylthioether with 2 equivs. of N-tert-butoxycarbonyl (BOC)-protected 2chloromethylbenzimidazole. Deprotection of the N-BOC group with concentrated HCl, followed by neutralization, afforded L 1 in 76% yield, see Figs. S1-S3 in the Electronic Supporting Information (ESI). The corresponding cupric complexes were synthesized under conditions reported for similar systems, with Cu(ClO4)2.6H2O or Cu(SO3CF3)2 as Cu II sources. [14] [L 1 Cu(NCCH3)(ClO4)]ClO4 (1) was thus obtained in 92% yield, Xray quality crystals were obtained by slow evaporation of an acetonitrile solution. The complex crystallizes in the orthorhombic space group Pbca, revealing a pseudo-octahedral environment around the Cu II center. The Cu1-N average distance of 1.958 Å to the benzimidazole donors, Cu1-N1 distance of 2.128( 5) Å to the central amine, and Cu1-N6 of 2.021( 6) Å to the acetonitrile molecule are not exceptional, as is the Cu1-S1 distance of 2.646(2) Å for an axial thioether donor bound to a Jahn-Teller distorted Cu II ion. Full characterization and crystallographic data are presented in Figs. S4-S5, Tables S1 andS2 (ESI). 4). [14b] Electrochemical characterization of 1 by cyclic voltammetry (CV) was performed in acetonitrile, affording a half-wave potential of E1/2 = 135 mV relative to the ferrocenium/ferrocene (Fc + /Fc) couple, corresponding to the highest value among the complexes in this study (Fig. 2). On the other hand, 2 has the lowest value at E1/2 = -115 mV (Table 1), thus favoring the oxidized Cu II form. Initial reactivity tests involved in situ reduction of 1, 3, and 4 with 1.5 equivs. of sodium ascorbate at room temperature in CH3CN, evidenced by the disappearance of d-d transitions from 550 to 800 nm in UV-vis absorption spectra. Cooling to -30°C in a cryostat was followed by O2 bubbling through the Cu I solutions for one min; studies with 2 were precluded by its low solubility. For the rest of the complexes, no reactive copper-oxygen species could be discerned in the UV-vis spectra (Figs. S7-S9, ESI), even if 3 and 4 should react with dioxygen based on their redox potentials. Alternatively, we tested the reactions of 1-4 with H2O2 as oxidant.

Hydrogen peroxide is suspected to be the true oxidant in LPMO catalysis, and in model systems it has commonly been employed in conjunction with triethylamine (NEt3) as base to promote the formation of cupric-hydroperoxo (Cu II -OOH) species. [15] The use of methanol as solvent allowed monitoring the reaction at -80°C, since the reactions at -30°C did not reveal significant changes in the absorption spectra. Thus, 1:1 H2O2/NEt3 methanolic solutions were added to the complexes at low temperature for analysis by UV-vis spectroscopy. Changes in the spectra were more evident than in the corresponding oxidations with O2, particularly after the addition of 3 or more equivs. of H2O2/NEt3, Figs. S10-S12. The most informative spectra correspond to those of 4 in the presence of 10 equivs. of oxidant, featuring absorption bands at 347 (e = 1280 M -1 cm -1 ) and 611 nm (e = 570 M -1 cm -1 ) that can be assigned to a putative Cu II -OOH, [16] Figs. 3 and4. Based on its intensity, DFT calculations (see below) and previous reports, the band at 347 nm was assigned as a hydroperoxo to Cu II Ligand to Metal Charge Transfer (LMCT) transition, while the band at 611 nm was assigned as a combination of charge transfer and d-d transitions. Further support for the formation of cupric hydroperoxo [L n Cu II -OOH] + species in the reactions between 1-4 and H2O2/NEt3 was provided by EPR spectroscopy. This technique can discern the presence of monometallic Cu II species in solution, relative to the bimetallic peroxo-bridged dicopper complexes that may also form under the experimental conditions described above. [17] The latter species are expected to be EPR-silent, since the bridging peroxo ligands give rise to antiferromagnetically coupled cupric centers. Instead, addition of 10 equivs. of H2O2/NEt3 to acetonitrile solutions of 1-4 resulted in EPR active species: the originally axial spectra give rise to isotropic ones at 77 K after addition of the oxidant, with giso S3-S7). Geometry-optimized [L 4 Cu II -OOH(ClO4)] (Figs. 4, S22-S23) afforded an EPR calculated spectrum with giso = 2.098 (exp: 2.132102, Table S9) and main UV-vis features at 344 and 629 nm (exp: 347 and 611 nm, Figs. 3 and S24-S26 and Table S10), with the former assigned as HOO→Cu II LMCT band. Once the potential reactive intermediate was identified as the putative cupric hydroperoxo complex, a protocol based on such conditions was adapted for testing the degradation of commercial cellulose. Thus, the use of 1-4 as catalyst precursors allowed us to undertake preparative scale degradation of 30 mg of cellulose in 2 mL of deionized water under benchtop conditions. CuSO4, H2O2/NEt3 mixture, and CuSO4/H2O2/NEt3 were tested as controls.

Stirring for 24 h was followed by filtration to recover the unreacted cellulose to determine the conversion into soluble oligosaccharide derivatives (Fig. S27). In none of the control reactions was cellulose consumed, based on the weight of insoluble material recovered. In contrast, cellulose degradation was observed when 5% mol of complexes 1-4 were used, even in the absence of oxidant mixture, albeit in low yields, entries 1-4 in Table 2. Only marginal differences were observed after 24 and 72 h.

Two possibilities may account for the reactivity in the absence of H2O2/NEt3: the first one corresponds to hydrolysis by the Lewis acidic Cu II centers. [18] The second one would entail conversion of the Cu II complexes to their cuprous analogs mediated by the reducing ends of cellulose. [19] Cu I complexes thus formed could react with O2 from air to oxidatively cleave the polysaccharide chain. The second option was discarded based on the reaction of complexes 1-4 with cellulose under inert atmosphere, resulting in similar degradation degree (4-15%); a hydrolytic mechanism may thus account for some percentage of the observed degradation in all cases. Complex 3 is most active for cellulose degradation in the absence of oxidant, with its ligand featuring a thiophene moiety that may be considered as poorly coordinating. [14b] This may result in a Cu II center with a hemilabile thiophene arm in 3, and a more active catalyst for cellulose hydrolysis (14% after 72 h, entry 3 in Table 2). Catalyst performance improved upon addition of H2O2/NEt3, confirming the predominantly oxidative nature of the cellulose degradation, entries 5-8 in Table 2 and Scheme 2. An advantage of this method is that no pretreatment of cellulose is necessary, [START_REF] Yang | Biofuels[END_REF] and that the reactions proceed at ambient temperature in open vessels. This contrasts with reports that require pretreatment, long reaction times, high temperature, pressure, and high catalyst loadings. [21] Table 2. Cellulose degradation (%) based on solid recovered.

Entry

Catalyst/Additives % Consumed after 24 h % Consumed after 72 h [c] 1 [a] 5% mol of catalyst used. [b] 5% mol of catalyst and 0.5 equiv. of H2O2/NEt3 relative to cellulose. [c] A second load of 0.5 equiv. of H2O2/NEt3 was added after 24 h for entries 5-8. Characterization of soluble products was undertaken by HPLC-MS. Filtration of the aqueous phase through a short plug of Celite to eliminate the catalysts resulted nonetheless in intense peaks corresponding to ligands and their fragments in some chromatograms, that are attributed to catalyst decomposition.

1 [a]
Despite these peaks, analysis of soluble degradation samples of 1/H2O2/NEt3 revealed chromatograms with byproducts such as ONEt3 as a result of the oxidation of NEt3 (detected as [HONEt3] + , Figs. S27, S28), along with the disodium adducts of aldonic acid and cellobiose at long retention times (rt = 23.88, Fig. S28, Scheme 2). The low activity of 2 under the conditions employed is likely due to its low aqueous solubility (Table 2), which did not make it amenable to HPLC-MS analysis.

In the case of the most active catalyst 3, HPLC analysis shows virtually no soluble compounds present in the aqueous phase without oxidant mixture H2O2/NEt3, Fig. S29a; the only peak identifiable by ESI-MS at rt = 14.58 (Fig. S29b) was assigned as protonated L 3 at m/z = 374. S30 and Scheme 2). Similar fragments and cellulose degradation products were detected when 4 was employed as catalyst (Fig. S31). Although these results that challenges remain, such as the higher sensitivity for species that are readily protonated like the amines employed as ligands, and that higher glucose oligomers that may be present in solution after hydrolysis or oxidative cleavage cannot be detected, [22] the feasibility to employ relatively simple cupric complexes as catalysts for cellulose degradation under benchtop conditions is encouraging.

In summary, benzimidazole-based copper complexes with coordination environments and redox potentials similar to those in LPMO monooxygenase enzymes are competent cellulose degradation catalysts under mild conditions, with Cu II -OOH species forming in the presence of H2O2/NEt3 as oxidant mixture.

Combined quantification of the remaining insoluble substrate that is not consumed during the reaction and analysis of the soluble portion by HPLC-MS provides information of the oxidative degradation of cellulose. The possibility to extend this strategy to recalcitrant substrates such as chitin and lignocellulose with the complexes herein reported, as well as LPMO-inspired derivatives, is currently under investigation in our laboratory.

Entry for the Table of Contents

LPMO-inspired benzimidazole-based copper complexes oxidatively degrade cellulose to cellobiose, aldonic acid and levoglucosan as soluble products in the presence of hydrogen peroxide/triethylamine as oxidant mixture under mild conditions through initially formed cupric hydroperoxo intermediates.

Institute and/or researcher Twitter usernames: Ivan Castillo @IvanCas68864020, Maylis Orio @orio52503713

Figure 1 . 4

 14 Figure 1. Mercury diagram of 1 at the 50% probability level, H atoms and perchlorate counterions (except for O1) omitted for clarity. Color code: C, grey; N, blue; O, red; Cl, green; S, yellow; Cu, turquoise.

Figure 2 .

 2 Figure 2. Cyclic voltammogram of 1 mM 1 in CH3CN with 0.1 M [n-Bu4N]PF6 as electrolyte; glassy carbon electrode, scan speed 0.1 V s -1 .

Figure 3 .

 3 Figure 3. UV-vis spectra of 1 mM 4 in MeOH at -80°C upon addition of 1:1 H2O2/NEt3 mixture (4-10 equivs.); inset: TD-DFT calculated UV-vis spectrum.

  = 2.102 to 2.107 (Figs. 4 and S13-S15). While no geometric information can be derived from the spectra, they are consistent with monometallic Cu II species. Investigation of the properties of [L 4 Cu II -OOH(ClO4)] by DFT calculations involved geometry optimization of the parent 4, validated by calculation of its redox potential at -70 mV (exp: -75 mV, Figs. S16-S21, Tables

Figure 4 .

 4 Figure 4. EPR spectrum of 2 mM 4 after addition of 10 equivs. H2O2/NEt3 mixture at -30°C in acetonitrile (acquired at 77 K), and DFT-minimized structure of [L 4 Cu II -OOH(ClO4)] formed in the presence of H2O2/NEt3.

Scheme 2 .

 2 Scheme 2. Oxidative depolymerization of cellulose catalyzed by 1-4; R and R' represent the polymer chains of cellulose.

Table 1 .

 1 Anodic (Ea), cathodic (Ec), DE (Ea -Ec), and half-wave (E1/2) potentials for Cu I salt and 1-4 (mV vs Fc + /Fc).

	Compound	Ea	Ec	DE	E1/2
	[Cu(NCCH3)4]ClO4	760	550	210	655
	1	210	60	150	135
	2 [a]	-60	-170	110	-115
	3 [a]	-10	-160	150	-85
	4 [a]	40	-190	230	-75

[a] From reference 13b.

  15. Similar results were obtained in the absence of oxidant with 4, revealing only protonated L 4 at m/z = 368.2. Soluble products obtained in the presence of H2O2/NEt3 were analyzed in the same manner, and the results lend further support for the oxidative cleavage of cellulose, as opposed to a hydrolytic transformation. Samples of cellulose treated with 3/H2O2/NEt3 resulted in chromatograms that differ significantly from those obtained from 3 alone: [HONEt3] + (rt = 1.86), [L 3 H] + (rt = 14.53) and degradation fragments at rt = 11.76, 22.37, were detected along with peaks assigned to protonated levoglucosan (rt = 4.90, m/z = 163.05), cellobiose (rt = 19.05, m/z = 343.13), and the disodium adduct of gluconic acid (rt = 23.86, m/z = 242.29, Fig.

Acknowledgements

The authors thank Simón Hernández-Ortega for crystallographic assistance, Virginia Gómez-Vidales for EPR spectroscopy, Lucero Ríos and María del Carmen García for mass spectrometry, María de la Paz Orta for combustion analysis, Everardo Tapia for HPLC-MS, DGAPA-PAPIIT (IN217020), Conacyt-ECOS Nord (291247) and Conacyt (A1-S-8682, Beca 778895) for financial support.


Coralie Triquet
email: triquet.coralie@gmail.com

Anthony Roume

Alexander Wezel

Vincent Tolon

Aurélie Ferrer

In-field cover crop

Keywords: Carabidae, mid-field vegetation strip, winter cover crop, edge effect, ecological traits, pest regulation, conservation biological control

come

Introduction

Biodiversity loss has been recorded in the last years, with entomofauna being particularly endangered [START_REF] Didham | Interpreting insect declines: seven challenges and a way forward[END_REF][START_REF] Sánchez-Bayo | Worldwide decline of the entomofauna: A review of its drivers[END_REF][START_REF] Wagner | Insect Declines in the Anthropocene[END_REF].

Agricultural activities are identified as one of the main drivers of arthropod biodiversity loss [START_REF] Seibold | Arthropod decline in grasslands and forests is associated with landscape-level drivers[END_REF][START_REF] Stoate | Ecological impacts of arable intensification in Europe[END_REF] with the intensification of land use and landscape simplification in agricultural landscapes [START_REF] Holland | Structure, function and management of semi-natural habitats for conservation biological control: a review of European studies: Structure, function and management of semi-natural habitats for biological control[END_REF]. However, the primary role of entomofauna and their associated ecosystem services provision (e.g. pollination, pest and weed regulation) for agricultural production has been widely demonstrated [START_REF] Albrecht | The effectiveness of flower strips and hedgerows on pest control, pollination services and crop yield: a quantitative synthesis[END_REF][START_REF] Martin | The interplay of landscape composition and configuration: new pathways to manage functional biodiversity and agroecosystem services across Europe[END_REF].

Decreasing detrimental disturbances and increasing plant diversity and semi-natural habitats inside and outside the agricultural production area is an efficient approach to mitigate the loss of vulnerable arthropod species [START_REF] Ekroos | Sparing Land for Biodiversity at Multiple Spatial Scales[END_REF]. In this respect, agri-environmental schemes have been developed in the EU policies and proposed to farmers, aiming to address this challenge and increase biodiversity and ecosystem services in agricultural landscapes [START_REF] Martin | The interplay of landscape composition and configuration: new pathways to manage functional biodiversity and agroecosystem services across Europe[END_REF]. Among them, different agroecological infrastructures (AEI) are targeted such as vegetation strips. Generally placed at the edge of cropped fields, they have a particular interest for multiple ecosystem services, e.g. water regulation and purification, and prevention of soil erosion. Many studies have shown the attractiveness of vegetation strips for a large range of beneficial arthropods [START_REF] Campbell | Wildflower plantings harbor increased arthropod richness and abundance within agricultural areas in Florida ( USA )[END_REF][START_REF] Holland | Structure, function and management of semi-natural habitats for conservation biological control: a review of European studies: Structure, function and management of semi-natural habitats for biological control[END_REF][START_REF] Kujawa | Annual Wildflower Strips as a Tool for Enhancing Functional Biodiversity in Rye Fields in an Organic Cultivation System[END_REF][START_REF] Triquet | Undestroyed winter cover crop strip in maize fields supports ground-dwelling arthropods and predation[END_REF], including pollinators when they contain flowers [START_REF] Amy | Flower Strips in Wheat Intercropping System: Effect on Pollinator Abundance and Diversity in Belgium[END_REF][START_REF] Haaland | Sown wildflower strips for insect conservation: a review: Wildflower strips for insect conservation[END_REF]. They can offer food resources, shelter [START_REF] Griffiths | Efficacy and economics of shelter habitats for conservation biological control[END_REF][START_REF] Landis | Manipulating plant resources to enhance beneficial arthropods in agricultural landscapes[END_REF] and overwintering habitat for ground-dwelling arthropods [START_REF] Landis | Habitat Management to Conserve Natural Enemies of Arthropod Pests in Agriculture[END_REF][START_REF] Pywell | Determinants of overwintering habitat quality for beetles and spiders on arable farmland[END_REF], and thus are expected to deliver ecosystem services to adjacent cropping [START_REF] Bianchi | Habitat functionality for the ecosystem service of pest control: reproduction and feeding sites of pests and natural enemies[END_REF].

The effectiveness of ecosystems delivery by entomofauna from vegetation strips thus relies on the spillover of the beneficial arthropods into adjacent fields [START_REF] Bianchi | Sustainable pest regulation in agricultural landscapes: a review on landscape composition, biodiversity and natural pest control[END_REF][START_REF] Blitzer | Spillover of functionally important organisms between managed and natural habitats[END_REF]. Such spillover and the effect of AEI in general on the adjacent crops have been mainly investigated through the measurement of the abundance and richness of beneficial arthropods. Some studies showed such spillover effect [START_REF] Gayer | Carabid functional diversity is enhanced by conventional flowering fields, organic winter cereals and edge habitats[END_REF][START_REF] Woodcock | Spill-over of pest control and pollination services into arable crops[END_REF], but in others this could not be clearly observed [START_REF] Kujawa | Annual Wildflower Strips as a Tool for Enhancing Functional Biodiversity in Rye Fields in an Organic Cultivation System[END_REF][START_REF] Triquet | Undestroyed winter cover crop strip in maize fields supports ground-dwelling arthropods and predation[END_REF]. Indeed, little is known about this process, and particularly about the impact of AEI on the composition and the ecological trait distribution of arthropod communities in adjacent fields at different distances from the AEI [START_REF] Boetzl | Agri-environmental schemes promote ground-dwelling predators in adjacent oilseed rape fields: Diversity, species traits and distance-decay functions[END_REF][START_REF] Gallé | Arthropod functional traits shaped by landscape-scale field size, local agri-environment schemes and edge effects[END_REF][START_REF] Gayer | Carabid functional diversity is enhanced by conventional flowering fields, organic winter cereals and edge habitats[END_REF][START_REF] Pecheur | The influence of ecological infrastructures adjacent to crops on their carabid assemblages in intensive agroecosystems[END_REF]). Yet, pointed out that taxonomic and functional diversity of natural enemies plays a crucial role to enhance and maintain natural pest control and weed regulation in crops [START_REF] Crowder | Organic agriculture promotes evenness and natural pest control[END_REF][START_REF] Northfield | Niche saturation reveals resource partitioning among consumers[END_REF][START_REF] Rusch | Predator body sizes and habitat preferences predict predation rates in an agroecosystem[END_REF].

Carabids have been particularly studied as they have a high contribution to pest and weed regulation in arable crops [START_REF] Bohan | National-scale regulation of the weed seedbank by carabid predators: Carabid seed predation[END_REF][START_REF] Kulkarni | The Role of Ground Beetles (Coleoptera: Carabidae) in Weed Seed Consumption: A Review[END_REF]. This contribution is driven by their feeding characteristics and dispersal ability, of which ecological traits such as body size, diet and wing status give an indication [START_REF] Bertrand | Spatial and temporal heterogeneity of the crop mosaic influences carabid beetles in agricultural landscapes[END_REF][START_REF] Emmerson | Predator-prey body size, interaction strength and the stability of a real food web[END_REF]. For example, smaller species with a higher dispersal ability seem more capable to penetrate into cropped areas and consume pests in the centre of fields, while larger predatory carabids are able to consume a higher amount of pests and bigger prey, but may also feed on smaller predators resulting in intra-guild predation [START_REF] Rusch | Predator body sizes and habitat preferences predict predation rates in an agroecosystem[END_REF][START_REF] Williams | Ground Beetles as Predators of Oilseed Rape Pests: Incidence, Spatio-Temporal Distributions and Feeding[END_REF]. Thus, the effect of AEI on carabids community composition and more particular on their traits repartition will have an impact on the ecosystem services delivered in the adjacent crop, and with this on the functionality of the AEI.

Beyond the lack of knowledge about their functionality, vegetation strips and wildflower strips remain poorly adopted in France because of technical and economic constraints such as the additional working time and costs needed for implementation and maintenance [START_REF] Uyttenbroeck | Pros and cons of flowers strips for farmers. A review[END_REF]. Agroecological practices such as integration of AEI, if they are based on common practices and easy to implement in existing cropping systems, can be easily adopted by farmers [START_REF] Pearsons | In-Field Habitat Management to Optimize Pest Control of Novel Soil Communities in Agroecosystems[END_REF][START_REF] Pe'er | Adding Some Green to the Greening: Improving the EU's Ecological Focus Areas for Biodiversity and Farmers[END_REF]. For example, maintaining a strip of a previous winter cover crop in the middle of a spring crop has been recently documented and showed potential for ground-dwelling arthropods support and the enhancement of predatory activities in maize fields [START_REF] Triquet | Undestroyed winter cover crop strip in maize fields supports ground-dwelling arthropods and predation[END_REF]. However, the effects of such mid-field cover crop strips on the composition of carabid communities and the distribution of their ecological traits remain to be explored. In this study, we tested these effects for the first year of implementation of mid-field cover crop strips, and compared the results with the community composition and distribution of ecological traits in permanent grassy field margins. We hypothesized that the strip, by providing an uncropped habitat in the middle of the field, could support a carabid community and traits distribution patterns reflecting a mix between the species from the surrounding cropped area and the permanent field margin. We also hypothesized an edge effect around the strip within the cropped area due to spillover or attractiveness of some species. We therefore predicted that:

1.

The carabid community in the preserved cover crop strip is composed of a mix of some species from the cropped area and the permanent field margin, with activity-densities in the same order of magnitude;

2.

In the cover crop strip, the distribution of ecological traits of carabids reflects the combination of the ecological traits of the field margin and of the cropped area;

3.

The distribution of ecological traits in the cover crop strip evolves during the season towards a margin-like ecological trait distribution (because the strip remains longer undisturbed and vegetation characteristics evolve more towards those of perennial herbaceous habitats);

4.

In the cropped area, distance from the strip influences the carabid community composition and the spatial distribution of their ecological traits, in particular the trait distribution in the close proximity to the cover crop strip will have some common points with the cover crop strip, due to spillover or aggregation of some species.

Materials and methods

Experimental design

The field trails took place on 12 conventionally managed fields, on commercial farms located in the region Auvergne-Rhône-Alpes (France), in the Rhône, Ain, Isère and Drôme districts. The climate in the study area is temperate semi-continental with Mediterranean influence during summer. The fields were located in landscapes dominated by conventional specialized arable cropping systems and mixed crop livestock systems. Field experiments were conducted on twelve different maize fields, five in 2019 and seven in 2020, which size ranging from 1.1 to 5.7 ha. The mean distance between strips was 46 km (from 0.2 to 95 km). No insecticides were applied on the fields during the experiment.

In the middle of each field, a strip of 5 m width and 110 m length of the previous winter cover crop sown in autumn the year before was preserved, and remained uncultivated and not mowed during the whole maize cultivation cycle, from sowing in April-May to harvest in October. This strip was connected to a grassy field margin (Fig. 1). The vegetation of the strip was composed of a commercial cover crop mixture sown with 37.5 % ray grass (Lolium

Arthropod sampling and ecological traits

Carabids were monitored using pitfall traps (10 cm opening diameter, 15 cm depth, filled with of a 30% propylene glycol solution with a drop of detergent) protected from rain by a plastic roof [START_REF] Woodcock | Pitfall trapping in ecological studies, in: Insect Sampling in Forest Ecosystems[END_REF]. The pitfall traps were placed in the cover crop strip, in the connected grassy field margin and in the cultivated area at 10 m, 25 m and 50 m from the strip (defined as the five studied "zones" in the article). Traps were placed in and around the strip along three parallel transects as shown in Figure 1. There were in total five sampling periods: before winter cover crop ploughing (initial stage, IS), at the maize seeding (S), at the 4 leaves (4L) and 8 leaves (8L) stages of maize development, and after maize harvest (final stage before the cover crop strip destruction, FS). Pitfall traps were active during 48 hours for each sampling period.

Carabids were identified at the species level. We selected three ecological traits of carabids linked with their feeding and dispersal characteristics: diet (predatory, polyphagous, phytophagous), wing status (macropterous, dimorphic, wingless; giving indication of dispersal ability), body size (size classes: class A ≤ 5 mm, class B ≤ 10 mm, class C ≤ 15 mm, class D ≤ 25 mm and class E > 25 mm). These ecological traits were determined according to the available literature [START_REF] Jeannel | Coléoptères Carabiques[END_REF][START_REF] Jeannel | Coléoptères Carabiques[END_REF][START_REF] Lindroth | The ground-beetles (carabidae) of fennoscandia. A zoogeographic study[END_REF][START_REF] Ribera | Effect of Land Disturbance and Stress on Species Traits of Ground Beetle Assemblages[END_REF].

Data analysis

We first explored graphically the activity-density variations of each species through sampling periods (IS, S, 4L, 8L and FS) in order to identify major changes in community compositions in the five sampled zones (margin, strip, 10 m, 25 m, 50 m). We conducted compositional dissimilarity analyses based on Bray-Curtis distances to assess the impact of site, sampling period and zone on the differences in community compositions. For this, analysis of similarity (ANOSIM) and Permutational analysis of variance (PERMANOVA)

were conducted with 9999 permutations, respectively using anosim and adonis2 functions of the R vegan package [START_REF] Faith | Compositional dissimilarity as a robust measure of ecological distance[END_REF][START_REF] Warton | Distance-based multivariate analyses confound location and dispersion effects[END_REF]. We selected the R value of ANOSIM analysis as an indicator of the dissimilarity between groups (from 0 to 1, 1 being the highest dissimilarity, R compares the mean of ranked dissimilarities between groups to the mean of ranked dissimilarities within groups). We used the R 2 values from the PERMANOVA models as quantification of the magnitude of change in communities among groups (explained by site, sampling period or zone). In addition, a pairwise PERMANOVA (multilevel comparison) analysis was conducted for the variable "zone" (Martinez Arbizu, 2020).

To detect the potential species significantly associated to groups of sites or zones, we computed indicator species analyses by calculating the Point Biserial correlation coefficient [START_REF] De Cáceres | Associations between species and groups of sites: indices and statistical inference[END_REF] with 9999 permutations, with the function multipatt from the R package indicspecies [START_REF] De Cáceres | Improving indicator species analysis by combining groups of sites[END_REF]. Each analysis was carried out using data from Initial Stage (IS) to Final Stage (FS), and for comparison was repeated using only data from the three sampling periods during maize development (S, 4L, 8L) which are close in time (from April to beginning of July) and avoid the major changes occurring after IS and before FS: ploughing and maize harvest. We also used this method to check if the main species were not associated with only one site.

Analyses looking at ecological traits instead of species composition of community were then performed to explore a potential similar distribution and evolution in time of ecological traits in the different zones among sites. In order to assess the relations between environmental variables (sampling period and zone) and ecological traits of carabids, co-inertia RLQ analysis [START_REF] Dray | Combining the fourth-corner and the RLQ methods for assessing trait responses to environmental variation[END_REF] was conducted with the ade4 R package. A trajectory scatter plot was used for the visualization of the spatio-temporal evolution of traits distribution in carabid community. Data used for the RLQ analysis were pooled by zones, by sampling periods, and by sites. A Monte Carlo randtest (9999 permutations, function randtest of ade4 package) was computed to test the overall association between ecological traits and environmental variables. The fourth corner method was used to determine pairwise significant associations between ecological traits and environmental variables [START_REF] Dray | Combining the fourth-corner and the RLQ methods for assessing trait responses to environmental variation[END_REF].

All statistical analyses were conducted using R, version 4.1.0 (R Development Core Team, 2021).

Results

A total of 4162 carabids were sampled in the 1352 pitfall traps taken into account all sampling periods. In total, 76 different carabid species were collected. The three most abundant species were Anchomenus dorsalis, Poecilus cupreus, and Pterosticus melanarius (see complete list of carabid species in Supporting Information A). From the delimitation of the strip (i.e. from the "S" sampling period), the strip harboured 35% A. dorsalis of total species abundance, 13% P. cupreus, and 5% P. melanarius, and in the cropped area there were 37% P. melanarius, 20% P. cupreus and 10% A. dorsalis. In the grassy margin, we counted 23% of A. dorsalis, 9% of P. melanarius and 2% of P. cupreus.

Community species composition

The carabid community composition was significantly different between sites (p<0.001), and the effect of site was stronger than the effect of zone or sampling period (R=0.269 from IS to FS, R=0.340 from S to 8L, Table 1). PERMANOVA analysis showed that the effect of the interaction between site and zone and between site and sampling period were highly significant (p<0.001, Table 1), indicating that the variation of community composition between zones and periods differed among sites, taking into account the full sampling period or only the main maize growing period. The R² outcomes of the PERMANOVA indicated that the site explained approximatively 21% of the community composition of carabids, and zone and sampling period together (with their interaction) explained only 6% of the variations in the community (Table 1). At the beginning of the experiment (IS), we could observe two distinct carabid communities: one in the margin and the other in the winter cover crop. Then, from the S sampling period, three different communities can be detected: in the margin, in the crop, and in the strip (the later with a mix of species present in the margin and in cropped area communities), which changed at 4L with the addition of carabid species (Fig. 2). The community composition was significantly different among zones (p<0.001) and among sampling periods (p<0.001, Table 1). The PERMANOVA analysis validated these significant effects (p<0.001 for both zone and period) and indicated a significant interactive effect between zone and sampling period (p<0.001 from IS to FS, p<0.001 from S to 8L, Table 1), highlighting that the community composition among zones changed differently through time, even during the main maize growth period (see Supporting Information C showing beta diversity). The pairwise multilevel comparison showed that the community was not significantly different between the 3 sampling zones within the crop (10 m, 25 m, 50 m; Table 1).

Looking at indicator species analyses by groups of zones, A. dorsalis was significantly associated with the strip zone, (p<0.001, Supporting Information B). It was indeed observed from IS sampling period in the margin, and then colonized during S also the strip where it became the most abundant until maize harvest (Fig. 2). P. cupreus was observed from S to 8L in the strip and in the cropped area, and was the most frequently found at 10 m from the strip. We further could observe a gradient of decreasing activity-density of P. cupreus from 10 m to 50 m from the strip in the cropped area (Fig. 2). This was validated by the indicator species analysis showing that P. cupreus was significantly associated with the "0 m + 10 m + 25 m" zone group (corresponding to strip and the closest part of the cropped area from the strip) (p<0.001, Supporting Information B). P. melanarius was significantly associated with the "10 m + 25 m + 50 m" zone group (corresponding to the cropped area, p<0.001 from IS to FS and p<0.001 from S to 8L, Supporting Information B), and was observed from S to FS in the strip and the cropped area, with an important peak at 4L in the cropped area. As for P.

cupreus, it was the most abundant at 10 m from the strip and with a gradient of decreasing activity-density from 10 m to 50 m from the strip (Fig. 2). Finally, the genera Harpalus was sampled in all three zones but some species were only found in the strip and in the margin.

Species from the genera Carabus were observed from S in the margin, and later mostly found in the strip at 4L sampling period. Other species such as Brachinus scolpeta and Amara similata were recorded mainly in the strip (Fig. 2, see Supporting Information A for the composition of the community), which were significantly associated only to the strip or the grassy margin (Supporting Information B).

Ecological traits composition

The carabid communities were dominated by strictly predatory species in every habitat (68% in the field margin, 72% in the strip, and 65% in the crop, see Figure 3). Cumulating the sampling periods from S to FS, the ecological trait distribution in the strip was more similar to the cropped area regarding the diet trait, but more similar to the margin regarding the morphological traits (wing status and body size, Fig. 3). The RLQ analysis showed a significant overall association between ecological traits composition and environmental variables (Monte Carlo randtest, p<0.05). The two first RLQ axes explained 74% of the total inertia (47% and 27%, respectively).

At the initial stage (IS), the trait distribution was quite similar in every zone (top right part of scatter plot in Fig. 4a), with a specific presence of small dimorphic and phytophagous species (right part of scatter plot in Fig. 4b). This period was also characterised by a lower abundance of the medium sized (size C) and polyphagous species. At the beginning of maize growth (S and 4L), a shift appeared in the cropped area (10 m, 25 m and 50 m) that hosted more medium sized and polyphagous species (bottom left part of Fig. 4a,b). This pattern also affected to a lower extent the strip and the margin (bottom right part of Fig. 4a,b). From the 8L sampling period and until the harvest (FS), the 25 m and 50 m zones were clearly occupied by large (class D) and wingless species (top left part of Fig. 4a,b). The trait distribution at 10 m did not change at 8L but followed the same shift toward large and wingless species at FS. From 8L, the margin remained associated to small and dimorphic species until the end of the harvest (FS), while the strip included more medium sized and polyphagous species than before (bottom left part of Fig. 4a, b, trait distribution close to what was observed during maize growth in the cropped area).

Fourth corner analysis applied to the carabids data showed a significant negative association between the body size class B and the zones at 25 m and 50 m from the strip in the crop (p = 0.0385, Fig. 5). This outcome confirmed the rapid shift toward large species of the farthest cropped area from the strip observed in the Figure 4, but the relatively small data set and the correction applied on p-values due to numerous tests probably explained a lack of significant results.

Figure 4. Ordination plot of the RLQ co-inertia analysis on carabids community, with a) trajectory scatter plot of each zone through time (IS, S, 4L, 8L and FS), b) ecological traits of carabids, and c) carabid species (code names correspondence see Supporting Information A)

. Note that by definition the RLQ analysis often highlights the traits that highly specific to a habitat or period (e.g. dimorphic, phytophagous or polyphagous), but the widespread traits appear to be non-discriminant (e.g. predatory, wingless or macropterous).

Figure 5. Results of fourth-corner statistics for the pairwise associations between environmental variables and carabids ecological traits. Coloured squares represent significant associations (red positive; blue negative) at the p < 0.05 level.

Discussion

Differences in communities of the field margin, the strip and the cropped area

The field margin, the cover crop strip and the cropped area had a significantly different community composition. This is consistent with previous studies showing that community species composition is different between uncropped and cropped areas [START_REF] Boetzl | Agri-environmental schemes promote ground-dwelling predators in adjacent oilseed rape fields: Diversity, species traits and distance-decay functions[END_REF][START_REF] Pecheur | The influence of ecological infrastructures adjacent to crops on their carabid assemblages in intensive agroecosystems[END_REF], and between perennial field margins and recently established vegetation strips [START_REF] Maas | Functional traits driving pollinator and predator responses to newly established grassland strips in agricultural landscapes[END_REF].

The differences of community composition between zones were significantly impacted by the period effect, even during the maize growth, showing the rapid evolution of carabid communities in the agroecosystem along with seasons and human interventions. The initial carabid community of the winter cover crop has been disturbed by the ploughing and sowing in the cropped area, and consequently showing a different community than in the undestroyed cover crop strip. The strip community did not comprise only a mix of the species of the field margin and the cropped area, as we predicted, but some species were also found mostly (e.g. Brachinus scolpeta, Amara similata, Carabus cancellatus) or only (e.g.

Syntomus obscuroguttatus

) in the strip. This observation is coherent with some ecological traits of these species. Indeed, Amara spp. are known to be mostly found in non-cropped habitats where the soil is less compacted and easier to dig galleries in [START_REF] Pywell | Determinants of overwintering habitat quality for beetles and spiders on arable farmland[END_REF][START_REF] Thomas | Aggregation and temporal stability of carabid beetle distributions in field and hedgerow habitats: Spatial distributions of carabid beetles[END_REF]. B. sclopeta is indicted to have an affinity with rapeseed [START_REF] Marrec | Crop succession and habitat preferences drive the distribution and abundance of carabid beetles in an agricultural landscape[END_REF], and its presence in the strips could potentially be explained by the plant composition of the cover crop, which includes turnip rape being a plant closely related to rapeseed. These different outcomes show that implementing such vegetation strips can not only support carabid species within fields from the first year of establishment, but also, by hosting a community different from a crop area or a perennial field margin, can support local diversity or species richness. In the same way, the singular characteristics of the margins in term of carabid community confirm the absolute importance of protecting the perennial field margins to preserve species that are dependent on stable habitats and their associated ecosystem functions [START_REF] Maas | Functional traits driving pollinator and predator responses to newly established grassland strips in agricultural landscapes[END_REF].

The effect of habitat type on carabid communities has also been observed on the distribution of ecological traits. First of all, we observed that the overall ecological trait distribution in the strip had similarities with both the margin and the cropped area, as we predicted. At the initial stage, the trait distribution was quite similar in the field margin and the cover crop field, with mainly very small species (less than 5 mm), and then this distribution started to have a different trajectory. During the maize growth (i.e. from S to 8L corresponding to the period from April to July), we recorded the highest activity-density of carabids, with polyphagous, wingless and large species (sized between 10 and 25 mm) more present in the cropped area. Likewise, previous studies have shown a higher proportion of polyphagous and more mobile species (i.e. larger and/or winged carabids, [START_REF] Homburg | Broad-scale geographic patterns in body size and hind wing development of western Palaearctic carabid beetles(Coleoptera: Carabidae)[END_REF] in cropped fields than in uncropped habitats [START_REF] Boetzl | Agri-environmental schemes promote ground-dwelling predators in adjacent oilseed rape fields: Diversity, species traits and distance-decay functions[END_REF][START_REF] Gayer | Carabid functional diversity is enhanced by conventional flowering fields, organic winter cereals and edge habitats[END_REF], probably linked with their higher ability to live in more frequently disturbed habitats [START_REF] Cardarelli | Effects of grass management intensity on ground beetle assemblages in rice field banks[END_REF] and with particular species known to breed in field centre areas such as P.

melanarius [START_REF] Holland | Contrasting the farm-scale spatio-temporal dynamics of boundary and field overwintering predatory beetles in arable crops[END_REF][START_REF] Jowett | Species matter when considering landscape effects on carabid distributions[END_REF]. During the same period, the cover crop strip and the field margin hosted more phytophagous and macropterous species. They hosted also more species of two distinct groups of size: small carabids (5 to 10 mm), and very large carabids (more than 25 mm) corresponding to species of the genera Carabus, mostly found in the cover crop strip. These results are partly consistent with previous studies that reported a higher proportion of phytophagous and smaller carabids in vegetation strips or field margins than in crops [START_REF] Baulechner | Converting arable land into flowering fields changes functional and phylogenetic community structure in ground beetles[END_REF][START_REF] Birkhofer | Grassy margins along organically managed cereal fields foster trait diversity and taxonomic distinctness of arthropod communities[END_REF][START_REF] Gallé | Arthropod functional traits shaped by landscape-scale field size, local agri-environment schemes and edge effects[END_REF].

The higher density and richness of plants in vegetation strips and grassy margins provide a more abundant and diverse food resource for phytophagous species and favour their presence [START_REF] Kulkarni | The Role of Ground Beetles (Coleoptera: Carabidae) in Weed Seed Consumption: A Review[END_REF]. The strip had a tall vegetation with large flowering plants (e.g. phacelia, turnip rape), which was more heterogeneous but less dense than the vegetation of the field margin (mainly composed of grasses). Consequently, the strip probably provided a more suitable habitat for the largest carabids (Carabus spp.) which cannot easily disperse in a dense vegetation [START_REF] Ng | Disentangling the effects of farmland use, habitat edges, and vegetation structure on ground beetle morphological traits[END_REF][START_REF] Rouabah | Response of carabid beetles diversity and size distribution to the vegetation structure within differently managed field margins[END_REF].

Effect of the distance from the strip into the adjacent crop

The two dominant species in the field (P. cupreus and P. melanarius) were the most abundant in the cropped area at 10 m from the strip, but not in the strip, validating our fourth prediction concerning species composition. This affinity with the proximity of the strip could be interpreted both as a spillover effect from the strip [START_REF] Holland | Contrasting the farm-scale spatio-temporal dynamics of boundary and field overwintering predatory beetles in arable crops[END_REF][START_REF] Schellhorn | Movement of Entomophagous Arthropods in Agricultural Landscapes: Links to Pest Suppression[END_REF], or as a phenomenon of aggregation towards the strip [START_REF] Bianchi | Spatial variability in ecosystem services: simple rules for predator-mediated pest suppression[END_REF]. However, the latter explanation fits better with our results as the activity-density of P. cupreus and P.

melanarius was not at a high level in the strip. Therefore, the strip cannot be considered as a preferred habitat acting as a source habitat generating a spillover effect into the adjacent crop for these two species [START_REF] Bianchi | Habitat functionality for the ecosystem service of pest control: reproduction and feeding sites of pests and natural enemies[END_REF][START_REF] Blitzer | Spillover of functionally important organisms between managed and natural habitats[END_REF][START_REF] Jowett | Species matter when considering landscape effects on carabid distributions[END_REF]. The cover crop strip comprised several flowering plant species, which attracted many arthropods, including flying insects which can be prey for many ground-dwelling predatory arthropods [START_REF] González-Chang | Habitat Management for Pest Management: Limitations and Prospects[END_REF][START_REF] Holland | Structure, function and management of semi-natural habitats for conservation biological control: a review of European studies: Structure, function and management of semi-natural habitats for biological control[END_REF]. Consequently, a high amount of prey in the proximity of the strip could have attracted these entomophagous carabids [START_REF] Anjum-Zubair | Influence of within-field position and adjoining habitat on carabid beetle assemblages in winter wheat[END_REF], potentially leading, in turn, to an increased pest regulation in the zone [START_REF] Triquet | Undestroyed winter cover crop strip in maize fields supports ground-dwelling arthropods and predation[END_REF][START_REF] Woodcock | Spill-over of pest control and pollination services into arable crops[END_REF]. However, unobserved but possible drawbacks of the attractiveness of the strip for many arthropods could be the increase of flying crop pests, and the concentration of local carabids in the strips, hence reducing their abundance in the cropped area and creating an ecological trap when the strip is destroyed after maize harvest [START_REF] Ganser | Sown wildflower strips as overwintering habitat for arthropods: Effective measure or ecological trap?[END_REF].

Regarding the distribution of ecological traits, the body size class was the most affected by the distance from the strip. During the maize growth, small carabids (between 5 mm and 10 mm) were less present in the farthest zones from the strip in the cropped area. Thus, in the vicinity of the strip, we recorded more small carabids such as these associated with the strip, indicating a possible spillover effect of some species from the strip [START_REF] Schellhorn | Movement of Entomophagous Arthropods in Agricultural Landscapes: Links to Pest Suppression[END_REF] in search for food resources [START_REF] Tscharntke | Conservation biological control and enemy diversity on a landscape scale[END_REF]. This effect of distance from the strip on carabids size validates our fourth prediction concerning ecological traits, and it is consistent with previous studies showing that the size of carabids increased with the distance of the field margin or an AEI [START_REF] Boetzl | Agri-environmental schemes promote ground-dwelling predators in adjacent oilseed rape fields: Diversity, species traits and distance-decay functions[END_REF][START_REF] Gallé | Arthropod functional traits shaped by landscape-scale field size, local agri-environment schemes and edge effects[END_REF][START_REF] Pecheur | The influence of ecological infrastructures adjacent to crops on their carabid assemblages in intensive agroecosystems[END_REF].

Influence of the experimental site and possible links with landscape context

The species composition of carabids community significantly differed among experimental sites, and the effect of the site was stronger than the effect of the zone or the sampling period.

In this context, the analysis of the communities through ecological traits allowed us to detect other relevant trends in the evolution of carabid communities in different habitats, beyond species identity. We found a distinct overall distribution of ecological traits of carabids in the three habitats, reflecting the capacity of different habitat to provide different resources more fitting with the needs and abilities of different carabids species to use these habitats (e.g. more phytophagous species where more weed seeds are available, smaller species where the density of vegetation is high). However, we did not find significant pairwise associations between ecological traits and zone or sampling period (except for size class B and 25 m + 50 m zones). This outcome prompts to take precautions in generalizing our observations. Indeed, the carabids communities are known to be strongly influenced by the local characteristics (e.g. in-field agricultural interventions) and the landscape compositional and configurational heterogeneity. At the field scale, the crop type [START_REF] Aguilera | Crop diversity benefits carabid and pollinator communities in landscapes with semi-natural habitats[END_REF][START_REF] Labruyere | Crop type, crop management and grass margins affect the abundance and the nutritional state of seed-eating carabid species in arable landscapes[END_REF][START_REF] Rusch | Management intensity at field and landscape levels affects the structure of generalist predator communities[END_REF], the cropping management type (e.g. organic vs. conventional), cropping practices [START_REF] Gallé | Arthropod functional traits shaped by landscape-scale field size, local agri-environment schemes and edge effects[END_REF][START_REF] Gallé | Landscape configuration, organic management, and within-field position drive functional diversity of spiders and carabids[END_REF][START_REF] Gayer | Carabid functional diversity is enhanced by conventional flowering fields, organic winter cereals and edge habitats[END_REF], and the local soil and climate conditions can modify local species pool and shape the traits distribution in carabids communities. In our case, cropping management and practices were generally similar, although farmers adapted the timing and intensity of interventions to their particular constraints.

At the landscape scale, the effect on arthropods regarding the proportion of semi-natural habitats (including field margins) and their spatial configuration has been widely studied [START_REF] Gallé | Arthropod functional traits shaped by landscape-scale field size, local agri-environment schemes and edge effects[END_REF][START_REF] Gallé | Landscape configuration, organic management, and within-field position drive functional diversity of spiders and carabids[END_REF][START_REF] Duflot | Landscape heterogeneity as an ecological filter of species traits[END_REF]. A more heterogeneous landscape, with more semi-natural habitats, smaller cultivated fields and a higher length of field margins is known to increase activity-density, species richness and functional diversity of carabids, [START_REF] Badenhausser | Increasing amount and quality of green infrastructures at different scales promotes biological control in agricultural landscapes[END_REF][START_REF] Landis | Manipulating plant resources to enhance beneficial arthropods in agricultural landscapes[END_REF][START_REF] Martin | The interplay of landscape composition and configuration: new pathways to manage functional biodiversity and agroecosystem services across Europe[END_REF][START_REF] Sirami | Increasing crop heterogeneity enhances multitrophic diversity across agricultural regions[END_REF].

Conclusion

Our study provides new insights into the species and ecological trait distribution in carabid communities within three different habitat types in agroecosystems: preserved cover crop strips, grassy margins and cropped maize field. Our results indicate that preserved cover crop strips in maize fields can help enhance the carabid diversity at least at field level, from the first year of establishment, but probably also in the larger agricultural landscape. This highlights the good dispersal capacity and adaptability of carabid communities in agroecosystems. The cover crop strips had an influence on the community of the adjacent cropped area, with more P. melanarius and P. cupreus, and more small carabids, in the proximity to the strip. However, spillover effects in our study were limited, an increased number of cover crop strip implementation in space (farm or landscape scale) and time (over years) could lead to a higher occurrence of some carabids and greater resilience of the carabid community in cultivated landscapes. Moreover, we found major predatory species that were associated with margins, but they did not always occur densely within them, probably because of the limited size of the margins. This might have limited also the spillover effect into crop areas. In this respect, it needs to be taken into account that the effects of such strips on carabids community composition and structure in agroecosystems can vary among cultivated fields or territories [START_REF] Concepción | Interactive effects of landscape context constrain the effectiveness of local agri-environmental management: Landscape constrains the effectiveness of local management[END_REF]Karp et al., 2018), and thus having varying spillover and potential resilience effects. Thus further research should investigate the influence of the interactions between local and landscape characteristics on the efficiency of agroecological practices regarding ecosystem functions and services. This could be of prime importance for the development of innovative agroecological practices, which are also realistic for implementation on farmers' fields, and understand the conditions of success for an increase of pest and weed seed control in the cropping systems.

  multiflorum, in 2019) and/or rye (Secale cereal, in 2020), 35 % Crimson clover (Trifolium incarnatum, in 2019) or Alexandria clover (Trifolium alexandrinum, in 2020), 15 % Turnip Rape (Brassica rapa subsp. Oleifera), and 12.5 % Phacelia (Phacelia tanacetifolia). The changes in the Poaceae and clover species composition between 2019 and 2020 was necessary to adapt the practice to farmers' constraints and expectations.

Figure 1 .

 1 Figure 1. Sampling design of the field experiment. Each black dot represents a sampling point where a pitfall trap was installed.

Figure 2 .

 2 Figure 2. Mean activity-density per trap of the 15 main species (> 50 individuals) and Amara similata in the 5 zones, for each sampling period. Red bars represent predatory species, blue bars polyphagous species and green bars phytophagous species. Code names correspondence is available in Supporting Information A.

Figure 3 .

 3 Figure 3. Mean activity-density by trap of carabids, grouped by ecological traits (diet, body size, wing status) in the five zones (strip, 10 m, 25 m, 50 m, margin). The activity-densities take into account all sampling periods with the preserved strip (from Sowing to Final Stage) and all experimental sites. Standard deviations are shown.

Table 1 .

 1 Outcomes of ANOSIM, PERMANOVA and pair-wise PERMANOVA analyses on carabids community composition, with all sampling periods (IS to FS) or grouped for maize growth period (S to 8L). R value of ANOSIM analysis compares the mean of ranked dissimilarities between groups to the mean of ranked dissimilarities within groups. A low R value indicates closer community composition between groups. Significant results are in bold(p-value<0.05).

		Full period (from IS to FS)	During maize growth (from S to 4L)
				ANOSIM
	Site	R: 0.269		R: 0.340
		p-value: 1e-04		p-value: 1e-04
	Period	R: 0.098		R: 0.041
		p-value: 1e-04		p-value: 1e-04
	Zone	R: 0.060		R: 0.082
		p-value: 1e-04		p-value: 1e-04
				PERMANOVA
		df	R 2	Pr(>F)	df	R 2	Pr(>F)
	Site	11	0.173	1e-04	11	0.214	1e-04
	Period	4	0.049	1e-04	2	0.025	1e-04
	Zone	4	0.032	1e-04	4	0.042	1e-04
	Site:Period	41	0.124	1e-04	22	0.087	1e-04
	Site:Zone	44	0.075	1e-04	44	0.096	1e-04
	Period:Zone	16	0.024	1e-04	8	0.013	2e-04
	residual	686	0.524		537	0.524
	total	806	1		628	1
			Pairwise PERMANOVA on Zone
		R 2		p-value	R 2		p-value
	margin vs 0m	0.0103		0.04	0.021		0.01
	margin vs 10m	0.0401		0.01	0.044		0.01
	margin vs 25m	0.0406		0.01	0.050		0.01
	margin vs 50m	0.0279		0.01	0.028		0.01
	0m vs 10m	0.0359		0.01	0.053		0.01
	0m vs 25m	0.0378		0.01	0.060		0.01
	0m vs 50m	0.0295		0.01	0.045		0.01
	10m vs 25m	0.0014		1.00	0.001		1.00
	10m vs 50m	0.0047		0.37	0.006		0.38
	25m vs 50m	0.0039		0.81	0.007		0.16

Supporting Information A: List of carabid species ranked by abundances in the three habitats (margin, strip and cropped area)

  For each species, rank, abundance (Ab.), and proportion in the habitat (%) are given. Bold species are the 3 main species; grey boxes highlight the species found exclusively in the strip (however in low abundances). These abundances take into account all sampling periods with an existing strip (from Sowing to Final Stage).

	Strip Supporting Information Brank Ab. % Margin rank Ab. %	Crop rank Ab.	%
	synobs	27	3	0,4	caraur	27	2	0,6 notqua	27	10	0,3
	agomue	28	2	0,3	haralb	28	2	0,6 ophazu	28	9	0,3
	amaaen	29	2	0,3	harrub	29	2	0,6 ciccam	29	4	0,1
	Code names correspondences: Strip rank Ab. % amabif 30 2 0,3	Margin rank Ab. % pladep 30 2 0,6	Crop rank Ab. psecal 30 4	% 0,1
	abapar: Abax parallelus (Duftschmid, 1812) ancdor 1 267 35,3 ancdor nebbre 31 2 0,3 amabif	lorpil: Loricera pilicornis (Fabricius, 1775) 1 80 23,2 ptemel 1 1078 37,3 31 1 0,3 amaaen 31 3 0,1
	acumer: Acupalpus meridianus (Linne, 1761) agoema : Agonum emarginatum (Gyllenhal, 1827) agomue : Agonum muelleri (Herbst, 1784) amaaen: Amara aenea (De Geer, 1774) amabif: Amara bifrons (Gyllenhal, 1810) amasim: Amara similata (Gyllenhal, 1810) poecup 2 98 13 ptemel acumer 32 1 amasim 32 metlam: Metallina lampros (Herbst, 1784) 2 31 9 poecup 2 577 1 0,3 calcin 32 3 0,1 metpro: Metallina properans (Stephens, 1828) 20 0,1 micmau: Microlestes maurus (Sturm, 1827) brascl 3 54 phyobt 3 20 5,8 hardis 3 140 4,8 badbul 33 1 0,1 asasti 33 1 0,3 haranx 33 3 0,1 7,1 micmin: Microlestes minutulus (Goeze, 1777) nebbre: Nebria brevicollis (Fabricius, 1792) ptemel 4 39 haraff 4 17 4,9 caraur 4 135 4,7 bemqua 34 1 0,1 braexp 34 1 0,3 harrub 34 3 0,1 5,2 nebsal: Nebria salina Fairmaire & Laboulbène, 1854 carcan 5 35 4,6 hardim 5 17 4,9 trequa 5 117 4 braexp 35 1 0,1 diager 35 1 0,3 harser 35 3 0,1
	ancdor: Anchomenus dorsalis (Pontoppidan, 1763) amasim 6 29 3,8 pseruf carnem 36 1 0,1 harsma	notqua: Notiophilus quadripunctatus Dejean, 1826 6 17 4,9 phyobt 6 116 36 1 0,3 micmau 36 3	4 0,1
	anisig: Anisodactylus signatus (Panzer, 1796) asasti: Asaphidion stierlini (Heyden, 1880) carmon 7 26 3,4 carcan carvio 37 1 0,1 metpro	notsub: Notiophilus substriatus G.R. Waterhouse, 1833 7 16 4,6 pseruf 7 37 1 0,3 stopum 37	78 3	2,7 0,1
	badbul: Badister bullatus (Schrank, 1798) bemqua: Bembidion quadrimaculatum (Linne, 1761) braele: Brachinus elegans Chaudoir, 1842 braexp: Brachinus explodens Duftschmid, 1812 brascl: Brachinus sclopeta (Fabricius, 1792) calcin: Calathus cinctus Motschulsky, 1850 micmin 8 23 3 hardis dryden 38 1 nebbre 0,1 pseruf 9 21 trequa metpro 39 1 0,1 polcon 2,8 hardim 10 17 carcor phyobt 40 1 0,1 pteovo 2,2 hardis 11 16 2,1 metlam 11 ocylat: Ocydromus latinus (Netolitzky, 1911) 8 13 3,8 micmin 8 73 2,5 38 1 0,3 badbul 38 2 0,1 ocytet: Ocydromus tetracolus (Say, 1823) ophazu: Ophonus azureus (Fabricius, 1775) 9 12 3,5 carmon 9 48 1,7 39 1 0,3 ocylat 39 2 0,1 ophcri: Ophonus cribricollis (Dejean, 1829) parbis: Paratachys bistriatus (Duftschmid, 10 11 3,2 ptemad 10 47 1,6 40 1 0,3 ophcri 40 2 0,1 1812) parmac: Parophonus maculicornis (Duftschmid, 11 3,2 hardim 11 43 1,5 ptenie 41 1 0,1 steteu 41 1 0,3 parbis 41 2 0,1
	calfus: Calathus fuscipes (Goeze, 1777) callun: Callistus lunatus (Fabricius, 1775) trequa 12 16 2,1 brascl semsig 42 1 0,1	1812) philun: Philochthus lunulatus (Geoffroy in 12 10 2,9 carcan 12 38 tacpar 42 2	1,3 0,1
	caraur: Carabus auratus Linne, 1761 carcan: Carabus cancellatus Illiger, 1798 braele 13 14 1,9 haranx stopum 43 1 0,1	Fourcroy, 1785) phyobt: Phyla obtusa (Audinet-Serville, 1821) 13 10 2,9 asasti 13 36 1,2 acumer 43 1 0
	carcor: Carabus coriaceus Linne, 1758 carmon: Carabus monilis Fabricius, 1792 carnem: Carabus nemoralis Müller, 1764 carvio: Carabus violaceus subsp. purpurascens Fabricius, 1787 ciccam: Cicindela campestris Linne, 1758 clifos: Clivina fossor (Linne, 1758) carcor 14 14 1,9 harser zabten 44 1 0,1 caraur 15 10 poecup 15 pladep: Platyderus depressus (Audinet-Serville, 14 10 2,9 cylger 14 35 1,2 agoema 44 1 0 1821) poecup: Poecilus cupreus (Linne, 1758) 7 2 haraff 15 34 1,2 amasim 45 1 0 1,3 poelep: Poecilus lepidus (Leske, 1785) polcon: Polistichus connexus (Geoffroy in haraff 16 10 micmin 16 6 1,7 anisig 16 33 1,1 callun 46 1 0 1,3 Fourcroy, 1785) psecal: Pseudoophonus calceatus (Duftschmid, metlam 17 9 1,2 carmon 17 5 1,4 metlam 17 29 1 carvio 47 1 0
	cylger: Cylindera germanica (Linne, 1758) dematr: Demetrias atricapillus (Linne, 1758) asasti 18 6 0,8 hartar	1812) psegri: Pseudoophonus griseus (Panzer, 1797) 18 5 1,4 ancdor 18 26 0,9 dematr 48 1 0
	diager: Diachromus germanus (Linne, 1758) dryden: Drypta dentata (Rossi, 1790) pteant 19 5 0,7 ophazu	pseruf: Pseudoophonus rufipes (De Geer, 1774) pteant: Pterostichus anthracinus (Illiger, 1798) 19 5 1,4 metpro 19 23 0,8 harpyg 49 1 0
	haraff: Harpalus affinis (Schrank, 1781) haralb: Harpalus albanicus Reitter, 1900 haranx: Harpalus anxius (Duftschmid, 1812) hardim: Harpalus dimidiatus (Rossi, 1790) hardis: Harpalus distinguendus (Duftschmid, 1812) harpyg: Harpalus pygmaeus Dejean, 1829 nebsal 20 4 0,5 cylger ptemad 21 4 micmau 21 ptemad: Pterostichus madidus (Fabricius, 1775) 20 4 1,2 pteant 20 20 harsma 50 1 0 0,7 ptemel: Pterostichus melanarius (Illiger, 1798) ptenie: Pterostichus niger (Schaller, 1783) 4 1,2 nebbre 21 19 0,7 lorpil 51 1 0 0,5 pteovo: Pterostichus ovoideus (Sturm, 1824) semsig: Semiophonus signaticornis (Duftschmid, anisig 22 3 ptemad 22 4 1,2 ptenie 22 17 0,6 ocytet 52 1 0 0,4 1812) steteu: Stenolophus teutonus (Schrank, 1781) calfus 23 3 0,4 dryden 23 3 0,9 calfus 23 15 0,5 philun 53 1 0
	harrub: Harpalus rubripes (Duftschmid, 1812) harser: Harpalus serripes (Quensel in cylger 24 3 0,4 nebsal	stopum: Stomis pumicatus (Panzer, 1796) synobs: Syntomus obscuroguttatus (Duftschmid, 24 3 0,9 bemqua 24 14 0,5 poelep 54 1 0
	Schönherr, 1806) harsma: Harpalus smaragdinus (Duftschmid, dematr 25 3 0,4 parmac	1812) tacpar: Tachyura parvula (Dejean, 1831) 25 3 0,9 nebsal 25 14 psegri 55 1	0,5 0
	1812) hartar: Harpalus tardus (Panzer, 1797) ophazu 26 3 0,4 amaaen 26 trequa: Trechus quadristriatus (Schrank, 1781) zabten: Zabrus tenebrioides (Goeze, 1777) 2 0,6 carcor 26 13 0,4 steteu 56 1 0
	1

: Indicator species analysis results by zone groups

	Multilevel pattern analysis: 9999 permutations
	Significance level (alpha): 0.05
	Significance codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
	First analysis:

full sampling period (from Initial stage to Final Stage)

	Total number of species: 76
	Selected number of species: 19
	Number of species associated to 1 group: 14
	Number of species associated to 2 groups: 2
	Number of species associated to 3 groups: 2
	Number of species associated to 4 groups: 1
	List of species associated to each combination:
	Group	species	stat	p.value
	Group margin	haranx	0.207	0.0001 ***
		hartar	0.186	0.0001 ***
		harser	0.155	0.0003 ***
		micmau	0.137	0.0037 **
		pladep	0.124	0.0167 *
		parmac	0.117	0.0137 *
	Group 0m	ancdor	0.334	0.0001 ***
		brascl	0.231	0.0001 ***
		amasim	0.191	0.0001 ***
		braele	0.160	0.0001 ***
		carmon	0.129	0.0075 **
		dematr	0.108	0.0497 *
		carcan	0.102	0.0500 *
	Group 50m	ptenie	0.124	0.01 **
	Group margin+0m	carcor	0.167	4e-04 ***
	Group 10m+25m	caraur	0.131	0.0059 **
	Group 0m+10m+25m	poecup	0.192	1e-04 ***
	Group 10m+25m+50m	ptemel	0.165	2e-04 ***
	Group margin+10m+25m+50m phyobt	0.136	0.0023 **

Second analysis: during maize growth (from Sowing to 8 Leaves stage)

	Total number of species: 76
	Selected number of species: 21
	Number of species associated to 1 group: 17
	Number of species associated to 2 groups: 1
	Number of species associated to 3 groups: 2
	Number of species associated to 4 groups: 1
	List of species associated to each combination:
	Group	species	stat	p.value
	Group margin	haranx	0.246	0.0001 ***
		hartar	0.195	0.0006 ***
		harser	0.183	0.0004 ***
		haralb	0.145	0.0137 *
		pladep	0.145	0.0137 *
		parmac	0.138	0.0167 *
		micmau	0.136	0.0150 *
		haraff	0.133	0.0158 *
		dryden	0.119	0.0444 *
	Group 0m	ancdor	0.448	0.0001 ***
		brascl	0.277	0.0001 ***
		amasim	0.218	0.0001 ***
		braele	0.183	0.0001 ***
		carmon	0.150	0.0053 **
		dematr	0.141	0.0235 *
		agomue	0.133	0.0401 *
	Group 50m	ptenie	0.147	0.0087 **
	Group 10m+25m	caraur	0.146	0.0068 **
	Group 0m+10m+25m	poecup	0.219	1e-04 ***
	Group 10m+25m+50m	ptemel	0.181	7e-04 ***
	Group margin+10m+25m+50m phyobt	0.148	0.006 **

Acknowledgments

We thank Lucile Toniutti, Thomas Lhuillery and Manon Bouvier for their technical assistance, and Sandrine Leblond for her help and support in the design of the study. We would like to thank the farmers of the study site for allowing us to conduct our experiment on their fields. This work was funded by Isara and by BASF France division Agro.

Data Availability Statement

Data available via Zenodo at [DOI] [START_REF] Triquet | Undestroyed winter cover crop strip in maize fields supports ground-dwelling arthropods and predation[END_REF].

Authors' contributions

C.T, A.F., A.R., and A.W. designed the study; C.T and A.R. conducted data collection; A.R. identified carabids; C.T. analysed data with the support of V.T.; C.T. wrote the first draft of the manuscript. All authors revised the manuscript and gave final approval for publication.

Declaration of Competing Interest

The authors declare that they have no conflict of interest.

Supporting information

Additional supporting information may be found online in the Supporting Information section.

Supporting Information C: Multivariate dispersion

Effect of zone and sampling period (IS, S, 4L, 8L, FS) on the multivariate dispersion of species composition. Taxonomic β-diversity is measured as the distance of factors to their group centroid using Bray-Curtis, represented on the first two axes of a PCoA. Additionnal PcoA showing only zones (without IS period) and sites are presented with the sd ellipses.