Alicia Valdés
email: aliciavaldes1501@gmail.com

Jonathan Roger Michel

Henri Lenoir

Pieter De Frenne

Emilie Andrieu

Jorg Brunet

Olivier Chabrerie

Sara A O Cousins

Marc Deconchat

Pallieter De Smedt

Martin Diekmann

Jonathan Lenoir

Jörg Brunet

Steffen Ehrmann

Emilie Gallet-Moron

Stefanie Gärtner

Brice Giffard

Karin Hansen

Martin Hermy

Annette Kolb

Vincent Le Roux

Jaan Liira

Jessica Lindgren

Ludmilla Martin

Tobias Naaf

Taavi Paal

Willem Proesmans

Michael Scherer-Lorenzen

Monika Wulf

Kris Verheyen

Guillaume Decocq

High ecosystem service delivery potential of small woodlands in agricultural landscapes

Keywords: Anthropocene, biodiversity, connectivity, habitat fragmentation, island biogeography, management practices, metacommunity dynamics, multifunctionality, woodlands

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

ABSTRACT

 Global forest loss and fragmentation have strongly increased the frequency of forest patches smaller than a few hectares. Little is known about the biodiversity and ecosystem service supply potential of such small woodlands in comparison to larger forests. As it is widely recognized that high biodiversity levels increase ecosystem functionality and the delivery of multiple ecosystem services, small, isolated woodlands are expected to have a lower potential for ecosystem service delivery than large forests hosting more species.

 We collected data on the diversity of six taxonomic groups covering invertebrates, plants and fungi, and on the supply potential of five ecosystem services and one disservice within 224 woodlands distributed across temperate Europe. We related their ability to simultaneously provide multiple ecosystem services (multiservice delivery potential) at different performance levels to biodiversity of all studied taxonomic groups (multidiversity), forest patch size and age, as well as habitat availability and connectivity within the landscape, while accounting for macroclimate, soil properties and forest structure.

 Unexpectedly, despite their lower multidiversity, smaller woodlands had the potential to deliver multiple services at higher performance levels per area than larger woodlands of similar age, probably due to positive edge effects on the supply potential of several ecosystem services.

 Biodiversity only affected multiservice delivery potential at a low performance level as well as some individual ecosystem services. The importance of other drivers of ecosystem service supply potential by small woodlands in agricultural landscapes also INTRODUCTION Forests deliver a wide range of ecosystem services to human society, and positive relationships between biodiversity and these services have been reported for large forested areas (Gamfeldt et al., 2013;[START_REF] Van Der Plas | Jack-of-all-trades effects drive biodiversity-ecosystem multifunctionality relationships in European forests[END_REF]. However, there is a lack of knowledge about the relationship between biodiversity and multiple ecosystem services in small woodland patches [START_REF] Decocq | Ecosystem services from small forest patches in agricultural landscapes[END_REF][START_REF] Mitchell | Forest fragments modulate the provision of multiple ecosystem services[END_REF]. Indeed, in many parts of the world, the original forest cover has been heavily fragmented [START_REF] Haddad | Habitat fragmentation and its lasting impact on Earth's ecosystems[END_REF][START_REF] Hansen | High-resolution global maps of 21st-century forest cover change[END_REF] due to human activities; more than 70% of the remaining global forest cover lies within 1 km of a forest edge [START_REF] Haddad | Habitat fragmentation and its lasting impact on Earth's ecosystems[END_REF]. Many of the remaining woodlands are smaller than 10 ha [START_REF] Estreguil | Forest landscape in Europe: pattern, fragmentation and connectivity[END_REF] and scattered in a human-transformed matrix of mostly arable lands, pastures and human settlements. Loss of area, increased isolation and greater exposure to human disturbances along forest edges are leading causes of biodiversity loss [START_REF] Haddad | Habitat fragmentation and its lasting impact on Earth's ecosystems[END_REF], although small patch size can sometimes be beneficial for biodiversity, as shown by the preponderance of positive significant responses to habitat fragmentation [START_REF] Fahrig | Ecological responses to habitat fragmentation per se[END_REF].

Small woodland patches in agricultural landscapes also vary in age, since many of them have developed on farmland that was abandoned at different points in time [START_REF] Flinn | Recovery of forest plant communities in post-agricultural landscapes[END_REF]. Consequently, small woodlands not only differ in size and degree of isolation, but also in age, quantified as the time since forest establishment, ranging from very ancient (several centuries or older) to more recent (less than a century). Despite their small size and isolation, patches of (semi-)natural habitats such as small and ancient woodlands can have an important role as refugia for biodiversity [START_REF] Decocq | Ecosystem services from small forest patches in agricultural landscapes[END_REF] and as providers of multiple ecosystem services [START_REF] Decocq | Ecosystem services from small forest patches in agricultural landscapes[END_REF][START_REF] Mitchell | Forest fragments modulate the provision of multiple ecosystem services[END_REF]. Therefore, small and ancient forest patches, analogously to other "small natural features" [START_REF] Hunter | Conserving small natural features with large ecological roles: An introduction and definition[END_REF] such as scattered and old trees in agricultural landscapes [START_REF] Herrera | The role of remnant trees in seed dispersal through the matrix: being alone is not always so sad[END_REF]Manning, Gibbons, & Lindenmayer, 2006;Manning, Fischer, & Lindenmayer, 2009), have an important conservation value. This suggests that the age of these elements in the landscape might also contribute to their conservation value. As it is unknown if the relationships among biodiversity and ecosystem services that emerged from research in large forests (Gamfeldt et al., 2013;[START_REF] Van Der Plas | Jack-of-all-trades effects drive biodiversity-ecosystem multifunctionality relationships in European forests[END_REF] apply to small and ancient woodlands, the conservation and management of such elements in agricultural landscapes would strongly benefit from a specific assessment of the drivers of biodiversity and ecosystem service delivery in small woodlands.

The delivery potential of multiple ecosystem services by small and ancient woodlands can be studied by linking the theory of island biogeography [START_REF] Macarthur | The theory of island biography[END_REF] to ecosystem functioning (Fig. 1). Large and spatially well-connected forest patches are expected to host more species compared to small and isolated woodlands [START_REF] Macarthur | The theory of island biography[END_REF]. Likewise, patches that have been forested for a long time will likely be more species-rich than recently established forests, which can remain floristically impoverished for centuries [START_REF] De Frenne | Interregional variation in the floristic recovery of post-agricultural forests[END_REF] because of the slow immigration rate of forest specialists [START_REF] Naaf | Colonization credit of post-agricultural forest patches in NE Germany remains 130-230 years after reforestation[END_REF]. Together with this variation in taxonomic diversity [START_REF] Valdés | The contribution of patch-scale conditions is greater than that of macroclimate in explaining local plant diversity in fragmented forests across Europe[END_REF], the functional diversity of patches is expected to vary as well since traits of species colonizing and persisting in, for instance, small, young or isolated patches will differ from those inhabiting large, ancient or well-connected patches [START_REF] Craven | Evaluating resilience of tree communities in fragmented landscapes: linking functional response diversity with landscape connectivity[END_REF][START_REF] Vanneste | Functional trait variation of forest understorey plant communities across Europe[END_REF]. These functional responses to patch characteristics can in turn affect their functioning and potential for ecosystem service delivery (cf. [START_REF] Suding | Scaling environmental change through the community-level: a trait-based response-and-effect framework for plants[END_REF].

Indeed, the effect of taxonomic or functional diversity on ecosystem functioning and service delivery is widely acknowledged and very well documented in the scientific literature [START_REF] Balvanera | Quantifying the evidence for biodiversity effects on ecosystem functioning and services[END_REF][START_REF] Balvanera | Linking biodiversity and ecosystem services: current uncertainties and the necessary next steps[END_REF][START_REF] Bastian | The role of biodiversity in supporting ecosystem services in Natura 2000 sites[END_REF]Gamfeldt et al., 2013;[START_REF] Lefcheck | Biodiversity enhances ecosystem multifunctionality across trophic levels and habitats[END_REF][START_REF] Van Der Plas | Jack-of-all-trades effects drive biodiversity-ecosystem multifunctionality relationships in European forests[END_REF][START_REF] Duffy | Biodiversity effects in the wild are common and as strong as key drivers of productivity[END_REF]. In a rigorous quantitative review covering 446 measures of biodiversity effects on ecosystem functioning and services, [START_REF] Balvanera | Quantifying the evidence for biodiversity effects on ecosystem functioning and services[END_REF] found clear evidence that biodiversity has positive effects on most of the ecosystem services assessed in their quantitative review. Similarly, a more recent quantitative review focusing on 67 field studies has demonstrated that biomass production, a key ecosystem service, increases with species richness in a wide range of wild taxa and ecosystems [START_REF] Duffy | Biodiversity effects in the wild are common and as strong as key drivers of productivity[END_REF]. This positive relationship between biodiversity and the delivery of single ecosystem services has even been extended to the delivery of multiple services (Gamfeldt et al., 2013;[START_REF] Van Der Plas | Jack-of-all-trades effects drive biodiversity-ecosystem multifunctionality relationships in European forests[END_REF][START_REF] Lefcheck | Biodiversity enhances ecosystem multifunctionality across trophic levels and habitats[END_REF]. For instance, higher levels of multiple ecosystem services are found in large forests with more tree species (Gamfeldt et al., 2013). If the positive relationship between biodiversity and the delivery of multiple ecosystem services reported for large forested areas (Gamfeldt et al., 2013;[START_REF] Van Der Plas | Jack-of-all-trades effects drive biodiversity-ecosystem multifunctionality relationships in European forests[END_REF] would be fully applicable to small woodlands, we could hypothesize that small, isolated and recent woodlands (cf. situation "a" in Fig. 1) would have a lower ecosystem service supply potential [START_REF] Bodin | The value of small size: loss of forest patches and ecological thresholds in Southern Madagascar[END_REF][START_REF] Decocq | Ecosystem services from small forest patches in agricultural landscapes[END_REF], because they harbor less biodiversity compared to large, connected and ancient forest patches (cf. situation "d" in Fig. 1).

Here, we assess the supply potential of multiple ecosystem services provided by small (both recent and ancient) woodlands as well as larger (both recent and ancient) forest patches (n = 224) in agricultural landscapes distributed along a 2500-km latitudinal gradient spanning the entire temperate forest biome in Europe (Fig. 2). We collected biodiversity data for six taxonomic groups with different functional roles and dispersal potential, as well as data on the supply potential of six important ecosystem (dis)services delivered by woodlands, representing a mixture of provisioning, regulating and cultural services.

MATERIALS AND METHODS

Study area and sampling design

We studied deciduous forest patches in seven regions along a transect spanning c. 2500 km across the entire temperate forest biome of Europe (Fig. 2). In each region, two 5 × 5 km² landscape windows (14 in total) with two contrasting connectivity levels were selected: (1) a "high-disturbance landscape" with isolated forest patches surrounded by an intensively cultivated matrix, dominated by large open fields with intensive use of pesticides and fertilizers; and (2) a "low-disturbance landscape" with forest patches more or less connected by different types of hedgerows and surrounded by a less disturbed matrix with fewer and smaller crop fields (see [START_REF] Valdés | The contribution of patch-scale conditions is greater than that of macroclimate in explaining local plant diversity in fragmented forests across Europe[END_REF] for more info). In each landscape window, sixteen woodlands were selected for sampling. When possible, these woodlands were chosen to be equally distributed among each of four combinations of size and historical age categories: small-old, small-young, large-old, large-young (see details on patch selection in Appendix S1, and summary statistics for each size and age category per landscape window in Table S1).

Data collection

Patch features

Patch size and historical age were calculated with a geographic information system (GIS, ArcGis 9.3, ESRI), using contemporary and historical maps of the landscape windows (recent aerial photographs, all taken after the year 2000, and maps from the 18th, 19th and 20th centuries). Patch size was calculated using digitized woodlands in each window. For calculating patch historical age, we digitized all woodlands in historical maps, and historical age was estimated using the date of the oldest map on which a patch appeared. As a given patch may contain a mosaic of fragments with different historical ages, we calculated a sizeweighted average of the historical age of all fragments composing an isolated patch.

We used a 500-m buffer centered on each patch to calculate metrics related to habitat connectivity (see also Valdes et al., 2015) and the habitat amount hypothesis sensu Fahrig [START_REF] Fahrig | Rethinking patch size and isolation effects: the habitat amount hypothesis[END_REF]: (1) the proportion of buffer surface covered by forest, with higher values indicating a higher amount of forest habitat availability within the landscape; and (2) the proportion of buffer surface covered by crops, with higher values indicating a lower habitat connectivity, as agricultural intensification of the matrix reduces forest connectivity and increases its ecological isolation [START_REF] Donald | Habitat connectivity and matrix restoration: the wider implications of agrienvironment schemes[END_REF].

Environmental drivers

We calculated values of three groups of environmental drivers that can affect both biodiversity and supply potential of multiple ecosystem services: macroclimatic, soil and forest structural drivers.

Macroclimatic drivers

We extracted five temperature variables from the EuroLST dataset (250-m resolution, http://www.geodati.fmach.it/eurolst.html) and five precipitation variables from the WorldClim global database (1-km resolution, http://www.worldclim.org/) and averaged each variable for each patch using all pixels intersecting with the patch area. We performed a principal components analysis (PCA, Fig. S1) on these 10 variables representing seasonality and extreme or limiting environmental factors (see details on Fig. S1 legend), and retained the first axis (explaining 74% of the total variance) as an explanatory variable in our models. This variable, called "macroclimate", indicated high seasonality and low temperature and precipitation.

Soil drivers

We took soil samples in each patch (see details on soil sampling in Appendix S1), and calculated mean values of depth of the forest floor, C:N ratio, total P content and pH of the mineral topsoil at the patch level and used them to perform a PCA (Fig. S2). We then retained axes 1 and 2 (explaining, respectively, 44 and 26 % of the total variance) as explanatory variables in our models. Axis 1, called "soil nutrients", was positively correlated with the total P in the mineral topsoil and negatively correlated with the depth of the forest floor. Axis 2, called "soil acidity & C/N" was positively correlated with the C:N ratio in the mineral topsoil and negatively correlated with the pH of the mineral topsoil.

Forest structural drivers

We calculated two variables describing variation in forest composition and structure, respectively: tree diversity, calculated as a stem number-based Shannon diversity index; and structural diversity, calculated as the coefficient of variation (CV) of tree diameters (see details on data collection in Appendix S1). These variables were selected based on data availability on the one hand and their relevance for explaining variation in tree and forestassociated biodiversity and in ecosystem functioning (e.g. [START_REF] Van Der Plas | Jack-of-all-trades effects drive biodiversity-ecosystem multifunctionality relationships in European forests[END_REF][START_REF] Penone | Specialisation and diversity of multiple trophic groups are promoted by different forest features[END_REF] on the other hand.

Biodiversity

In order to assess patch biodiversity (hereafter multidiversity sensu [START_REF] Allan | Interannual variation in land-use intensity enhances grassland multidiversity[END_REF], we surveyed species richness for six different taxonomic groups that are representative of forestassociated ground-dwelling biota: ground beetles (i.e. Insecta); spiders (i.e. Arachnida); millipedes (i.e. Myriapoda); woodlice (i.e. Crustacea); understory herbs (i.e. focusing on vascular plant species only); and mushrooms (i.e. focusing on species with visible and aboveground fruiting bodies such as stem, cap and gills). The selected groups cover different trophic levels (primary producers, predators and decomposers) and are assumed to directly or indirectly influence the potential delivery of the six studied ecosystem services. We refer to Appendix S1 for more information on field surveys specific to each taxonomic group.

Proxies for supply potential of multiple ecosystem services

In addition to patch multidiversity, we assessed the potential of a given patch to simultaneously provide multiple services (hereafter multiservice delivery potential). Here we included two proxies for provisioning services (abundance of usable plants and stemwood volume), three for regulating services (pest control potential, tick-borne disease risk and topsoil carbon storage), and one for a cultural or recreational service (game production potential). See Appendix S1 for more information on each service/disservice and details on data collection.

Calculation of multidiversity and multiservice delivery

For each patch i, we calculated a multidiversity index (MD) according to [START_REF] Allan | Interannual variation in land-use intensity enhances grassland multidiversity[END_REF] using the raw species richness values of understory herbs (UH), mushrooms (MU), carabid beetles (CB), spiders (SP), millipedes (MI) and woodlice (WO). Each raw species richness value for a given taxonomic group was divided by the mean of the five highest values among all studied patches to account for extreme values [START_REF] Allan | Interannual variation in land-use intensity enhances grassland multidiversity[END_REF]. MD was calculated as:

(Equation 1)

Based on the raw values of each individual ecosystem service/disservice, we used a multiple threshold approach [START_REF] Byrnes | Investigating the relationship between biodiversity and ecosystem multifunctionality: challenges and solutions[END_REF] to define service-specific threshold values representing low, intermediate and high performance levels of ecosystem service supply potential (see details in Table S2). The performance level is the amount of the provided service (or disservice) supply potential per area unit and could either be low, intermediate or high. Examples of high performance levels are, for instance, small woodland patches that simultaneously provide a large total stemwood volume (> 300 m 3 per ha) and have a low density of ticks (< 50 nymphs per 100 m 2 ) and thus minimize the prevalence of tick-borne diseases in the human population. For each of the three performance levels, we calculated multiservice delivery potential as the proportion of ecosystem service proxies (sometimes less than six measured ecosystem proxies per patch if the data was not available for a given proxy within a focal patch) for which the amount of the provided service (or disservice) proxy per area unit exceeded (or was lower than) a proxy-dependent threshold (Table S2). For instance, if one service proxy exceeded its high-threshold value and two exceeded their respective lowthreshold values within a given patch for which five services were measured in total, then the high-performance multiservice delivery potential is 0.2 and the low-performance multiservice delivery is 0.4 for this patch. This threshold approach is considered as the state-of-the-art standard method to evaluate multiservice delivery [START_REF] Byrnes | Investigating the relationship between biodiversity and ecosystem multifunctionality: challenges and solutions[END_REF][START_REF] Gamfeldt | Multiple functions increase the abundance of biodiversity for overall ecosystem functioning[END_REF][START_REF] Lefcheck | Biodiversity enhances ecosystem multifunctionality across trophic levels and habitats[END_REF]. Multiple thresholds are used because it has been shown that the sign of the relationship between biodiversity and ecosystem multifunctionality can differ when low-performance versus high-performance thresholds are applied (van der [START_REF] Van Der Plas | Jack-of-all-trades effects drive biodiversity-ecosystem multifunctionality relationships in European forests[END_REF].

Statistical modeling

We used piecewise structural equation modeling (piecewise SEM, [START_REF] Lefcheck | piecewiseSEM: Piecewise structural equation modelling in r for ecology, evolution, and systematics[END_REF] to study the response of low-, intermediate-and high-performance multiservice delivery potential to multidiversity, patch characteristics (size, age and the interaction between size and age) as well as habitat availability and connectivity within the landscape, while accounting also for macroclimate, soil and forest structural characteristics. We chose a piecewise approach (instead of the traditional variance-covariance based SEM) because of its ability to fit multiple separate linear models with non-normal distributions and random effects, which was wellsuited for our data. In our models, we considered both direct responses of multiservice delivery potential to the different predictors, and indirect responses mediated by effects of the different predictors on multidiversity. The piecewise SEM consisted of four component models:

1. A linear mixed-effects model (LMM) with tree diversity as the response variable and patch size (log-transformed), historical age, habitat availability (proportion of forests) and connectivity (proportion of crops), macroclimate (PC1) and soil (PC1 and PC2) as predictor variables.

2. A LMM with structural diversity as the response variable and patch size (logtransformed), historical age, habitat availability (proportion of forests) and connectivity (proportion of crops) and tree diversity as predictor variables.

3. A LMM with multidiversity as the response variable and patch size (logtransformed), historical age (and their interaction term to account for the speciestime-area relationship; [START_REF] Adler | Evidence for a general species-time-area relationship[END_REF], habitat availability (proportion of forests) and connectivity (proportion of crops), macroclimate (PC1), soil (PC1 and PC2), tree diversity and structural diversity as predictor variables.

A generalized linear mixed-effects model (GLMM) with a binomial error distribution

with multiservice delivery potential as the response variable and multidiversity, patch size (log-transformed), historical age, habitat availability (proportion of forests) and connectivity (proportion of crops), macroclimate (PC1), soil (PC1 and PC2), tree diversity and structural diversity as predictor variables.

LMMs were used for response variables with approximately normal distributions (tree diversity, structural diversity and multidiversity), and a binomial GLMM was used for multiservice delivery potential (proportional). We used "region" and "window type" nested within "region" as random effect terms in all four component models to account for the hierarchical, nested structure of the sampling design along the studied gradient. This piecewise SEM model structure was tested for the three levels of performance of multiservice delivery potential considered (low, intermediate and high), and for each individual ecosystem service, separately. Overall fit of the piecewise SEM models was assessed using Shipley's test of direct separation [START_REF] Shipley | Confirmatory path analysis in a generalized multilevel context[END_REF], which evaluates the probability that none of the paths missing from the hypothesized causal network contain useful information, and yields the Fisher's C statistic. A χ 2 -value of Fisher's C below the significance level (p < 0.05) indicates that the model is inconsistent with the data, and should be rejected. Statistical analyses were carried out in R 3.4.2 (R Core Team, 2018) using the packages piecewiseSEM [START_REF] Lefcheck | piecewiseSEM: Piecewise structural equation modelling in r for ecology, evolution, and systematics[END_REF] and nlme [START_REF] Pinheiro | nlme: Linear and Nonlinear Mixed Effects Models[END_REF].

RESULTS

Tree species diversity increased with patch historical age, while structural diversity, in turn, increased with tree species diversity (Fig. 3). Multidiversity was higher in the larger and more ancient patches, as shown by the positive interaction effect (p = 0.03) between patch size and historical age (Fig. 3).

The response of multiservice delivery potential to multidiversity, patch characteristics, habitat availability and connectivity and environmental drivers depended on the level of performance considered. Multiservice delivery potential at a low performance level increased with multidiversity and was also indirectly positively affected by the interaction between patch size and historical age as larger and more ancient patches hosted higher levels of multidiversity (Fig. S3). Multiservice delivery potential at an intermediate performance level was not affected by any of the studied drivers (Fig. S3). Multiservice delivery potential at a high performance level increased directly with patch historical age and decreased directly with patch size, i.e. it was maximal in the smallest and most ancient woodlands (Fig. 3). It also decreased with soil acidity & C/N, i.e. it was maximal in less acidic soils with lower C:N ratio (Fig. 3).

Each individual ecosystem service/disservice showed a particular response to the array of drivers studied (Fig. 4). Patch size affected three ecosystem service proxies and patch age only one. Smaller woodlands exhibited a higher game production potential and topsoil carbon storage capacity, but lower tick-borne disease risk than larger patches. More ancient woodlands showed a higher topsoil carbon storage. Multidiversity, mediated by the positive interaction effect of patch size × patch age, had a significant positive impact on three out of the six individual ecosystem services studied: abundance of usable plants, pest control potential and game production potential, while tick-borne disease risk, topsoil carbon storage, and stemwood volume were unaffected by multidiversity. The landscape variables only had a limited effect on the ecosystem service delivery potential, with only the abundance of usable plants positively related to habitat connectivity. The abundance of usable plants was also the only service proxy affected by macroclimate; enhanced seasonality had a positive effect. Soil drivers affected several individual ecosystem services. Soil nutrient availability increased the abundance of usable plants and decreased tick-borne disease risk, game production potential and topsoil carbon storage. Tick-borne disease risk and game production potential increased in more acidic soils with higher C:N ratio, while the abundance of usable plants and stemwood volume decreased. Forest structure affected two proxies: game production potential increased with tree species diversity and decreased with structural diversity, and stemwood volume, which decreased with tree species diversity and increased with structural diversity.

All piecewise SEM models reported here were consistent with the data (p > 0.05 in all cases).

DISCUSSION

We showed that multidiversity was highest in large and ancient forest patches. Multiservice delivery potential at high performance levels per area was maximal in the smallest and most ancient woodlands and was not affected by multidiversity, which only influenced multiservice delivery potential at low performance levels, as well as some of the individual ecosystem services.

The interplay between patch biodiversity, age and size and their effects on multiservice delivery potential

Our results show that, although larger and more ancient woodlands hosted a higher biodiversity, as predicted by the species-time-area relationship [START_REF] Adler | Evidence for a general species-time-area relationship[END_REF], this was unrelated to a high-performance multiservice delivery potential. Contrary to our expectations, high-performance multiservice delivery potential decreased as patch size increased, independent of the positive effect of patch size on multidiversity. This means that smaller woodlands potentially deliver multiple services at higher performance levels on a per area basis than larger woodlands of a similar age, even if the larger woodlands harbor a higher biodiversity. This unexpected result may be explained by the positive edge effect on the delivery potential of some ecosystem services [START_REF] Bodin | The value of small size: loss of forest patches and ecological thresholds in Southern Madagascar[END_REF][START_REF] Mitchell | Forest fragments modulate the provision of multiple ecosystem services[END_REF]. As smaller woodlands have a relatively high edge-to-core ratio [START_REF] Ewers | Fragmentation impairs the microclimate buffering effect of tropical forests[END_REF][START_REF] Weathers | Forest edges as nutrient and pollutant concentrators: potential synergisms between fragmentation, forest canopies and the atmosphere[END_REF], they are highly exposed to external influences; they receive, for example, proportionally more light penetrating through the edge, a warmer and drier microclimate and more nutrient input from surrounding agricultural lands compared to large forests. These conditions promote, among others, higher patch-level microclimate heterogeneity [START_REF] Frey | Spatial models reveal the microclimatic buffering capacity of old-growth forests[END_REF] as well as a more dense vegetation cover and an increased biomass production at forest edges [START_REF] Remy | Driving factors behind litter decomposition and nutrient release at temperate forest edges[END_REF][START_REF] Remy | Altered microbial communities and nitrogen availability in temperate forest edges[END_REF] in small woodlands. This altered functioning in turn increases the delivery potential of some services, such as game production potential, due to an increased quantity of food available for game, and topsoil carbon storage, due to the faster incorporation of organic matter in the soil. Tick-borne disease risk is, however, lower, likely due to decreased larval densities in the unfavorable (e.g. hotter and drier) microclimatic conditions at the edge (Ehrmann et al., 2017).

On the other hand, the positive and indirect interaction effect of patch size and age, mediated by a higher multidiversity, increased multiservice delivery potential at a lowperformance level, as well as some individual ecosystem services. Therefore, it seems that the higher biodiversity hosted by larger and more ancient woodlands is able to maintain a minimal level of multiservice supply potential, while the maintenance of higher levels is less dependent on the amount of biodiversity. The supply potential of several individual ecosystem services indirectly increased in larger and more ancient woodlands because it was dependent on higher levels of biodiversity. For example, abundance of usable plants and game production potential might have increased due to a positive correlation with vascular plant diversity, while pest control potential probably increased due to bottom-up effects through the trophic chain [START_REF] Scherber | Bottom-up effects of plant diversity on multitrophic interactions in a biodiversity experiment[END_REF]. On the contrary, tick-borne disease risk, topsoil carbon storage and stemwood volume were unrelated to multidiversity, probably because they depended on particular environmental conditions or on the presence and abundance of specific species [START_REF] Winfree | Abundance of common species, not species richness, drives delivery of a real-world ecosystem service[END_REF] rather than on species richness per se.

Finally, it should be noted that we focused on the service delivery potential on a per area basis and that the total amount of services provided by large patches might still be larger than that of small patches. Our findings should therefore not be interpreted as a trade-off between large, biodiverse patches versus small patches that have a higher potential to deliver services, but rather as an observation that small woodlands in agricultural landscapes have the potential to deliver a high flow of services relative to their size.

The effect of other drivers on woodland multiservice delivery potential

The amount of forest cover around each woodland (cf. the habitat amount hypothesis, [START_REF] Fahrig | Rethinking patch size and isolation effects: the habitat amount hypothesis[END_REF] had no effect on multidiversity, multiservice delivery potential and individual services, which indicates that habitat availability within the landscape did not influence the service supply potential. This lack of effect may be a consequence of the disruption of metacommunity functioning in highly fragmented systems, with the dispersal of species among small forest patches dramatically reduced [START_REF] Jamoneau | Fragmentation alters beta-diversity patterns of habitat specialists within forest metacommunities[END_REF]. Both models [START_REF] Thompson | Ecosystem multifunctionality in metacommunities[END_REF] and experiments [START_REF] Haddad | Habitat fragmentation and its lasting impact on Earth's ecosystems[END_REF] predict reduced multifunctionality in such highly fragmented systems. The delivery of some ecosystem services may decline with low habitat connectivity, and with an intensively managed landscape matrix, as we have observed for the abundance of usable plants, which decreased with the proportion of crops surrounding the forest patch. Such an effect most likely results from a greater exposure of the forest edges to the biocides and fertilizers used in the adjacent croplands [START_REF] Chabrerie | Maturation of forest edges is constrained by neighbouring agricultural land management[END_REF].

Macroclimatic conditions affected neither multidiversity nor multiservice delivery potential, although we studied a large climatic gradient covering the entire European temperate biome. Only one of the individual services, the abundance of usable plants, was positively affected by macroclimate seasonality, increasing towards northern latitudes. This overall lack of effects of macroclimate suggests that the expected major biogeographic gradients influencing ecosystem service delivery potential are lost in highly fragmented forest ecosystems, at least partly due to the loss of macroclimate-driven biodiversity gradients [START_REF] Valdés | The contribution of patch-scale conditions is greater than that of macroclimate in explaining local plant diversity in fragmented forests across Europe[END_REF][START_REF] Vanneste | Functional trait variation of forest understorey plant communities across Europe[END_REF] and a decoupling between sub-canopy and free-air temperatures [START_REF] De Frenne | Microclimate moderates plant responses to macroclimate warming[END_REF], 2019;[START_REF] Frey | Spatial models reveal the microclimatic buffering capacity of old-growth forests[END_REF][START_REF] Lenoir | Climatic microrefugia under anthropogenic climate change: implications for species redistribution[END_REF].

Similarly, other potentially influential microclimate factors such as soil moisture and relative air humidity, not assessed in this study, could contribute to the observed lack of effects of macroclimate on multidiversity and multiservice delivery.

Future research avenues

This study is a first step towards a better understanding of the factors that influence the biodiversity and multiservice delivery potential of woodland patches in agricultural landscapes. Obviously, not all possible services were considered here (see [START_REF] Decocq | Ecosystem services from small forest patches in agricultural landscapes[END_REF] for an overview) and this raises the question of whether inclusion of other services, like erosion control and water quality regulation, both acting at a landscape scale, would alter the results. This is not unlikely, since it has been shown that different services are underpinned by different forest attributes [START_REF] Felipe-Lucia | Multiple forest attributes underpin the supply of multiple ecosystem services[END_REF]. Next, inclusion of more driving variables, e.g. those that quantify the past and current management and the microclimate in the patches, may help to further clarify how human actions in these patches influence their biodiversity and ecosystem service delivery potential. Another research avenue along the same lines is making the step from ecosystem service delivery potential towards actual ecosystem delivery. Indeed, not all services play an equally important role in all landscapes or for all stakeholders, and potential ecosystem service assessments should ideally be complemented by socio-economic analyses quantifying the importance and value of the studied services (e.g. [START_REF] Bagstad | From theoretical to actual ecosystem services: mapping beneficiaries and spatial flows in ecosystem service assessments[END_REF][START_REF] Zoderer | Stakeholder perspectives on ecosystem service supply and ecosystem service demand bundles[END_REF]. Finally, such integrated analyses will require the use of more sophisticated indicators that better reflect the actual ecosystem services that are delivered than the indicators we used.

CONCLUSIONS

Our study responds to the call for empirical research into the nature of the relationships between fragmentation and ecosystem service supply potential [START_REF] Mitchell | Reframing landscape fragmentation's effects on ecosystem services[END_REF]. We have shown that both large and small woodland patches in agricultural landscapes fulfill important roles. The large, ancient woodlands host the highest biodiversity and are particularly important for conservation. They also have the potential to deliver a considerable flow of services, but smaller woodlands have a higher potential to deliver multiple ecosystem services on a per area basis than the larger woodlands, especially when they are ancient. This higher potential of small woodlands might be caused by edge effects that can enhance the delivery potential of several services. Preserving the small woodlands is hence important to increase the multifunctionality of agricultural landscapes. This is an important finding because especially the small woodlands are currently not included in major national and international policies affecting biodiversity and ecosystem services in agricultural landscapes, such as the EU Natura 2000 network, the EU water Directive and the Common Agricultural Policy [START_REF] Pe'er | EU agricultural reform fails on biodiversity[END_REF]. Our findings advocate for the development of relevant policy instruments in order to ensure their future conservation. Next, we also found that the importance of drivers vary depending on performance levels of the ecosystem service and on the individual (dis-)service considered. A given driver may thus affect individual services in opposite directions, which leads to trade-offs among services (Gamfeldt et al., 2013;[START_REF] Lefcheck | Biodiversity enhances ecosystem multifunctionality across trophic levels and habitats[END_REF][START_REF] Felipe-Lucia | Multiple forest attributes underpin the supply of multiple ecosystem services[END_REF]. Consequently, different management strategies of these woodlands would be required to achieve multiservice delivery at high performance levels or to maximize (respectively minimize) the delivery of a specific ecosystem service (respectively disservice). For some services and performance levels, biodiversity conservation and ecosystem service delivery go hand in hand, but in other cases there may be a trade-off between both. This should be taken into account when designing policies and management strategies for small woodlands in agricultural landscapes.

AUTHORS' CONTRIBUTIONS

Author contributions: AV, JL and PDF contributed equally to the work. KV and GD share the last authorship. AV, JL, PDF, KV and GD designed the study. All authors collected the data. JL, AV, PDF and KV analyzed the data. AV, JL, PDF, KV and GD wrote the first draft of the paper. All authors contributed substantially to the writing and gave final approval for publication. connected and ancient forest patches (a higher species richness is expected). The green line and the green shaded polygon depict the average expected relationship between species richness and supply potential of multiple ecosystem services as well as the hypothetic confidence interval of all possible shapes we can expect for this relationship, respectively. Soil samples were taken during the period from August to October 2012 before leaves were shed and when the soil was well moisturized. For each patch, we sampled soil evenly along parallel transect lines located 50-m apart from each other. In the smallest patches (less than 7000 m2) a total of 3-5 soil samples were sampled and subsequently pooled. In bigger patches, the number of soil samples were related to patch size, ranging from 5 to 31 soil samples per patch (mean±SE = 5.99±0.26 samples per patch). We used a frame of 25 × 25 cm2 to collect forest floor samples. Care was taken not to mix forest floor with mineral soil when collecting the samples. The mineral soil was sampled using a metal soil core with a diameter of 4.2 cm. One core sample was taken down to 10 cm in mineral soil at all places where forest floor was removed. A total of 1315 forest floor samples and 1315 mineral soil samples were taken.

The samples were dried at 40°C and subsequently weighed. Ten percent of the samples were later dried at 105ºC to establish a correction factor for weight loss. The soil was passed through a 1 mm sieve to remove stones and gravel. Fine and coarse roots were removed by hand and weighed. All samples were analyzed for pH, total carbon (C) and nitrogen (N) as well as total phosphorous (P) in a single laboratory provider. Soil pH (CaCl2) was measured using a glass electrode (Orion, Orion Europe, Cambridge, England, model 920A) following the procedure described in ISO 10390:1994(E). The concentration of C and N was measured by high temperature combustion at 1150°C using an elemental analyzer (Vario MACRO cube CNS, Elementar, Germany). Total P concentration was measured after complete destruction with HClO4 (65%), HNO3 (70%) and H2SO4 (98%) in Teflon bombs for 4 hours at 150°C. P concentrations were measured according to the malachite green procedure [START_REF] Lajtha | Soil phosphorus: characterization and total element analysis[END_REF].

For each sample, we calculated the average depth of the forest floor (cm), and the total C and N contents (Mg ha-1), the C:N ratio, the total P content (kg ha-1)

and the pH (CaCl2) of the mineral topsoil (0-10 cm).

Details on data collection for forest structural drivers

Data were collected at 1-9 randomly chosen points per patch (number of points depending on patch size, mean±SE = 2.34±0.13) using an adaptation of the Point-Center-Quarter [START_REF] Cottam | The use of distance measures in phytosociological sampling[END_REF] method. Instead of one tree per quarter, two trees per quarter were measured, to gain an increased degree of detail per sample point.

Trees were chosen to be of small and large diameter per quadrant and would thus represent different layers/historical age classes of the sampled forest patches. Small trees were chosen to be between 7 cm and 30 cm in diameter at breast height (d130) and larger trees were chosen to be larger than 30 cm at d130. For each tree we recorded the species, tree height, number of stems, d130, the distance and angle to the central sampling point and whether the tree was alive. Sampling was restricted to a 20-m radius around the central point and if no tree of either group was found per quadrant, this was recorded.

Details on data collection for biodiversity

Understory herbs

All patches were comprehensively surveyed for all understory herb (i.e. non-woody)

species by walking along parallel transects located 10-m apart from each other. Along these transects, we recorded all species seen and thus obtained a quasi-exhaustive value of understory herb species richness for each patch.

Arthropods

Species richness of arthropods belonging to four taxonomic groups, including ground beetles (Insecta), spiders (Arachnida), millipedes (Myriapoda) and woodlice (Crustacea) was evaluated using pitfall trap sampling. We used pitfall traps with a diameter of 10 cm and a depth of 11 cm. Traps were filled with 200 ml of ethylene glycol and water

(1:1). A drop of detergent was added to remove water surface tension and the traps were equipped with aluminum roofs. In order to sample as much of the whole species community a sample point was set-up in the center of each patch and a second one was placed at the south-facing edge of the patch. If the south-facing edge was not suitable e.g. bordered by a ditch, road or other physical barrier (38 % of the edges), we used the eastern (16 % of the edges), western (18 %) or northern edge (4 %) instead. Each sample point consisted of two subsample units spaced five meters from each other. Each subsample unit consisted of two pitfall traps each separated by a plastic barrier (100 cm × 30 cm), parallel to the forest edge. This setup was used originally to identify species fluxes. Thus in each forest patch, eight pitfall traps were set up (four in the core and four at the southern edge). Arthropods were collected twice between April and August 2013. To make data comparable among regions, the two field sampling campaigns started at Growing Degree Hours values of ca. 10,000 and 20,000 (based on data of local weather stations in 2008 and 2009), respectively. Traps were open for 14 consecutive days, after which the trap content was collected and transported to the lab for sorting and subsequent identification. All individuals were identified to the species level.

Mushrooms

In each patch, mushroom species richness was evaluated by recording the presence of all fruiting bodies encountered along a 6-m wide strip-transect disposed along the widest diameter of the patch. Under the general name "mushrooms", we considered only those fungus producing fruiting bodies (i.e. epigeous sporocarps). Mushroom sampling was performed in autumn 2013, and in order to standardize the sampling timing we used six mushroom species with a wide European distribution as phenological indicators: mushroom sampling started as soon as the sporocarps of one of the following taxa were observed: Armillaria sp.; Collybia butyracea; C. dryophila;

Hypholoma fasciculare; Laccaria amethystina; or Lactarius quietus. These species frequently occur during the peak of sporocarp production of most species which coincides with a peak in autumn rainfall [START_REF] Bonet | Empirical models for predicting the production of wild mushrooms in Scots pine (Pinus sylvestris L.) forests in the Central Pyrenees[END_REF]. Local and European encyclopedias and guides [START_REF] Bon | Champignons de France et d'Europe[END_REF][START_REF] Courtecuisse | Champignons de France et d'Europe[END_REF][START_REF] Eyssartier | Le Guide des champignons -France et Europe (3 e édition)[END_REF] as well as databases (Société Mycologique du Nord de la France;

http://www.smnf.fr/) were used to identify mushroom species in the field. When the species could not be identified in the field, pictures of the whole sporocarps and, when possible, details of the cap, pores, stipe, ring, and gill were taken with a digital camera for species identification.

Details on data collection for proxies for ecosystem services

Abundance of usable plants

We performed a second, more detailed vegetation sampling in each patch, where we walked along parallel transects located 50-m apart from each other and established sampling plots every 100 m along a given transect. Number of sampling plots per patch ranged from 1 (for patches smaller than 0.5 ha) to 128 (mean±SE = 5±0.41). We recorded all vascular species present in the understory within a 10-m radius sampling plot and their abundance in an ordinal scale (1 = rare, 2 = common, 3 = dominant); i.e. a simplified version of the Tansley-scale (Tansley, 1935). We used several bibliographic sources to determine if each of the herbaceous species was of potential use by humans (see usable species list and references in Table S3). We then summed the abundances of usable species for each sampling plot, and calculated the average abundance of usable plants per patch by averaging among all sampling plots within each patch. Note that this variable is completely independent from the variable measuring understory plant diversity, which was obtained from a different survey at a different spatial resolution, i.e. abundance of usable plants was recorded at the plot level whereas understory plant diversity was recorded at the patch level.

Game production potential

Game production potential was assumed to be correlated with the abundance of palatable plants for large herbivores (Gamfeldt et al., 2013) and thus calculated as the average abundance per patch (calculated in a similar way as for usable plants) of the species eaten by roe deer (considering all vascular species, including herbs, shrubs and trees). We checked bibliography on roe deer diet across Europe and selected the vascular plant species (or genus) cited in at least three publications (see species list and references in Table S4). Roe deer was selected since this is an important game species that occurs in all our study regions, which is not the case for, e.g. red deer or wild boar.

Roe deer needs woody plant species in its diet [START_REF] Lovari | Woodland features determining home range size of roe deer[END_REF] and thus requires woodlands or hedgerows within its foraging range. Although there is no direct evidence in the scientific literature to support our hypothesis that roe deer density is positively related to the abundance of palatable plant, we argue that such a positive relationship is very likely and that our approach of using palatable plants as a proxy for game production potential is robust. Indeed, during the hunting season (mostly autumn and winter), when food has become rare for roe deer, both in croplands (harvests are completed and winter wheat has not emerged yet) and grasslands (cattle are still grazing in the meadows at that time of year), roe deer concentrate in woodlands where most of the palatable woody plants for roe deer are still available. Besides, roe deer use woodlands as a shelter against hunters during the hunting season in open farmlands (which starts before the hunting season in woodlands). This increases the probability for roe deer diet to solely rely on woody palatable plants from woodland habitats. Besides, we are not interested in assessing the density of roe deer per se here but rather the potential of a given woodland to provide hunting recreational activities and thus game production potential should be seen here as a recreational service rather than as a pure provisioning service for food supply.

Pest control potential

Pest control potential was assessed by the total abundance of malacophagous carabid beetles per patch from trapping data (see above and Table S5 for the list of malacophagous species and references). The rationale is that the selected malacophagous carabid beetles will prey on slugs in the adjacent fields; i.e. they use the forest patches for overwintering and refuge against agricultural disturbances and migrate to the agricultural land to prey (see references in Table S5). Note that abundance values from a very restricted set of species (malacophagous beetles) were used here to compute pest control potential whereas the variable measuring arthropod diversity is based on occurrence data across four different arthropod groups.

Tick-borne disease risk

Tick-borne disease risk (a disservice) was assumed to be correlated with tick density because a higher tick density would result in a higher risk for humans to be infected by tick-borne diseases such as Lyme Borreliosis. For each patch, tick density (i.e. mean density of nymphs per 100 m²) was assessed using a standardized sampling protocol.

All ticks stages were collected via drag sampling [START_REF] Tack | The effects of sampling method and vegetation type on the estimated abundance of Ixodes ricinus ticks in forests[END_REF] with a 1 × 1 m 2 piece of white flannel fabric attached to a wooden pole on a 100-m long transect (resulting in a 100 m² sampled area). Ticks were collected in the close vicinity (less than 25 m distance) of the central sampling point used for forest structure (see above). Ticks were picked off the cloth after 25 m of dragging and preserved in ethanol before being identified at the species level and counted in the lab (Ehrmann et al., 2017).

Stemwood volume

Stemwood volume (m 3 ) was determined from published species-and region-specific allometric equations based on tree height and diameter at breast height (d130) measurements. Species-specific equations to calculate stemwood volume are not available for all regions, and some species found in forests are not represented by any equation in the literature. Thus, either generalized regional equations (regional Quercus equation for all Quercus trees in that region, irrespective of species), generalized equations irrespective of the region, equations from substitute species (Quercus robur for Castanea sativa) or generic equations based merely on d130 and tree height, if no equations were available for the respective species [START_REF] Deleuze | Estimer le volume total d'un arbre, quelles que soient l'essence, la taille, la sylviculture, la station[END_REF], were used in order to calculate stemwood volume (see https://github.com/EhrmannS/tree-allometry for a detailed description of the selection of utilized equations).

Topsoil carbon storage

We used the soil C content (Mg/ha) in the combined forest floor and mineral topsoil (10 cm) for each patch as a measure of the potential for topsoil carbon storage. were determined after consultation of the literature for pest control potential [START_REF] Rouabah | Response of carabid beetles diversity and size distribution to the vegetation structure within differently managed field margins[END_REF], tick-borne disease risk [START_REF] Ruyts | Year-to-year variation in the density of Ixodes ricinus ticks and the prevalence of the rodent-associated human pathogens Borrelia afzelii and B. miyamotoi in different forest types[END_REF]), stemwood volume (FAO, 2015a[START_REF] Tansley | The use and abuse of vegetational concepts and terms[END_REF] and topsoil carbon storage [START_REF] Vesterdal | Carbon and nitrogen in forest floor and mineral soil under six common European tree species[END_REF]. The thresholds for the abundance of usable plants and the game 275 production potential were based on expert judgment by the paper's core author 276 team (AV, JL, PDF, KV, and GD) since literature data were lacking.

Topsoil carbon storage

Forest floor + topsoil (0-10 cm) (Mg/ha). We assume that higher topsoil stocks are the result of higher carbon sequestration rates Table S4: List of species present in the sampled forest patches and considered to be palatable for roe deer, i.e. cited in at least three of the references consulted hereafter: [START_REF] Barančeková | The roe deer diet: is floodplain forest optimal habitat[END_REF][START_REF] Barančeková | Annual changes in roe deer (Capreolus capreolus L.) diet in the Bohemian Forest, Czech Republic/Germany[END_REF][START_REF] Bartolomé | Diet composition of roe deer (Capreolus capreolus) in the Natural Park of the Garrotxa volcanic zone (Catalonia, Spain)[END_REF][START_REF] Cederlund | Seasonal differences between moose and roe deer in ability to digest browse[END_REF][START_REF] Czernik | Fast and efficient DNA-based method for winter diet analysis from stools of three cervids: moose, red deer, and roe deer[END_REF][START_REF] De Jong | Diet selection by roe deer Capreolus capreolus in Kielder Forest in relation to plant cover[END_REF][START_REF] Helle | Food composition and feeding habits of the roe deer in winter in central Finland[END_REF][START_REF] Henry | Dispersion, diet and criteria of age of roe deer (Capreolus capreolus L.)[END_REF][START_REF] Jackson | The annual diet of the roe deer (Capreolus capreolus) in the New Forest, Hampshire, as determined by rumen content analysis[END_REF][START_REF] Kamler | Faecal nitrogen: a potential indicator of red and roe deer diet quality in forest habitats[END_REF][START_REF] Maizeret | Effects of population density on the diet of roe deer and the availability of their food in Chizé forest[END_REF][START_REF] Moser | Resource selection by roe deer: Are windthrow gaps attractive feeding places?[END_REF][START_REF] Mysterud | Scale-dependent trade-offs in foraging by European roe deer (Capreolus capreolus) during winter[END_REF][START_REF] Schmidt | Dispersal of vascular plants by game in northern Germany. Part I: Roe deer (Capreolus capreolus) and wild boar (Sus scrofa)[END_REF][START_REF] Storms | Seasonal variation in diet composition and similarity of sympatric red deer Cervus elaphus and roe deer Capreolus capreolus[END_REF][START_REF] Tixier | Food selection by European roe deer (Capreolus capreolus): effects of plant chemistry, and consequences for the nutritional value of their diets[END_REF][START_REF] Wallach | Feeding activity and dietary composition of roe deer at the southern edge of their range[END_REF].

Species name

Figure 1 :

 1 Figure 1: Linking island biogeography (MacArthur & Wilson, 1967) and ecosystem

Figure 2 :

 2 Figure 2: Sampling biodiversity and ecosystem service supply potential in small forest

Figure 3 :

 3 Figure 3: Linkage between the environment, biodiversity and high-performance multiservice

Figure 4 :

 4 Figure 4: Linkage between the environment, biodiversity and individual ecosystem service

Figure 1 :

 1 Figure 1: Linking island biogeography (MacArthur & Wilson, 1967) and ecosystem functioning for studying the supply potential of multiple ecosystem services by small forest patches. Hypothetical framework on the effects of forest continuity across space and over time on immigration and extinction rates, and thus on the level of biodiversity (i.e. total number of species per forest patch) and the associated supply potential of multiple ecosystem services. (A) MacArthur & Wilson's theory of island biogeography adapted to forest spatiotemporal characteristics (effect of patch connectivity and age on immigration rate and of patch size on extinction rate). (B) Theoretically expected relationship between species richness and supply potential of multiple ecosystem services. The letters along the x-axis (a, b, c and d) depict different landscapes with different expectations on species richness and thus on supply potential of multiple ecosystem services, consisting of: (a) small, isolated and recent forest patches (a lower species richness is expected); (b) large but isolated and recent forest patches (intermediate levels of species richness are expected); (c) small but connected and ancient forest patches (intermediate levels of species richness are expected); and (d) large, connected and ancient forest patches (a higher species richness is expected). The green line and the green shaded polygon depict the average expected relationship between species richness and supply potential of

Figure 3 :

 3 Figure 3: Linkage between the environment, biodiversity and high-performance multiservice delivery in small woodlands across Europe. Results of the piecewise structural equation model (piecewise SEM, p = 0.512) studying the response of high-performance multiservice (MS) delivery to multidiversity, patch size, age and connectivity (proportion of forests and crops in a 500-m surface around each patch), while accounting for macroclimate, soil and forest structural characteristics as environmental drivers. Blue arrows indicate positive effects and red arrows indicate negative effects. Arrow thickness is proportional to the effect size. Only significant paths (p < 0.05) are shown, while variables not significantly contributing to the models are attenuated and represented with grey shading. Grey crossed arrows have been included to highlight the lack of effects of multidiversity on multiservice, and of tree species diversity and stand structure on multidiversity. The blue square depicts the positive interaction effect (p = 0.03) between patch size and historical age on multidiversity, as predicted by the species-time-area relationship. Marginal (cf. fixed effects) and conditional (fixed and random effects) R2 values are 0.33 and 0.48, respectively. The color ramp from red to blue depicts different historical patch ages ranging from the youngest (10 yrs) to the oldest (270 yrs) sampled patch (n = 200). All covariates (connectivity, macroclimate, soil and stand conditions) in the model except patch size and historical age were set to their mean values. 181x119mm (300 x 300 DPI)

Figure S1 :

 S1 Figure S1: Diagram of the principal component analysis (PCA) used to calculate the

Figure S2 :

 S2 Figure S2: Diagram of the principal component analysis (PCA) used to calculate

Figure S3 :

 S3 Figure S3: Linkage between the environment, biodiversity and multiservice delivery

(Coleoptera) in relation to their feeding habits. EntomologicalReview, 48,[462][463][464][465][466][467][468][469][470][471]

ACKNOWLEDGEMENTS

We acknowledge Catherine Bataillon, Déborah Closset-Kopp, Katharina Filzen, Kent Hansson, Aurélien Jamoneau, Tom Macheleidt, Willem Proesmans, Laurent Raison, Robert Saguez, Ilka Strubelt, Johan Van Keer, Rieneke Vanhulle, Stephan Wehling, Jérôme Willm and Christina Wolterink for fieldwork, Filip Moldan, Luc Willems, Greet de Bruyn and Lars Rosenqvist for help with soil analyses, Régis Courtecuisse and Pierre-Arthur Moreau for help with mushroom identification, Jérôme Buridant for help with patch history, Justine Louvel for database elaboration, Peter Manning for comments on an earlier version of the manuscript and Margot Vanhellemont for checking the English language. Funding: This research was funded by the ERA-Net BiodivERsA project smallFOREST, with the national funders ANR (France), MINECO (Spain), FORMAS (Sweden), ETAG (Estonia), DFG (Germany), BELSPO (Belgium) and DLR (Germany) part of the 2011 BiodivERsA call for research proposals.

DATA ACCESSIBILITY Should the manuscript be accepted, the data supporting the results will be archived in an appropriate public repository (e.g. Dryad) and the data DOI will be included at the end of the article.

Confidential Review copy

Journal of Applied Ecology multiple ecosystem services as well as the hypothetic confidence interval of all possible shapes we can expect for this relationship, respectively.

159x319mm (300 x 300 DPI)

Confidential Review copy

Journal of Applied Ecology S1 to S5.

Appendix S1: Supplementary Methods.

Details on patch selection

We wanted the subset of patches selected for sampling to be representative of the variation of patch size and historical age within each window. For that purpose, we divided all patches occurring within a given landscape window into two categories of size (small vs. large patches) and historical age (historically old vs. historically young patches), by using the median values of, respectively, patch size and historical age as division points between categories. Next, we selected 16 patches per window (i.e., 224 patches in total). Our aim was to select four patches for each of the combinations of size times historical age categories (small-old, small-young, large-old, large-young), although due to various reasons (e.g. some patches being unsuitable for sampling) this was not always possible. Summary statistics for each category per landscape window are shown in Table S1). and considered to be of potential use for humans. We used several bibliographic sources [START_REF] Couplan | Le régal végétal: plantes sauvages comestibles[END_REF][START_REF] Ducerf | L'encyclopédie des plantes bio-indicatrices alimentaires et médicinales: guide de diagnostic des sols[END_REF][START_REF] Mabey | Food for free[END_REF][START_REF] Mears | Wild food[END_REF][START_REF] Tobyn | The Western herbal tradition: 2000 years of medicinal plant knowledge. Singing Dragon[END_REF] to determine if each of the understory herb species found in the vegetation surveys was of potential use by humans. We used [START_REF] Couplan | Le régal végétal: plantes sauvages comestibles[END_REF] as the primary source, as it was the most complete, providing detailed indications on plant uses. Uses were divided in 3 categories: food use, medicinal use and other uses. Plant use was only considered when used in Europe (from France to Scandinavia, from the United Kingdom to Poland). Supposed or potential uses were not considered and only realized uses were considered. Plants that are used but which are not forest or forest edge species have not been considered, as forest populations of these species will never be used because there are much larger populations outside forests (e.g. Papaver rhoeas, Hypochoeris radicata, Chenopodium album, Lamium album, Plantago spp., etc.).

Details on soil sampling

Species name Food Medicinal Other

Adoxa moschatellina


Marianne Marchioni

Giulia Veronesi

Isabelle Worms

Wai-Li Ling

Thomas Gallon

Didier Léonard

Christelle Gateau

Mireille Chevallet

Pierre-Henri Jouneau

Laura Carlini

Marchioni

Wai Li

Didier Leonard

Chiara Battocchio

Pascale Delangle

Isabelle Michaud-Soret
email: isabelle.michaud-soret@cea.fr

Aurélien Deniaud
email: aurelien.deniaud@cea.fr

Safer-by-design biocides made of tri-thiol bridged silver nanoparticle assemblies

Introduction

Silver nanoparticles (AgNPs) are widely used in consumer products for their biocidal activity. AgNP-containing medical devices is also a growing market with applications in dentistry, prosthesis, catheters or wound dressings. Their action is due to Ag(I) release that is highly toxic for microorganisms inducing alterations of lipid bilayers, proteins and nucleic acids. This wide spectrum of effects makes them a good choice to avoid specific mutations in bacterial strains that could confer resistance. However, resistant strains have been observed 1 thanks to acquisition of the ability to chelate and excrete or to trap Ag species. 2- 4 The reactivity of Ag in general and AgNPs in particular is very complex and depends on the surrounding media 5 including living species such as Bacillus subtilis that secretes molecules to hinder their activity. 6 Ag(I) release from AgNPs is based on an oxidative dissolution mechanism and thus requires oxygen. 7 In aerobiosis, the metallic Ag(0) core of AgNPs is covered by surface Ag(I) species, and the dissolution of the NP into ions is favored by pH decrease 8 and/or by some thiol molecules. 9,10 The behavior of AgNPs in presence of thiol molecules is highly versatile either inducing time-dependent dissolution into ions 9,10 or forming a coating that protects the NP. 8,11 These phenomena depend on the architecture of the molecules and the number of thiols 8,9,11 but remains unpredictable to date. In order to solve concerns about NP use, next generation nanomaterials are being developed following a safer-by-design approach. 12 In the case of AgNPs, it is crucial to limit Ag(I) release to the strictly required amount for the biocidal activity. Besides, it would also be interesting to avoid AgNP release from the product in the environment or in the human body for medical devices. This approach would therefore limit bacterial resistance against silver and toxicity for mammals and other living species. In order to reach these objectives, we developed an innovative strategy that consists in bridging AgNPs together using a trithiol molecule, which favors the formation of larger and stable species that we decided to name AgNP assemblies. In these assemblies, AgNPs keep their high specific surface area without being released from the final product. The growth of the AgNP assemblies is mediated by thiol functions and can be controlled using a thiol-reactive molecule to stop the process and thereby to obtain a stable nanomaterial of defined size. Hence, AgNPs are entrapped into a tightly bound organic coating, which enables the long-lasting, slow dissolution into Ag(I). Therefore, the release from AgNP assemblies is reduced to an amount necessary to keep the biocidal activity, unlike isolated AgNPs, which undergo uncontrolled ion release. Altogether, using a smart and innovative process we reached the objectives needed for the future production of safer biocides to be grafted on nano-enabled biocidal products.

Results and discussion

NTA(CysOEt)3, abbreviated L 3S in the following, is a tri-thiol molecule with C3 symmetry inspired from a single metallothionein Cu(I) binding site (Fig. 1a). 13 This is a high affinity ligand for Cu(I) and Ag(I) that forms AgS3 complexes. 14 In the presence of 20 nm diameter citrate-coated AgNPs in equimolar concentration of Ag and S, L 3S induced a change of the solution color over time (Fig. 1b), due to a modification of the surface plasmon resonance (SPR) properties (Fig. 1c). Indeed, a decrease of the specific AgNP SPR signal around 400 nm was observed, along with the appearance of a broad peak centered at higher wavelengths that increased with time. This phenomenon is explained by the formation of non-symmetric dipole and is due to nanoparticles in contact without interpenetration between them. 15 These results suggested that L 3S induced an AgNP agglomeration-like mechanism, which is confirmed by the time-dependent increase of the hydrodynamic diameter of the species in solution, reaching about 250 nm with an homogenous distribution after 1h of reaction and more than 1 µm in an heterogeneous distribution after 75 min (Fig. 1d). However, several experimental evidences allow us to rule out the occurrence of simple agglomeration, meant as the formation of clusters of NPs held together by weak physical interactions in an unpredictable way, and resulting in a bulk material with no nano-properties.

Thiol molecules were previously demonstrated to favor AgNP dissolution into Ag(I) soluble species with a kinetic increasing with the number of thiol per molecule. 9 Indeed, phytochelatin-3, a tri-thiol molecule, induced an almost four times faster dissolution than glutathione, our standard one thiol molecule. Surprisingly, similar experiments performed with the tri-thiol molecule L 3S showed a slightly slower AgNP dissolution compared to the reaction with glutathione leading to lower amount of Ag(I) released after 24h incubation (Fig. 1e). In order to have a complete view of the different species produced over time, asymmetrical flow-field flow-fractionation (AF4) experiments were performed with online detection of Ag by inductively coupled plasma -mass spectrometry (ICP-MS) (Fig. 1f). Pristine AgNPs alone are eluted at 15 min retention time and remained stable in solution over 24h. The incubation with L 3S led to i) the decrease of AgNP peak (15 min) over time, ii) the appearance of a peak at lower retention time around 11 min that corresponds to Ag(I) soluble species and iii) the appearance of a broad peak corresponding to assemblies at a higher retention time, the latter increasing with the incubation duration. These results highlight the time-dependent formation of large assemblies of AgNPs triggered by L 3S . Moreover, the kinetics of the process can gradually be slowed down by decreasing the S to Ag molar ratio to 0.25 and 0.05, as shown by SPR spectra and dynamic light scattering (DLS) distributions (Fig. S1, ESI †). To control the assembly process, further experiments were performed with an S to Ag molar ratio of 0.25 and using iodoacetamide, a thiol-reactive molecule that alkylates and blocks free thiol functions. When added after 1h incubation between AgNPs and L 3S , the evolution of both SPR spectra and DLS distributions were stopped and the assemblies were stable for at least 120h at 25°C (Fig. 2a-b vs Fig. 1c-d). This proves that the AgNP assembly is thiol-driven and is not an agglomeration mechanism, which would be insensitive to iodoacetamide but sensitive to dilution, as observed with oxidized glutathione (Fig.

S2, ESI †).

In order to prepare assemblies of different sizes, L 3S and AgNPs were incubated during 1 hour before addition of iodoacetamide. Thiol-blocked AgNP assemblies were then fractionated over discontinuous sucrose gradient centrifugation into assemblies of different average hydrodynamic diameters (Fig. 2c). We obtained populations of assemblies of 40, 60 and 200 nm based on DLS measurement with different SPR spectra and colors (Fig. S3, ESI †) and zeta potentials of -28.3, -21.0 and -24.8 mV for the assemblies of 40, 60 and 200 nm, respectively. The DLS diameter distributions showed a clear shift towards larger diameter for the main peak but a second low intensity peak was observed at smaller values for both 60 and 200 nm assemblies (Fig. 2c). Therefore, the diameters determined by DLS are not accurate and the values 40, 60 and 200 nm are used as labels of the different population of assemblies in the following. The morphology of the assemblies and the number of particles per assembly were analyzed by electron microscopy: scanning transmission electron microscopy (STEM) and cryo-transmission electron microscopy (cryo-TEM) (Fig. 2d andS4, ESI †). Despite the different preparation protocols, both techniques clearly showed clusters of AgNPs comprising a number of NPs that increased with the expected size of the assemblies. Statistical analysis (Fig. S4b) showed that 40 nm assemblies are mainly formed by 1 to 4 NPs, 60 nm assemblies contained between 5 and 40 NPs with most assemblies containing 15 NPs and 200 nm assemblies are made of 16 to 60 NPs with most assemblies containing 40. Indeed, the larger is the hydrodynamic diameter of an assembly the more diverse are the possibilities to make it. Cryo-TEM also showed very short distances between AgNPs within an assembly, which is consistent with the small size of L3S with maximum distances between two sulfur atoms in the nanometer range (Fig. 2d andS4c).

Thiols are at the core of the assembly mechanism; therefore, we probed sulfur bonds with synchrotron radiation -induced X-ray photoelectron spectroscopy (SR-XPS) on assemblies of small (40 nm diameter) and medium (80 nm diameter) sizes (Fig. 3). Between 56 and 64% of the sulfur atoms were found to be bound to Ag atoms at the NP surface, with two distinct geometries in similar proportions. S 2p3/2 components at binding energies (BE) about 161 eV and 162 eV are attributed to sulfur atoms covalently bonded to metals in a monolayer or sub-monolayer regime, with the S atom in two different hybridizations, sp (161.3 eV) and sp3 (162.1 eV),16,17 coherently with what was already observed for organic thiols chemisorbed onto gold surfaces. 18 The low BE signal is reported as occurring characteristically in self-assembling monolayers of low degree of coverage. 19 Indeed, hybridization change is likely to cause a peak shift,20 with a BE of 162.1 eV for a sp3 S atom, a BE of about 161.3 eV (nearly 1 eV lower) would be expected for an sp-hybridized S atom.20 This proves the direct binding of the thiolate functions of L3S to the AgNP with two different geometries, linear and angular, corresponding to sp and sp3 hydridization, respectively (Scheme S1, ESI †).20,21

Due to iodoacetamide reaction, 25 to 29% of the sulfur atoms were involved in bonds with carbon (thioethers), while the remaining 10 to 15% formed disulfide bridges. This minor fraction of S species might belong to L3S molecules grafted to the NP surface with two thiolate groups, whereas the third thiolate forms an S-S bond with another partially grafted L3S molecule. Besides, the absence of a signal component below 161 eV suggests that the assemblies do not contain silver sulfide.22,23 XPS Ag 3d spectra showed that the fraction of Ag(I) remained constant around 6.6-6.8% of total Ag in the assemblies (Fig. S5, ESI †), which is close to the expected proportion of surface Ag for a 20 nm diameter NP (5.8%) confirming the limited dissolution of AgNPs. Therefore, AgNP assemblies are thiol-mediated and their structure is stabilized by Ag-S bonds involving L3S that has the capability to bridge AgNPs thanks to its three thiol functions in a tripodal architecture. Besides, ToF-SIMS positive mode analysis enabled the detection of significant amounts of Ag atoms (both isotopes 107 and 109, Fig. S6,ESI †) for citrate-coated AgNPs but not for AgNP assemblies of 40 or 200 nm. Since ToF-SIMS information depth is limited to the first monolayers at the surface of the sample, these results showed that L3S forms a layer that completely covers AgNPs in the assemblies.

The biological activity of assemblies of different diameters was then compared with citrated-coated AgNPs in order to gain insights into their potential as biocide and their toxicity for Human. All assemblies possess antibacterial activity against Escherichia coli (Gram-negative), with minimal inhibitory concentration (MIC) of 5.2, 5.9 and 12.7 µM Ag for 40, 60 and 200 nm assemblies, respectively. These values are higher than those measured for citrate-coated 20 to 60 nm AgNPs (MIC between 0.7 and 2.9 µM) (Fig. 4a), but still much lower than the values reported in literature for GSH-capped AgNPs (139 µM). 24 The biocidal activity was also tested against a Gram-positive bacteria, Bacillus subtilis, known to be difficult to be killed by AgNPs notably because of its ability to modify AgNP surface.6,25 As expected, the Ag concentration required to observe a growth delay was much higher than for E. coli: 113, 138 and 33 µM for assemblies of 40, 60 and 200 nm, respectively. Very interestingly, these concentrations are lower than the values determined for citrate-coated AgNPs (260 µM) (Fig. 4a). Therefore, the thick and tight coating afforded by the tri-thiol molecule L3S may protect assemblies against AgNP surface modifications that could prevent biocidal activity, rendering our assemblies more robust. Besides, toxicity was assessed in the human hepatocarcinoma cell line HepG2 since AgNPs accumulate in liver. 26 We were not able to reach a significantly toxic concentration (200 µM Ag higher concentration tested) for any of the assemblies, while the lethal dose for 50% of the cells is about 75 µM for citrate-coated AgNPs (Fig. 4b andS7, ESI †). Therefore, AgNP assemblies possess a biocidal activity against diverse strains but their cytotoxicity for eukaryote cells is drastically decreased.

Conclusions

In conclusion, we described an assembly mechanism of AgNPs bridged together by a bio-inspired tri-thiol molecule. This process, driven by thiol binding to surface Ag(I), results in the formation of a scaffold that protects the NPs from massive transformations induced by various environmental conditions normally responsible for their fast dissolution, sulfidation or agglomeration (see review 5). Moreover, these architectures do not prevent Ag(I) release and only slow it down (Fig. 4c), providing a nanomaterial that can be regarded as a novel promising safer-by-design biocide. Its tunable and controlled ion release and low sensitivity to the surrounding medium make it a longlasting biocide with drastically reduced hazard for Human and the environment with respect to currently employed technologies. The controlled Ag(I) release cannot only be explained by surface coverage since PVP-coated AgNPs are more toxic for prokaryotes and eukaryotes than assemblies.27 Therefore, the mixed sp3-sp hydridization of L3S thiols bound to surface Ag is likely to be responsible for the particular behavior of AgNPs involved in assemblies. The two hybridizations could mirror ligand binding on two different chemisorption sites, as hollow and on-top sites that favor stabilization of the assembly and dissolution into ions, respectively.16,28

The current work therefore describes the proof of concept for the development of novel biocidal AgNP assemblies. An original nanomaterial with promising properties has been developed thanks to a tripodal tri-thiol molecule, whose thiolate functions interact with surface Ag(I) ions, thereby bridging the NPs. Finally, further development of the organic molecule will enable the covalent grafting of the assemblies into objects that require efficient and long-lasting biocide such as medical devices.

Experimental section

Experimental details are available in the ESI †.

Conflicts of interest

There are no conflicts to declare Fig. 2 AgNP assemblies: production, purification and characterization. (a) time-dependent analysis of AgNP SPR spectra in the presence of L 3S between 0 and 120 hours of incubation in 0.25:1, S:Ag molar ratio. The reaction between AgNPs and L 3S occurs during 1 hour, then iodoacetamide is added at 100 µM for a thiol concentration of 90 µM. SPR spectra were acquired up to 120 hours total incubation time. (b) in a set-up similar to (a), hydrodynamic diameter distribution of the species in solution were determined for the AgNPs alone (t=0), at t=1h (time for the addition of iodoacetamide) and then at t=3, 4 and 96 hours. (c) hydrodynamic diameter distribution of different populations of assemblies separated on discontinuous sucrose gradient centrifugation. Three different populations were recovered: ''Assemblies-40nm'' corresponds to assemblies of 40 nm average diameter by DLS, ''Assemblies-60nm'' corresponds to assemblies of 60 nm average diameter by DLS and ''Assemblies-200nm'' corresponds to assemblies of 200 nm average diameter by DLS. (d) analysis by cryo-TEM of the different population of assemblies compared to initial AgNPs, for which a proper dispersion on the grid is obtained. Assemblies constituted of larger number of particles with larger diameter of the assemblies were observed on the grids. Clusters of AgNPs considered as an assembly are encircled to help the understanding of our statistical analysis (Fig. S4b, ESI †). The scale bar corresponds to 200 nm. "Iodo" stands for iodoacetamide. Fig. 3 Characterization of AgNP -L 3S interaction. SR-XPS S 2p core-level spectra collected on 40nm AgNP assemblies (a) and 80nm AgNP assemblies (b). For each signal, two spin-orbit components are reported: S 2p3/2 (thick curve) and S 2p1/2 (thin curve), with fixed intensity ratio S 2p3/2 / S 2p1/2 = 2/1 and doublet separation 1.2 eV. (c) spectral assignment based on BE values of S 2p3/2 components and semiquantitative analysis resulting in the estimation of the relative amount of the identified S species. * the statistic error in semiquantitative XPS analysis is of about 5% of the estimated value, 23 ** as reported in 16,19,20,22,[24][25][26] . pipetting layer by layer. Assemblies of average DLS diameter of 40, 60 and 200 nm were thus obtained.

Each population of assemblies were then rinsed three times in HEPES-Citrate buffer by centrifugation.

Scanning transmission electron microscopy. Sample (5 µL) was deposited on a glow-discharged nickel grid coated with a carbon film (Mesh 300, Agar Scientific / S160N3) to avoid particle agglomeration and enable even dispersion of objects on the grid. The drop was then dried under air. STEM micrographs were taken on a Zeiss MERLIN microscope operated at 30 kV, using the solid-state bright-field detector.

Micrographs were analyzed in the Fiji software. 4 For each sample, the number of NPs per assemblies was determined with a total counting of at least 600 individual NPs.

Cryo-transmission electron microscopy.

A small volume (2-4 µL) of the sample was deposited on a glowdischarged holey carbon grid and vitrified using an FEI Vitrobot. Frozen grids were observed on an FEI F20 Tecnai microscope at 200 kV and images were recorded on an FEI Ceta detector.

Synchrotron radiation X-ray photoelectron spectroscopy. Synchrotron radiation X-ray Photoelectron

Spectroscopy (SR-XPS) experiments were carried out at the BACH (Beamline for Advanced DiCHroism) beamline at the ELETTRA synchrotron facility (Basovizza, IT). XPS data were collected in fixed analyzer transmission mode (pass energy = 30 eV). A photon energy (PE) of 596 eV was used to acquire C 1s, S 2p, Ag 3d, O 1s and N 1s core-levels. The achieved resolving power was of 0.22 eV. Calibration of the energy scale was made by referencing to the gold Fermi-edge, and the metallic Ag 3d5/2 signal was always found at 368.20 eV, as expected. 5 XPS data analysis of S 2p and Ag 3d experimental spectra was performed via curve-fitting, by using a combination of Gaussian peaks, after subtraction of a Shirley background. The S 2p3/2 -S 2p1/2 and Ag 3d5/2 -Ag 3d3/2 doublets were fitted by using the same FWHM (full width half maximum) for the two spin-orbit components of the same signal, a spin-orbit splitting of 1.2 eV for S 2p and 6.0 eV for Ag 3d, and branching ratios S 2p3/2 / S 2p1/2 = 2, Ag 3d5/2 / Ag 3d3/2 = 3/2, respectively. In case of identifying presence of many chemically different species of the same element, the same FWHM value was used for all individual photoemission bands in order to reduce the number of refinement parameters then improving the reliability of the results.

Time-of-Flight Secondary Ion Mass Spectroscopy. ToF-SIMS analysis was carried out at Université

Claude Bernard-Lyon 1 (Villeurbanne, France) using a Physical Electronics TRIFT III ToF-SIMS instrument operated with a pulsed 22 keV Au + ion gun (ion current of 2 nA) rastered over a 300 µm x 300 µm area. Ion dose was kept below the static conditions limit. Data were analysed using the WinCadence TM software. Mass calibration was performed using hydrocarbon secondary ions.

Zêta potential. Zêta potential measurements were performed in a ZetaSizer (Malvern) at a 0.1 g.L -1 silver concentration in DTS1060 cuvettes (Malvern) and at 25 °C. The final value is based on 200 acquisitions using the Smoluchowski model. 6 Biocide assays. The E. coli strain K12 1655 was grown overnight in MD minimal medium (3.07 mM K2HPO4, 1.57 mM KH2PO4, 7.57 mM NH4(SO4)2, 1.94 mM sodium citrate, 0.1 g.L -1 glucose, 0.1 g.L -1 magnesium sulfate, pH 7.4). For MIC determination, E. coli cells were then inoculated at 0.01 OD in 96well plates in which assemblies or AgNPs were added in conditions allowing to screen for a wide range of Ag concentration. The plates were incubated at 37 °C with 900 rpm shaking in a Tecan reader plate and bacterial growth was continuously followed by measuring absorbance at 600 nm every 15 min. For B. subtilis (strain 3610), the set up was similar except that bacterial cells were grown in LB media. Besides, growth delays were determined instead of MIC that require too high Ag concentration.

Hepatocyte culture and cytotoxicity. HepG2 cells were grown in MEM media supplemented with fetal calf serum, glutamine and antibiotics (penicillin/streptomycin) and exposed to citrate-coated AgNPs or assemblies at the indicated concentrations of total Ag and for 24h. After exposure, HepG2 cells were harvested, suspended in PBS and mixed in one volume of Trypan Blue. Total and viable cells were counted in a TC20 Automated Cell Counter (Bio-Rad). Spectral assignments are based on Ag 3d5/2 component BE values. The main signal (Ag 3d5/2 = 368.2 eV, red curves) is attributed to metallic silver atoms at the NP core, while the signal at higher BE values (Ag 3d5/2 = 369.0 eV, blue curves) is due to partially positively charged silver atoms at the NP surface, Ag(δ + ), chemically interacting with thiol end-groups of the ligand L 3S . 5,7 (b) semiquantitative analysis and spectral assignment. * the statistic error in semiquantitative XPS analysis is of about 5% of the estimated value. 8 Scheme S1. Schematic representation of sulfur atoms (yellow circles) interacting with the Ag atoms on the nanoparticle surface in sp and sp 3 hybridizations. AgNP sp (linear) sp 3 (angular)

Fig. 1 L

 1 Fig. 1 L 3S -induced AgNP assemblies. (a) chemical structure of the tripodal molecule L 3S . (b) time-dependent evolution of the color of AgNP-L 3S mixture between 0 and 24 hours. (c) time-dependent analysis of AgNP SPR spectra in the presence of L 3S between 0 and 75 min of incubation in 1:1, S:Ag molar ratio. (d) timedependent evolution of the hydrodynamic diameter distribution of the species in a solution containing AgNP and L 3S at a 1:1, S:Ag molar ratio and between 0 and 75 min of incubation. The average diameter is written on top of each peak. (e) AgNP dissolution into Ag(I) species in the presence of GSH (noted "S-GSH") or L 3S (noted "S-L 3S "). Time-dependent measurement of the amount of Ag into the soluble fraction determined by ICP-AES. This amount corresponds to the fraction of the sample recovered in the supernatant of the sucrose cushion centrifugation (see experimental section in ESI †). (f) AF4 analysis of AgNPs in the presence of L 3S . Fractograms of species from AgNPs incubated alone for 0 or 24 hours and with L 3S for 1, 2 or 4 hours. The detection is based on 107 Ag by ICP-MS. In all experiments, t=0 corresponds to the AgNP alone in solution.

Fig. 4

 4 Fig. 4 Biological activity of AgNPs versus assemblies. (a) table presenting the MIC for AgNPs and the different assemblies determined based on overnight growth in liquid minimal media for E. coli and the concentration inducing growth delay in LB medium for B. subtilis. (b) HepG2 cell viability. Hepatocytes were exposed to 20 nm citrate-coated AgNPs or assemblies of 40, 60 or 200 nm for 24h before being harvested. Total and viable cells were counted in a TC20 Automated Cell Counter (Bio-Rad) using Trypan Blue. (c) amount of Ag(I) released from AgNPs or assemblies in 10 days. Each sample diluted in HEPES-citrate buffer at 370 µM in Ag was incubated for 10 days at 25 °C with constant steering at 400 rpm. The mixtures were separated on sucrose cushion centrifugation. Ag soluble species were in the supernatant and were quantified by ICP-AES. All the experiments were carried out at least three times independently.

Fig. S5 .

 S5 Fig. S5. Ag 3d XPS analysis. (a) SR-XPS Ag 3d core level spectra of 40 nm assemblies, 80 nm assemblies,

Acknowledgements

The authors acknowledge Cécile Lelong for providing the B. subtilis strain, Delphine Truffier-Boutry for providing access to the AF4 instrument and Elisabeth Mintz for discussions. This work was funded by the CEA-Toxicology Transversal Program through the NanoSilverSol grant and by the CEA Transversal Programs Toxicology and Nanoscience through the NanoTox-RX grant. This research is part of the LabEx SERENADE (grant ANR-11-LABX-0064) and the LabEx ARCANE and CBH-EUR-GS (grant ANR-17-EURE-0003). MM and IW were supported by the LabEx SERENADE. The authors also acknowledge Alejandro Fernandez Martinez for its help with Zêta potential measurements that have been performed within the analytical platform of ISTerre (OSUG-France). The AF4-ICP-MS and NanoZS were funded by the EQUIPEX NanoID (grant ANR-10-EQPX-39-01). This work is also supported by the Université Grenoble Alpes -AGIR grant NanoSilverSafe and IRS NanoBIS grant. This work used the electron microscope platform of the Grenoble Instruct-ERIC Center (ISBG: UMS 3518 CNRS-CEA-UGA-EMBL) with support from FRISBI (ANR-10-INBS-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). The EM facility is supported by the Auvergne-Rhône-Alpes Region, the Fondation Recherche Medicale (FRM), the fonds FEDER and the GIS-Infrastrutures en Biologie Sante et Agronomie (IBISA). We thank Dr. Guy Schoehn for support and access to the EM platform.

Experimental Section

Chemicals. GSH was purchased from Sigma. Synthesis and characterization of the molecule L 3S was previously described. 1 L 3S was dissolved in HEPES buffer (10 mM, pH 7.5) / acetonitrile, 6/4, v/v. AgNP synthesis. 20 nm diameter AgNPs coated with citrate were synthesized based on 2 . Briefly, AgNPs were produced by reduction of AgNO3 by tannic acid in presence of citrate. An aqueous solution (100 mL) containing 2 mM sodium citrate and 50 nM tannic acid were heated at 90 °C under 800 rpm stirring. When this temperature was reached five sequential additions of AgNO3 (200 µL) at 25 mM were done every 5 min. Upon AgNP synthesis the solution becomes yellow. AgNPs are finally recovered by 1 hour centrifugation at 9000 g. The pellet containing AgNPs was washed twice with an aqueous solution of 2 mM sodium citrate.

AgNP assembly process.

AgNPs at a final concentration of 370 µM in Ag were mixed in HEPES-Citrate buffer (10 mM HEPES, 2 mM sodium citrate pH 7) with L 3S at various concentrations in order to reach S:Ag molar ratio of 1:1, 0.25:1 or 0.05:1. The solutions were then incubated at 22 °C, under 400 rpm stirring for the desired incubation time before Ag quantification, AgNP SPR signal or DLS measurements. The process was also followed by AF4-ICP-MS. All these analyses were performed as described in 3 . The experiments performed with GSH and GSSG were done using the same protocol according to S:Ag ratio mentioned in the different figure legends.

AgNP assembly production.

For large scale assembly production, a S:Ag molar ratio of 0.25:1 was chosen.

After 90 min reaction at 22 °C, the reaction was stopped by addition of 100 µM iodoacetamide. The mixture was further incubated in the same conditions for 2 hours. Assembly populations of different average diameters were fractionated over discontinuous gradient centrifugation made of 50 µL of buffer containing showed the appearance of higher wavelengths peaks in presence of GSSG that are partially lost upon 6-fold dilution. Hydrodynamic diameter distribution of the species in solution (c, d) showed the formation of large size species in presence of GSSG that are lost upon 6-fold dilution. GSSG induces the agglomeration of AgNPs. The optical properties of agglomerates are similar to those of assemblies but agglomerates are unstable upon dilution contrary to assemblies. The reaction have been performed at a 0.25:1, S:Ag molar ratio. Besides, the reactions were performed in presence of iodoacetamide to prove that this agglomeration is independent of reduced thiolate. "dilution 2" and "dilution 6" stands for 2-fold and 6-fold dilution of the sample, respectively.