S Baronti

G Alberti

C Rumpel

M Giordan

F Camin

L Ziller

C Martinez

E Pusceddu

F Miglietta

I Criscuoli
email: irene.criscuoli@gmail.com

Anthropogenic charcoal-rich soils of the XIX century reveal that biochar leads to enhanced fertility and fodder quality of alpine grasslands

Keywords: biochar, charcoal, alpine grasslands, fertility, N:P ratio, fodder nutritional value

Anthropogenic charcoal-rich soils of the XIX century

reveal that biochar leads to enhanced fertility and fodder quality of alpine grasslands

Introduction

Alpine grasslands are considered critical biodiversity hotspots [START_REF] Väre | Taxonomic Diversity of Vascular Plants in the European Alpine Areas[END_REF] providing a series of ecosystem services [START_REF] Fontana | Comparing land-use alternatives: Using the ecosystem services concept to define a multi-criteria decision analysis[END_REF]) and sustaining the production of typical food resources with added value [START_REF] Bovolenta | Dairy production systems in the Italian alpine area[END_REF]. The persistence of these semi-natural ecosystems is based on ancient and specific management techniques adapted to the local needs ensuring their productivity for thousands of years [START_REF] Poschlod | The historical and socioeconomic perspective of calcareous grasslandslessons from the distant and recent past[END_REF] and the accumulation of one of the highest soil carbon stock [START_REF] Gamper | Short-time effects of land-use changes on O-horizon in subalpine grasslands[END_REF][START_REF] Ciais | The greenhouse gas balance of European grasslands[END_REF]. Traditional management techniques include intense land uses based on different stocking rates (different breeds and grazing period lengths), mowing frequency, organic and inorganic fertilization [START_REF] Maurer | Restoration and Rehabilitation of Species-Rich Grassland Ecosystems in France : a Review[END_REF], periodic re-sowing [START_REF] Tozer | Pasture renewal on Bay of Plenty and Waikato dairy farms : impacts on pasture production and invertebrate populations post-establishment[END_REF] and more rarely irrigation [START_REF] Reid | Effects of different irrigation systems on the biodiversity of species-rich hay meadows[END_REF]. Major threats to these ecosystems arise from climate change, land abandonment and inadequate intensification. In particular, abandonment has substantially reduced the surface area occupied by mountain grasslands throughout Europe [START_REF] Tasser | Land-use changes and natural reforestation in the Eastern Central Alps[END_REF], mainly leading to the expansion of secondary forests [START_REF] Tasser | Impact of land use changes on mountain vegetation[END_REF]. Inadequate management techniques are instead increasing the risks of soil erosion, landslides and avalanches [START_REF] Tasser | Effects of land use in alpine grasslands on the probability of landslides[END_REF], often causing the diffusion of unpalatable plant species [START_REF] Krahulec | Vegetation changes following sheep grazing in abandoned mountain meadows[END_REF], loss of biodiversity [START_REF] Dullinger | A resampling approach for evaluating effects of pasture abandonment on subalpine plant species diversity[END_REF], decreasing touristic attractiveness [START_REF] Hunziker | The spontaneous reafforestation in abandoned agricultural lands : perception and aesthetic assessment by locals and tourists[END_REF] and, in some instances, the loss of important soil organic carbon stocks [START_REF] Poeplau | Sensitivity of soil organic carbon stocks and fractions to different land-use changes across Europe[END_REF]. In the context of these changes, appropriate management of mountain pastures and meadows is becoming a societal priority that should be based on novel techniques ensuring the socio-economic viability of mountain communities, the conservation of their beauty as well as their ecosystem services.

The use of biochar, a carbon rich co-product of pyrolysis, as soil amendment is currently receiving a lot of attention as it can improve the physico-chemical properties of soils, reduce nutrient leaching [START_REF] Glaser | The "Terra Preta" phenomenon: a model for sustainable agriculture in the humid tropics[END_REF], increase water infiltration and water holding capacity [START_REF] Liebig | Die organische chemie in ihrer answendund auf agrikultur und physiologie[END_REF][START_REF] Novak | Biochars impact on water infiltration and water quality through a compacted subsoil layer[END_REF], enhance cation exchange capacity [START_REF] Liang | Black Carbon Increases Cation Exchange Capacity in Soils[END_REF], soil aeration [START_REF] Case | The effect of biochar addition on N2O and CO2 emissions from a sandy loam soil -The role of soil aeration[END_REF], thus stimulating biomass yields [START_REF] Jeffery | A quantitative review of the effects of biochar application to soils on crop productivity using meta-analysis[END_REF]Biederman and Harpole 2012). Moreover, because of its substantial recalcitrance to microbial degradation, biochar application to soil is also considered as an effective way to sequester atmospheric carbon [START_REF] Lehmann | Bio-char Sequestration in Terrestrial Ecosystems -A Review[END_REF][START_REF] Sohi | Biochar , climate change and soil : A review to guide future research[END_REF][START_REF] Criscuoli | Carbon sequestration and fertility after centennial time scale incorporation of charcoal into soil[END_REF][START_REF] Wang | Biochar stability in soil: meta-analysis of decomposition and priming effects[END_REF]. On the other hand, the use of biochar as a soil amendment entails also some risks which are not yet fully understood [START_REF] Kuppusamy | Agronomic and remedial benefits and risks of applying biochar to soil: Current knowledge and future research directions[END_REF]. In fact, biochar can be a source of toxicants such as polycyclic aromatic hydrocarbon (PAHs) [START_REF] Kessler | Mineralstoffversorgung der Milchkuh auf einen Blick rap aktuell (Mineral nutrient supply of dairy cows at a glance)[END_REF], can retain heavy metals, can suppress the efficacy of applied pesticides and may induce changes in soil microbial community composition and structure [START_REF] Jenkins | Biochar alters the soil microbiome and soil function: results of next generation amplicon sequencing across Europe[END_REF] with possible influence on the microbial-mediated transformation of nutrients. Moreover, its role in offsetting C and the other greenhouse gases' emissions has been questioned because of the possible priming effect on the native soil organic matter [START_REF] Ventura | Biochar stability and priming effect on SOM decomposition in two European short rotation coppices[END_REF][START_REF] Fang | Effect of temperature on biochar priming effects and its stability in soils[END_REF], the induced changes in the surface albedo [START_REF] Genesio | Surface albedo following biochar application in durum wheat[END_REF] and the possible associated C aerosol emissions [START_REF] Genesio | Black Carbon aerosol from biochar threats its negative emission potential[END_REF]).

The large majority of studies involving biochar applications, including those made on lowland grasslands (Van de [START_REF] Van De Voorde | Soil biochar amendment in a nature restoration area : effects on plant productivity and community composition[END_REF][START_REF] Schimmelpfennig | Biochar, hydrochar and uncarbonized feedstock application to permanent grassland-Effects on greenhouse gas emissions and plant growth[END_REF][START_REF] Schimmelpfennig | Changes in macro-and micronutrient contents of grasses and forbs following Miscanthus x giganteus feedstock, hydrochar and biochar application to temperate grassland[END_REF], are based on short-term experiments, mostly made immediately after biochar application. Only the work of [START_REF] Hernandez-Soriano | Long-term effect of biochar on the stabilization of recent carbon : soils with historical inputs of charcoal[END_REF] showed that 150 years old charcoal kiln areas located in agricultural fields were still able to increase the productivity of maize (+10%). As biochar properties were shown to change with time of exposure (i.e. ageing), in particular through surface oxidation [START_REF] Liang | Black Carbon Increases Cation Exchange Capacity in Soils[END_REF], short-term studies may be insufficient to assess its impact on soil fertility in the long-term. The productivity of mountain grassland ecosystems is the result of a complex set of interactions between grazing pressures and nutrients export, plant species composition and the establishment of soil microorganisms and soil micro-fauna communities, which are related to specific functional traits, whose dynamics necessarily require long-term studies [START_REF] Grigulis | Relative contributions of plant traits and soil microbial properties to mountain grassland ecosystem services[END_REF].

To properly address this critical issue, this paper assesses the effects of 158 years old anthropogenic charcoal rich soils [START_REF] Criscuoli | Carbon sequestration and fertility after centennial time scale incorporation of charcoal into soil[END_REF], derived from carbonization of larch wood (Larix decidua L.) on flat terraces [START_REF] Backmeroff | Precisely dating iron-ore mining and its effects on an alpine valley: Summary of a dendrochronological investigation of charcoal hearths and relict woodland stands[END_REF], on germination, growth and nutritive value of a typical grass and mountain leguminous species (Festuca nigrescens Lam. and Trifolium pratense L. subsp. nivale (Koch)) in comparison to native (control) soil and control soil amended with fresh biochar derived from larch wood. The presence of multiple charcoal rich soils gave us the opportunity to work on a fully replicated scheme, to be considered as an analogue of a deliberate centennial timescale application of biochar to a mountain grassland soil. The hypothesis is that plant productivity and nutritional value of both plant species, and especially of T. pratense, are higher in charcoal hearth soils compared to control soils and to soils amended with fresh larch charcoal because of char ageing [START_REF] Pusceddu | Morphological investigation and physical characterization of ancient fragments of pyrogenic carbon[END_REF]. The effects on seeds germination are expected to be less evident.

Materials and methods

Study site, soil sampling and charcoal production

Several charcoal-enriched anthropogenic soils (charcoal hearths) derived from the carbonization of larch (Larix decidua L.) wood on flat terraces, dating back to between 1500 and 1858 [START_REF] Backmeroff | Precisely dating iron-ore mining and its effects on an alpine valley: Summary of a dendrochronological investigation of charcoal hearths and relict woodland stands[END_REF], were identified in Val di Pejo (Trentino, Northern Italy) at an altitude of 2150 m a.s.l., in a larch alpine grassland grazed in summer. A complete description of the site, soil characteristics and historical charcoal production can be found in [START_REF] Criscuoli | Carbon sequestration and fertility after centennial time scale incorporation of charcoal into soil[END_REF]. In brief, the charcoal hearths' soils are made of a surface organic horizon (~2 cm) and a thicker black anthropogenic layer (~20 cm) rich in charcoal residues left after the carbonization 158 years ago and today well mixed with the pre-existing soil layer. The control soils are sandy-loam brown acid soils with some podsolization (Lithic Dystrudept and Entic Haplorthod) [START_REF] Smith | Techniques in pedology[END_REF]. Both control and hearths' soils have a pH of 5.1. The hearths' soils are very rich in carbon (26.2 ± 5.3 kg C m -2 ), 90% of which is contained in the charcoal, and are also richer in nutrients than the surrounding control soils (Tab. 1).

Three paired sites (i.e. charcoal hearth and native soil as control) were selected on the basis of common aspect (SE) and conservation state (no significant geo-morphological dynamics or recent anthropogenic disturbances). In September 2014, 20 L of soil were sampled with shovels from the center of the three hearths and in the corresponding adjacent control areas, after removing the top organic layer.

Fresh charcoal was produced from larch wood at 450°C, the average temperature occurring in traditional carbonization wood piles (FAO 1987). Fragments of larch wood were carefully wrapped in Aluminum foil and placed in a preheated muffle furnace at a temperature of 450°C for 10 minutes. The carbon content of the freshly produced charcoal was 76±7 gC kg -1 , the nutrient composition is reported in the Tab. 1 and the specific surface area (total BET) was 239.5 m 2 g -1 .

Germination test

Seeds of Festuca nigrescens Lam. and Trifolium pratense L. subsp. nivale (Koch) were obtained from a nursery. Total germination and germination rates for each species were assessed through a test in petri dishes. Three different substrates were considered: control soil (C), charcoal hearth soil (H) and control soil mixed with a fraction of fresh larch wood charcoal (CC) to reach the soil-charcoal ratio of 0.6 equivalent to a charcoal dose of 39 kg m -2 , which was estimated as the initial char input at the three charcoal hearths 158 years ago [START_REF] Criscuoli | Carbon sequestration and fertility after centennial time scale incorporation of charcoal into soil[END_REF]. 54 dishes were prepared (2 species x 3 soil types x 3 sampling areas x 3 replicates). In each petri dish, 10 seed of one species were sown and dishes were maintained in the dark. Germination was determined when the coleoptile was ≥ 2 mm for F. nigrescens and when the two cotyledons were visible and free from the seed coat for T. pratense.

Growth experiment

The three soil types were also used as substrates for a plant growth experiment in small pots of 135 cm 3 . Prior to sowing, the pots were left for two months in a greenhouse with regular irrigation in order to allow the seeds contained in the seed bank to germinate spontaneously. Naturally germinated plants were then periodically removed manually. To eliminate possible confounding effects due to differences in the soil temperature, caused by different light reflection/absorbance properties (albedo), the soil surface of each pot was covered with an uniform thin layer of black inert quartz granules of 1.5 mm diameter (Granulati Zandobbio, Bergamo). The plants were grown in monoculture for a total of 144 pots (2 species x 3 soil types x 3 sampling areas x 8 replicates). Seeds were sown at an initial density of 20 seeds pot -1 on 7 th January 2015 and the pots were stored on trays in a greenhouse, following a fully randomized experimental design. Air temperature and light in the greenhouse were left to fluctuate following external conditions, however temperature never dropped below 15°C by means of an automated heating system, while a minimum day length of 12 hours was ensured using artificial illumination during the winter months. Soil temperature was regularly checked at 3 cm depth using a digital thermal probe (109SS-L, Campbell scientific, Inc.) and no significant difference was observed among the soil treatments.

Nebulized irrigation provided water automatically at regular intervals, four times a day. Seven weeks after sowing, plant density was reduced to six plants per pot. Subsequently, three growth cycles were considered with complete aboveground biomass harvest made 17 (1 st cycle), 22 (2 nd cycle) and 30 weeks (3 rd cycle) after sowing. At each harvest, the plants were oven dried at 80°C for 48 hours and weighed. At the end of the 3 rd cycle, the roots were manually separated from the soil, washed and dried for 48 hours at 80°C and weighed.

Plant, charcoal and soil physico-chemical analysis

Plant, soil and charcoal samples were dried and finely ground prior to analysis. Isotopic ratios were determined with an Isotope Ratio Mass Spectrometer (Thermo Fischer Scientific, Delta V Plus). Natural abundances of stable carbon (δ 13 C) and nitrogen (δ 15 N) isotopes were measured for both plant species and the fine soil fraction (<2 mm) of hearth and control soils as well as for both the ancient and the freshly produced charcoal fragments. The carbon isotope discrimination (Δ) was then calculated according to [START_REF] Farquhar | Isotopic Composition of Plant Carbon Correlates With Water-Use Efficiency of Wheat Genotypes[END_REF]:

p p a        1
where δa is the δ 13 CO2 in the air and δp is that of plant carbon.

Total Ca 2+ , K + , Mg 2+ , Na + , P, Cu, Fe were determined on oven-dried plant subsamples (105°C for 24 h) from the second harvest, according to the EPA method 3052 (USEPA 1996) using an ICP-OES spectrophotometer (Varian Inc., Vista MPX). Total carbon and nitrogen concentrations were determined via total combustion in an elemental analyzer (EA Flash 1112 ThermoFinnigan).

Soil and charcoal pH were measured in a soil-charcoal/distilled water solution (1:4 ratio as reported in Di [START_REF] Lonardo | Biochar successfully replaces activated charcoal for in vitro culture of two white poplar clones reducing ethylene concentration[END_REF][START_REF] Vaccari | Biochar stimulates plant growth but not fruit yield of processing tomato in a fertile soil[END_REF].

The surface area of fresh charcoal was calculated by using the BET (Brunauer-Emmet-Teller) method and Langmuir method applied to nitrogen adsorption data in the relative pressure (P/P°) range of 0.1-0.44.

Statistical analyses

Data were analyzed using R (version 2.15.3). The effects of the 3 treatments (C, H and CC) and of the 3 blocks (3 charcoal hearths and 3 control soils) were analyzed by a two-way ANOVA for each plant species. Individual comparisons were based on the Tukey's HSD post-hoc test. The data were checked for normality and homogeneity of variances by inspection of the residuals vs. fitted values and the Normal Q-Q plot. When data did not fulfill these requirements, they were log-transformed or square root-transformed. In the case of negative data (δ 13 C), the data were standardized. Germination rates were compared using a binomial test (test for equality of proportions). The p-values of individual comparisons were corrected with the Holm method. All data in the text, figures and tables are reported as mean ± standard deviation, if not differently stated.

Results

Germination test

Plants started to germinate after 96 hours of incubation. The percentage of germinated seeds after 96 and 120 hours from sowing was higher in CC compared to C and H both for F. nigrescens and T. pratense (Tab. 2), while early germination rates were similar or the same in C and H. Looking at the total number of germinated plants, almost all seeds germinated in all soil types (between 78 and 94%). The plant germination rates at the end of the experiment for both plants species were higher in both CC and H than in C (F. nigrescens: CC vs. C +14%, p=0.08 and H vs. C +13%, p=0.09; T. pratense: CC vs. C +13%, p=0.05 and H vs. C +12%, p=0.07).

Plant growth

At the end of the first growth cycle (week 1-17), both plant species grew more in H than in C and CC (Fig. 1) and the lower growth observed in the CC was highly statistically significant if compared to both H and C. The relative changes in plant biomass in comparison to C were +21, +9% for H and -35, -71% for CC, for T. pratense and F.

nigrescens, respectively. The mean plant growth rate (MGR) was much lower in CC (T. pratense: 1 mg day -1 pot -1 ; F. nigrescens: 1.5 mg day -1 pot -1 ) than in C (T. pratense: 2.3 mg day -1 pot -1 ; F. nigrescens: 3.2 mg day -1 pot -1 ) and H (T.

pratense: 2.8 mg day -1 pot -1 ; F. nigrescens: 3.5 mg day -1 pot -1 ).

At the end of the second growth cycle, biomass was again significantly higher in H than in C and CC for both species (Fig. 1). T. pratense and F. nigrescens plants grown on H produced almost 3 and 1.5 times more than on C, respectively. Plants grown on CC performed better than on the control soil (both T. pratense, F. nigrescens= +37%), even though the difference was significant only in the case of T. pratense (p=0.01). MGR was accelerated compared to the first cycle and rose to 10.7 and 41.1 mg day -1 pot -1 for T. pratense and to 16.6 and 40.7 mg day -1 pot -1 for F.

nigrescens grown in C and H, respectively.

Similarly to the second cycle, at the end of the third cycle plant biomass was significantly higher in H than in the other two treatments (Fig. 1). F. nigrescens plants grown on H produced almost 1.5 times more than plants grown on C and T. pratense 0.6 times more. Plants grown on CC performed better than on C (F. nigrescens = +51%, T. pratense = +22%) even though the difference was significant only in the case of F. nigrescens (p=0.01). The MGR decreased compared to the second cycle to 24.1, 9.9 and 15 mg day -1 pot -1 for F. nigrescens and to 19.1, 12.1 and 14.9 mg day -1 pot -1 for T. pratense grown in H, C and CC, respectively.

Overall, the total amount of biomass produced over the three cycles (above and belowground) was 88% and 108% higher in H compared to CC (p<0.001) and 114% and 148% higher in H compared to C (p<0.001) for F. nigrescens and T. pretense, respectively (Fig. 2). Total biomass was slightly higher in CC compared to C for both plant species, but the differences were not significant (F. nigrescens: p=0.29, T. pretense: p=0.25).

The root:shoot ratio, was significantly higher for T. pratense plants grown on H than C (p=0.02), while for F. nigrescens no differences in the ratio were detected among treatments (Fig. 3).

Soil and plant nutrients

The content of Ca 2+ , Mg 2+ , Na + and P (mg g -1 ) in the plants tissues (Tab. 3) was the highest in F. nigrescens and T. pratense grown on H followed, in most cases, by plants grown on CC. The differences in the nutrient content of T. pratense between H and C were significant at p=0.10 for all the nutrients, while no significant differences between H and CC in T. pratense and among all treatments for F. nigrescens were detected (Tab. 3). The K + content of T. pratense was higher in plants grown on H (H vs. CC: p=0.18; H vs. C: p=0.06), but for F. nigrescens plants it was higher when plants were grown on CC, even though not significantly. An opposite trend was observed for N, which was higher for plants grown on C for both species, followed by CC and H. Fe content showed a very high variability in plants grown on the same soil type, while soil Cu content was very similar for all the three treatments. The nutrient content of T.

pratense was usually higher than that of F. nigrescens with the exception of P that was higher for F. nigrescens plants when grown on H and C.

The N:P ratio of F. nigrescens and T. pratense plants grown on H was 10.4 and 10.7, respectively, while plants grown on C showed a ratio of 18.3 and 21.5 and plants grown on CC soils 23.3 and 16.3 (Fig. 4).

Soil nutrient content data were taken from [START_REF] Criscuoli | Carbon sequestration and fertility after centennial time scale incorporation of charcoal into soil[END_REF] and are here reported in the Tab. 1. The soil N:P ratio in soil was 13.3, 5.7 and 11 in C, H and CC, respectively (Fig. 4).

Plant P concentration increased with increasing soil concentration. The highest values were observed for the H soil and in the plants grown on it, while soil and plants grown on CC and C gave very similar results (Fig. 5). Plant K + concentration correlated positively with the soil nutrient contents. Concentrations of both soils and plants decreased in the order H>CC>C (Fig. 5). We observed a similar behavior for Ca 2+ in the case of T. pretense, while the content of F.

nigrescens was insensitive to the soil content variations (Fig. 5). Na + content was the lowest for H soils. The concentration of the plants tissues decreased for T. pratense with increasing soil concentrations, while values observed for F. nigrescens did not reflect the Na + soil content (Fig. 5). Mg 2+ concentration of the soil was not reflected in the content of plants for both species. The Mg 2+ content of T. pratense was double compared to F. nigrescens (Fig 5,Tab. 1 and Tab. 3).

Isotopic signature

The 13 C discrimination (Δ) at the end of the first growth cycle was significantly higher for F. nigrescens and T.

pratense plants grown on CC (24.5 and 23.6‰, respectively) than on C (21.8‰ with p=0.0006 and 22.1‰ with p=0.051) and H (21.5‰ with p=0.0004 and 22.1‰ with p=0.051).

At the end of the second growth cycle, the Δ of both plant species grown on H and C slightly increased in comparison with the first one (T. pratense: H +0.74‰, C +0.42‰; F. nigrescens: H +1.02‰, C +0.56‰), while, when they were grown on CC, the Δ decreased (T. pratense: -0.56‰; F. nigrescens: -1.55‰) to similar values for both species (22.99‰) and to the other two soil treatments. At the third harvest, plants showed smaller Δ values and no differences among treatments (Fig. 6).

At the end of the first growth cycle, the δ 15 N was very similar for T. pratense and F. nigrescens plants grown on C soils (6.9 and 7‰, respectively closer to the signature measured in the other treatments (ranging between 7.1 and 7.7‰; Fig. 7).

The control soil had a δ 15 N equal to 3.9±1.3‰, markedly higher than H (2.1±1.1‰), while the ancient and the freshly produced charcoal had very similar δ 15 N signatures (1.1±1.6‰ and 0.9±0.2‰, respectively).

Discussion and conclusions

The three substrates had differential effects on germination. For both plant species, early germination rates were higher in the control soils amended with fresh charcoal (CC) than in hearths soils (H) and control soils (C).

Moreover, the total number of germinated seeds 10 days after sowing was stimulated for both species by the presence of old (H) and fresh charcoal (CC) amendment (Tab.2). Previous research on T. pratense by [START_REF] Van De Voorde | Soil biochar amendment in a nature restoration area : effects on plant productivity and community composition[END_REF] found no effect of biochar addition to the soil substrate on the germination and Solaiman et al. ( 2011) found a dosedependent negative germination effect on another Trifolium species. In contrast, we did not observe any inhibitory effect on seeds germination, but a tendency to an increase in germination in the presence of both fresh and old charcoal.

These positive effects could be related to the presence of specific compounds such as karrikins, a family of butenolides, which are known to be contained in charcoal [START_REF] Flematti | Germination stimulant in smoke: isolation and identification[END_REF]Nelson et al. 2012). Karrikins act as germination stimulants and have been recently isolated, identified [START_REF] Flematti | A compound from smoke that promotes seed germination[END_REF]) and successfully synthesized [START_REF] Flematti | Production of the seed germination stimulant karrikinolide from combustion of simple carbohydrates[END_REF]) even though the mechanisms by which they might trigger seed germination are far from being completely described. Another possible mechanism explaining the higher germination rates in CC and H could be related to the ability of charcoal to adsorb biogenic phyto-toxins eventually present into the soil [START_REF] Garnett | Control of pitch pine seed germination and initial growth exerted by leaf litters and polyphenolic compounds[END_REF][START_REF] Hille | Charcoal and activated carbon as adsorbate of phytotoxic compounds a comparative study[END_REF].

In the pot experiment, at the end of the first growth cycle, both species grew better on H than on C and CC (Fig. 1). Reduced growth on CC was remarkable and attributable to a stress effect caused by the addition of fresh char.

Such a stress caused a decrease in the photosynthetic capacity of plant leaves, which was reflected in an increase in their Δ 13 C (Fig. 6) [START_REF] Brugnoli | Correlation between the Carbon Isotope Discrimination in Leaf Starch and Sugars of C3 Plants and the Ratio of Intercellular and Atmospheric Partial Pressures of Carbon Dioxide[END_REF]. A possible source of stress affecting plant growth on CC could have been the difference in the pH between the fresh larch charcoal (pH=7.1) and the control soil to which it was added to (pH=5.1).

Soil alkalization is known to have detrimental effects on plants as high pH generally causes metal ions to precipitate, thus affecting the absorption of inorganic ions and disrupting the ionic balance of tissues [START_REF] Chen | Comparative effects of salt and alkali stresses on organic acid accumulation and ionic balance of seabuckthorn ( Hippophae rhamnoides L .)[END_REF]). In our case, the bulk soil pH of CC rose to 5.8 and we cannot exclude that plant roots were in direct contact with charcoal fragments with a higher pH. However, 7.1 is a value close to neutrality and, moreover, [START_REF] Gell | Residues of bioenergy production chains as soil amendments: immediate and temporal phytotoxicity[END_REF] did not find a clear relationship between pH and short-term phyto-toxicity on lettuce, radish and wheat plants. Thus, the toxic effect observed in the first growth cycle is very likely not to be linked to a pH stress. On the contrary, there could be a direct phytotoxic effect of charcoal on plant growth, both on germination and root and shoot growth as observed in previous studies that related the phyto-toxicity to the presence of chemicals in the biochar. For example, [START_REF] Rombolà | Relationships between Chemical Characteristics and Phytotoxicity of Biochar from Poultry Litter Pyrolysis[END_REF] made the hypothesis that the phyto-toxicity was due to NH3, Volatile Fatty Acids and Benzoic acids contained in the charcoal, while Gell et al. ( 2011) associated the toxic effect of different kinds of biochar with their high water-soluble salts content, such as Chloride and Sodium, and possibly aliphatic and aromatic hydrocarbons such as phenols.

Observations made during the first growth cycle have important implications regarding the possibility of using biochar as a soil amendment in the framework of mountain pastures and meadows management and restoration. The data showed that while long-term exposure of pyrogenic carbon in soil, as occurred in H, buffers any possible toxic effect, the sudden addition of biochar could have detrimental effects on plant growth. Even if the exact temporal dynamics of the transition between toxic and non-toxic conditions following biochar incorporation remains unknown, these data already suggest that the negative effect of charcoal incorporation into the soil is transitory as better environmental conditions certainly develop during biochar ageing. Moreover, transitory toxic effects can be avoided if is a) washed with water or an organic solvent, b) degraded via a biological activity through composting or mixing it with activated sludge [START_REF] Bargmann | Hydrochar and Biochar Effects on Germination of Spring Barley[END_REF][START_REF] Rombolà | Relationships between Chemical Characteristics and Phytotoxicity of Biochar from Poultry Litter Pyrolysis[END_REF] or c) dried at temperatures between 100 and 300°C

for 24 hours [START_REF] Kołtowski | Toxicity of biochars after polycyclic aromatic hydrocarbons removal by thermal treatment[END_REF] before being amended to soil. It is also likely that lower doses than used in the present experiment, could reduce detrimental effects on the plant growth, even in the very early stages.

The hypothesis of a transitory toxic effect of biochar on plant growth is also confirmed when looking at biomass production during the second and the third growth cycles (Fig. 1). In fact, at the end of the second cycle, biomass was again significantly higher for plants grown on H compared to those grown on C. T. pratense plants grown on H produced almost threefold if compared to C and F. nigrescens 1.5-times more. On the other hand, conversely to what was observed in the first cycle, plants grown on CC performed better than those grown on C (both T. pretense and F. nigrescens= +37%). The recovery of both species grown in CC was also confirmed by the Δ 13 C, which was not significantly different from the other two treatments (Fig. 6). The mean plant growth rate measured for plants grown on H was 4 and 3 times higher than those of plants grown on C for T. pratense and F. nigrescens and 2 and 3 times higher compared to those grown on CC, respectively, indicating a clearly higher fertility in charcoal hearths soils than in their natural counterparts and soils with fresh biochar. Similar results were obtained by [START_REF] Naisse | Effect of biochar addition on C mineralisation and soil organic matter priming in two subsoil horizons[END_REF], who observed much higher (micro-)biological activity in charcoal hearths as compared to control soils without ancient charcoal. The increase in the charcoal hearth soils' fertility of our study site can be explained by the accumulation over time of soil nutrients, their higher plant availability as well as improved physical characteristics of charcoal hearth soils compared to control (Criscuoli et al., 2014, Tab. 1).

Similarly to the second cycle, also at the third harvest, plant biomass was significantly higher for H than for the other two treatments (Fig. 1). However, the biomass measured for T. pratense grown on H at the end of the third cycle was lower in comparison to the second one. This could be due to the higher temperatures registered during the third growth cycle (summer) compared to the second one (spring), which caused the spread of necrotic tissues in the leguminous species (data not shown).

Overall, the total plant biomass produced over the three cycles (above and belowground) was significantly higher when plants were grown on H compared to CC and C both for F. nigrescens and T. pratense (Fig. 2). No significant differences were detected between plants grown on CC and C, making the positive impact of charcoal on the long term productivity of alpine grasslands even more evident than the results of one single growth cycle.

The root:shoot ratio, a widely used indicator of plants health, gave differential results according to the species.

It was significantly higher for T. pratense plants grown on H than for those grown on C, while for F. nigrescens no difference in the ratio was detected among treatments (Fig. 3). The different behavior between the two species is related to the high interspecific variation in root:shoot ratios as reported by [START_REF] Koerner | Dry matter partitioning and root length / leaf area ratios in herbaceous perennial plants with diverse altitudinal distribution[END_REF]. The results observed for T. pratense seem to be in contrast with previous literature. In fact, for another species of clover (Solaiman et al. 2012), inconsistent effects of biochar on root:shoot ratio were observed. At high application rates charcoal had a negative effect. Moreover, it is known that the development of the root compartment may be smaller compared to aboveground biomass in nutrient rich soils [START_REF] Agren | Root : Shoot Ratios , Optimization and Nitrogen Productivity[END_REF], but according to our results T. pratense had a much higher root to shoot ratio in H soil, which shows the highest nutrient contents, both total and available (Criscuoli et al., 2014, Tab. 1). A possible explanation of high root to shoot ratios for T. pratense grown on H may be that roots development was more driven by hormonal factors rather than nutrients concentration. In fact, an increase in ethylene production, a plant hormone with important implication for plant growth and development, has been previously observed from biochar and biochar-amended soil [START_REF] Spokas | Ethylene: potential key for biochar amendment impacts[END_REF].

Soil and plants nutrient contents provided useful information to further examine and interpret our results. The idea that the ratios of N, P and K in plant tissues provides an indication of the relative availability of these nutrients in the soil has often been discussed since the Von Liebig's Law of the Minimum (Von Liebig 1840). [START_REF] Koerselman | The vegetation N : P ratio : a new tool to detect the nature of nutrient limitation[END_REF], among others, assumed that the N:P ratio of plant tissues is a reliable indicator of soil fertility and proposed, on the basis of a meta-analysis of experimental data, that N:P thresholds of plant tissues can be used as indicators of N-limitations (N:P < 14) or P limitations (N:P > 16) or their co-limitation (14 < N:P < 16) to plant growth.

Accordingly, plants of both F. nigrescens and T. pratense species grown on H soils can be considered mostly N-limited (leaves N:P ratios of 10.4 and 10.7, respectively), while they were P-limited when grown on C and CC soils (N:P ratios > 16; Tab. 3 and Fig. 4). Thus, the lower plant biomass production in C and CC could be related to a P-limitation. In fact, total and available P-concentrations of these soils were similar and about one third compared to that of H soils [START_REF] Criscuoli | Carbon sequestration and fertility after centennial time scale incorporation of charcoal into soil[END_REF]Tab. 1). The soil P concentration for C and CC was reflected in the plants concentration which was similar (Tab. 3 and Fig. 5), showing that P added via charcoal was available for plants. This has been previously demonstrated specifically for larch wood (Larix gmelinii Rupr.) charcoal produced at 400°C (available P: 42.7 mg kg -1 biochar) and for the larch seedlings grown on a mixture of sand larch biochar which had higher foliar P concentrations in the presence of higher rates of char [START_REF] Makoto | The growth of Larix gmelinii seedlings as affected by charcoal produced at two different temperatures[END_REF]. Thus, the higher P content observed in the H soils and plants growing on it have to be explained via other processes taking place in the long term other than the initial P input via charcoal addition. It has been shown that P availability in charcoal amended soils is enhanced also via an increased mycorrhizal colonization and a change in soil P fractionation [START_REF] Graber | A humic substances product extracted from biochar reduces Arabidopsis root hair density and length under P-sufficient and P-starvation conditions[END_REF]. Moreover, charcoal hearth soils might have accumulated the P atmospheric depositions due to desert dust transport and atmospheric pollution [START_REF] Bergametti | Source, transport and deposition of atmospheric phosphorus over the northwestern Mediterranean[END_REF] or dung released from cattle over the 158 years of exposure in soil.

The higher plant N:P ratio observed in CC soils for F. nigrescens compared to T. pratense (Fig. 4) is due to the higher N content in T. pratense (19.2 vs. 14.2 mg g -1 in F. nigrescens), while P content is almost the same in the two species (0.9 vs. 0.8 mg g -1 , respectively; Tab. 3). The higher N content of the leguminous species might be explained by its nitrogen fixing capacity and this is also confirmed by the lower δ 15 N that we observed for plants grown on CC in the first and second growth cycle, much closer to the atmospheric value (0 ‰) as compared to F. nigrescens (Fig. 7). These results are in line with several previous studies reporting an increase in nitrogen fixation in biochar amended soils [START_REF] Rondon | Biological nitrogen fixation by common beans (Phaseolus vulgaris L.) increases with bio-char additions[END_REF][START_REF] Ogawa | Pioneering works in biochar research , Japan[END_REF][START_REF] Güereña | Partitioning the contributions of biochar properties to enhanced biological nitrogen fixation in common bean (Phaseolus vulgaris)[END_REF][START_REF] Van Zwieten | Enhanced biological N2 fixation and yield of faba bean (Vicia faba L.) in an acid soil following biochar addition: dissection of causal mechanisms[END_REF] which has been mainly related to a greater B, Mo and P avaliability. The δ 15 N signature of F. nigrescens plants grown on CC was higher than the signature of T. pratense grown on the same soil, but was lower than the signature of plants of the same species grown on the other soil types along the three growth cycles. This might be explained by the uptake of nitrogen contained in the fresh charcoal fragments (De la Rosa and Knicker, 2011), as the nitrogen isotopic signature of charcoal fragments is close to 1‰, while control soils have a signature of 3.9±1.3‰.

Similar to P, the concentration of K + in tissues of both plant species and of Ca 2+ in T. pratense plants correlated positively with soil nutrient content (Fig. 5), with higher concentrations observed in the case of higher biomass production (H>CC>C). Similar observations were made by [START_REF] Schimmelpfennig | Changes in macro-and micronutrient contents of grasses and forbs following Miscanthus x giganteus feedstock, hydrochar and biochar application to temperate grassland[END_REF]. Even if the Ca 2+ content is much lower in C compared to H and CC, it cannot be considered as limiting factor as its concentration is well above the critical deficiency levels (0.5-1.5 cmolc kg -1 ) reported by [START_REF] Kopittke | A Review of the Use of the Basic Cation Saturation Ratio and the "Ideal" Soil[END_REF]. The concentration of Ca 2+ was doubled in the leguminous species compared to grass, in agreement with literature results [START_REF] Juknevičius | The content of mineral elements in some grasses and legumes[END_REF].

Na + content of the plants did not reflect the concentration of the nutrient in the soil (Fig. 5). Sodium is toxic at high concentrations for most of the plant species [START_REF] Greenway | MECHANISMS OF SALT TOLERANCE IN NONHALOPHYTES[END_REF], so the adsorption through the plant roots' cell is limited as much as possible by mechanisms of selective uptake and ion exclusions [START_REF] Schachtman | Molecular pieces to the puzzle of the interaction between potassium and sodium uptake in plants[END_REF]. The behavior of Na + is opposite to what was observed for K + . The two ions are very similar for their ionic radius and ion hydration energies, factors determining their movement through the cell's membrane [START_REF] Hille | Ionic Channels of Excitable Membranes[END_REF], but the competition between the two ions in soils which are not saline is usually in favor of K + because it is not toxic. On the contrary, it is fundamental for plant growth of all species and it is the cation with the highest concentration in plant tissues [START_REF] Mäser | Molecular mechanisms of potassium and sodium uptake in plants[END_REF]. As for Ca 2+ , the concentration of T. pratense plants is double, or more, compared to F.

nigrescens even if T. pratense is less tolerant to salinity than F. nigrescens (FAO 2002). However Na + concentration in the leaves of T. pratense did not reach toxic levels, in fact no detrimental effects were observed on the plant growth.

Similarly to Na ++ , higher soil concentrations of Mg 2+ did not correspond to any increase in the magnesium content in plant tissues. The concentrations of Na + and Mg 2+ were lower (H<CC<C) in the soils with elevated K + and Ca 2+ concentrations (H>CC>C). This may be related to the competition between cations given a certain cation exchange capacity of the soil. The fact that lower Mg 2+ soil content was not reflected by a lower concentration in plants tissues might be seen as the evidence of a proper cations balance in the soil. As for Na + and Ca 2+ , Mg 2+ content of T. pratense plants is twice than that of F. nigrescens and this is in line with previous works on these two plant species [START_REF] Juknevičius | The content of mineral elements in some grasses and legumes[END_REF].

The micro-and macro-nutrient contents of pasture and meadow plants are fundamental for the quality of the fodder. The German Society of Nutritional Physiology, among others, provides specific recommendations at this regard (Flachowsky et al. 2001, Online Resource Tab. 1). In our experiment the P content of fodder improves when plants were grown on H compared to those grown on C and CC but did not fulfill the cattle requirements.

K + content of plants increased for both charcoal treatments compared to C where both plant species had lower concentrations than recommended. K + content in F. nigrescens was in line with recommendations when grown on H and CC, while K + concentrations in T. pratense plants exceeded the recommendations when grown on H. However, the value did not reach the limit of ≥35 g kg -1 dry biomass, which is considered to be the cause of the so called "grass tetany", a Mg 2+ deficiency in ruminants which implies cattle to be fed with supplementary magnesium [START_REF] Kessler | Mineralstoffversorgung der Milchkuh auf einen Blick rap aktuell (Mineral nutrient supply of dairy cows at a glance)[END_REF].

Ca 2+ and Mg 2+ concentrations were very high for both plant species grown on all soil types compared to the recommendations, especially for those grown on soils amended with charcoal and for T. pratense plants. But an adequate nutritive balance between K + , Ca 2+ and Mg 2+ was guaranteed, as the ratio K:(Ca+Mg) was lower than 2.2 [START_REF] Reid | Effects of different irrigation systems on the biodiversity of species-rich hay meadows[END_REF].

Fe measurements showed a large variability in our experiment so they have to be considered with caution, but it is clear that Fe content of plant tissues exceeded the recommendation in all treatments and species, with highest values for those grown on C. It is very common for pastures and meadows plants to exceed the iron supply recommendations for cattle and values up to 500 mg kg -1 are usually tolerated [START_REF] Flachowsky | Empfehlungen zur Energie-und Nahrstoffversorgung der Milchkuhe und Aufzuchtrinder[END_REF]). This threshold is only exceeded in the case of F. nigrescens plants grown on C, showing that charcoal application has been beneficial.

Na + content was higher than recommended for T. pretense, especially when grown on soils amended with charcoal, but these concentrations are not considered harmful for cattle. The concentrations we found were lower than 2.5 g kg -1 Dry Matter (DM) (Tab. 3), while the maximum level of sodium tolerated in forage for dairy cattle is 15.73 g Na kg -1 DM [START_REF] Johansson | Salt to ruminants and horses[END_REF] Cu was higher than recommended only for T. pratense plants but with no difference among the treatments.

Chronic copper poisoning is possible in cattle with a dietary concentration of 40 mg kg -1 (National Research Council 2001), but this threshold was far from being reached in our samples with values around 17 mg kg -1 (Tab. 3).

Charcoal application to the alpine grasslands in this study, both in the short and in the long term (CC and H), overall improved fodder quality in terms of P, K + and Fe content compared to the control soils. Ca 2+ , Mg 2+ , Na + and Cu contents were found to be higher than recommendations in all treatments, including control, but they did not reach harmful levels, thus guaranteeing cattle health.

The positive results observed for charcoal amended soils in terms of germination, plant growth, root:shoot ratio and fodder quality, both directly after application and after ageing, support the idea that larch wood charcoal is a soil amendment suitable for alpine pastures and meadows even at an application rate of 390 t ha -1 . From a management point of view, biochar incorporation into soil implies ploughing, a technique which is very rarely used in alpine management because of multiple factors: the soil layer is thin to bedrock, with very irregular profiles [START_REF] Stanchi | The influence of Alpine soil properties on shallow movement hazards, investigated through factor analysis[END_REF]); ploughing will increase soil erosion which is already a main problem in mountain soils because of slope, soil depth, climate and soil low resilience which makes them almost no renewable [START_REF] Tasser | Effects of land use in alpine grasslands on the probability of landslides[END_REF][START_REF] Stanchi | The influence of Alpine soil properties on shallow movement hazards, investigated through factor analysis[END_REF]; most of the grasslands are located in areas very difficult to reach with machines that is needed for biochar spreading and incorporation into soil. Thus, biochar cannot be used for standard management, but can be helpful in the framework of grassland restoration and rehabilitation, as defined by [START_REF] Aronson | Restoration and Rehabilitation of Degraded Ecosystems in Arid and Semi-Arid Lands . I . A View from the South[END_REF]. The restoration and rehabilitation of abandoned pastures and meadows in the Alps is usually based on the manual removal of trees and invasive species, sometimes applying herbicides locally, and very rarely destroying the soil layer because of the reasons listed above [START_REF] Belleri | L'Oasi di Protezione faunistica del Baremone: analisi e sviluppi[END_REF]. For these reasons, biochar application is worth considering in cases of severely damaged grasslands (ski tracks openings) or where the modifications to the environment have been so profound that the reconstitution of the former ecosystem is no longer possible (roads or dumps construction) [START_REF] Maurer | Restoration and Rehabilitation of Species-Rich Grassland Ecosystems in France : a Review[END_REF]. However, possible impacts on plant biodiversity remain to be explored and a deeper examination of nutrient cycles, microbial biodiversity and the role played by hormones should be a research priority for next experiments in this unique environment to better understand and quantify the overall impact of biochar application on nature conservation and the important ecosystem services that mountain grasslands provide. Different letters indicate significant differences among soil treatments within each plant species.

Tab. 3 FIGURESFig. 1

 31 FIGURES

Fig. 2

 2 Fig.2Total belowground (black) and aboveground biomass (grey) per pot accumulated over the three growth cycles for T. pratense and F. nigrescens plants grown on control (C), control + fresh charcoal (CC) and charcoal hearth (H) soils.

Fig. 3

 3 Fig.3Root:shoot ratio of total biomass accumulated over the three growth cycles for T. pratense and F. nigrescens grown on C (light grey), CC (grey) and H (black) soils. Different letters indicate significant differences among soil treatments within each plant species.

Fig. 4

 4 Fig. 4 N:P ratio of soils (plot a) and of T. pratense and F. nigrescens (plot b) grown in C (light grey), CC (grey) and H(black) soils. The two lines correspond to N:P ratios of 14 (dashed) and 16 (continuous). Different letters indicate significant differences among soil treatments.

Fig. 5

 5 Fig.5Relationship between plant and soil P, K + , Ca 2+ , Na + , Mg 2+ content (mg g -1 ) (plot a, b, c, d and e, respectively) in T. pratense (circles) and F. nigrescens (triangles) grown in C (white), CC (grey) and H (black) soils.

Fig. 6

 6 Fig. 6 13 C discrimination (Δ) of T. pratense and F. nigrescens plants after the first (a), second (b) and third growth cycle (c) in C (light grey), CC (grey) and H (black) soils. Different letters indicate significant differences among soil treatments within each plant species.

Fig. 7

 7 Fig. 7 Isotopic signature (δ 15 N) of F. nigrescens and T. pratense plants after the first (a), second (b) and third growth cycle (c) in C (light grey), CC (grey) and H (black) soils. P-values of the individual comparisons are reported in the two tables. Different letters indicate significant differences among soil treatments within each plant species.

  ). The isotopic signature of plants grown on H was lower and very similar for the two plant species as well (T. pratense: 4.9‰; F. nigrescens: 5.1‰). Similar data were observed at the end of the second growth cycle (T. pratense C: 6.5‰, H: 4.8‰; F. nigrescens C: 6.4‰, H: 5.8‰), while, at the third harvest, the isotopic signature rose in both species and became slightly higher for plants grown on H as compared to those grown on C (T.

pratense C: 7.2‰, H: 7.7‰; F. nigrescens C: 7.1, H: 7.3‰; Fig.

7

). A very different trend was observed for plants grown on CC where, at the end of the first growth cycle, T. pratense plants showed a δ 15 N of -0.07‰, much lower than the other treatments, especially compared to C (CC vs. H p=0.19; CC vs. C p=0.08). F. nigrescens had a δ 15 N equal to 3.3‰ (CC vs. H p=0.24; CC vs. C p=0.04). After the second growth cycle, the isotopic signature of both T. pratense and F. nigrescens increased (1.4‰ and 4.8‰, respectively), but did not equal the values observed for the other two soil treatments (T. pratense: CC vs. H p=0.06, CC vs. C p=0.02; F. nigrescens: CC vs. H p=0.25, CC vs. C p=0.07). The δ 15 N of both species increased also during the third growth cycle (T. pratense: 5.3‰ and F. nigrescens: 5.8‰), getting

  Total nutrient content (mg kg -1 ) in control soil, charcoal hearth soil, fresh char and control soil + fresh char at the beginning of the experiment. In parenthesis, data on available nutrient concentration (mg kg -1 ) for control and charcoal hearths soils are reported. Data for hearths, control soils and fresh charcoal are taken from[START_REF] Criscuoli | Carbon sequestration and fertility after centennial time scale incorporation of charcoal into soil[END_REF], while data for the control soil + fresh charcoal are calculated. Asterisks indicate significant differences between control and charcoal hearth soils at p≤0.05. Germinated seeds (%) of T. pratense and F. nigrescens after 96 and 120 hours and 10 days from sowing in control (C), control + fresh charcoal (CC) and charcoal hearth (H) soils. Different letters indicate significant differences among soil treatments within each plant species and time frame at p≤0.10.

	TABLES Tab. 2 Trifolium pratense Element Control soil Charcoal hearth soil Fresh char Ca 993±233* (278±61)* 3301±321* (1006±274)* 5909±86 K 1604±201* (147±52) 2464±120* (279±208) 2906±54 Mg 2739±128 (80±21)* 2379±665 (245±7)* 1522±56 Na 298±88* (34±2) 93±15* (33±45) 210±4 P 321±13* (7±3)* 921±357* (12±7)* 308±1 N 4307±1402 5189±1756 2139±340 time C CC T. pratense 96 hours 2a 7a 120 hours 18ab 29a 10 days 83a 94b F. nigrescens 96 hours 6a 13a 120 hours 11a 19a 10 days 78a 90b Element (mg g -1 ) C CC Cu 0.017 ± 0.002a 0.016 ± 0.001ab Control soil + fresh char 2870±150 2100±130 2274±82 264±54 316±8 3480±870 H 2a 16b 93b 6a 12a 89b H 0.017 ± 0.001b Fe 0.4 ± 0.3a 0.3 ± 0.3a 0.2 ± 0.3a Ca 2+ 23.9 ± 3.3ab 26.7 ± 1.8a 28.6 ± 2.1b K + 8.4 ± 0.5ab 10.1 ± 1.9a 13.6 ± 3.5a Mg 2+ 5.8 ± 0.7a 4.8 ± 0.3b 6.6 ± 0.7b Na + 1.6 ± 0.2ab 0.2 ± 0.3a 2.5 ± 0.7b P 0.9 ± 0.1ab 0.9 ± 0.3a 1.4 ± 0.2b N 19.6 ± 0.3a 19.2 ± 3.7a 15.1 ± 2a Festuca nigrescens Element (mg g -1 ) C CC H Cu 0.008 ± 0.0002a 0.007 ± 0.0a 0.008 ± 0.002a Fe 0.5 ± 0.6a 0.2 ± 0.0a 0.2 ± 0.1a Ca 2+ 12.8 ± 0.3a 13.2 ± 0.1a 15.2 ± 0.1a Tab. 1 plant species K + 7.6 ± 2.5a 10.6 ± 1.6a 10.1 ± 2.5a
	Mg 2+	2.6 ± 0.2a	2.7 ± 0.2a	2.9 ± 0.4a
	Na +	1.2 ± 0.2a	1.2 ± 0.05a	1.3 ± 0.2a
	P	1.1 ± 0.3a	0.8 ± 0.2a	1.5 ± 0.5a
	N	18.5 ± 0.9a	14.2 ± 2.5b	14.1 ± 2.1b

Acknowledgments

The authors would like to thank the Stelvio National Park for allowing sampling campaigns; Ce.Spe.Vi. s.r.l., Pistoia for the experiment in the greenhouse; Dr. Luisa Andrenelli and Dr.Adriano Baglio (DISPAA Dipartimento di Scienze delle Produzioni Agroalimentari e dell'Ambiente -University of Florence) for laboratory analysis; Roberta Franchi (Fondazione Edmund Mach) for her important advise on alpine pastures and meadows management and the Italian Biochar Association (ICHAR).

Funding: This study was financed by: EuroChar project (FP7-ENV-478 2010ID-265179) funded by the European Commission and AgroPyroGas funded by Regione Toscana, Italy. G.A. was partially supported by a German Academic Exchange Service (DAAD, Germany) scholarship for a three-month research period at University of Freiburg, Germany. I.C. was supported by a Ph.D. grant from Fondazione Edmund Mach, Trento. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Online Resource

Tab. 1 Recommended nutrients supply of a dairy cow with a mean performance of 30 kg milk day -1 and a daily intake of 20 kg biomass dry matter [START_REF] Flachowsky | Empfehlungen zur Energie-und Nahrstoffversorgung der Milchkuhe und Aufzuchtrinder[END_REF].

Conflict of Interest:

The authors declare that they have no conflict of interest.


Juan Sorribas

Sandra González

Alfons Domínguez-Gento

Rosa Vercher

Abundance, movements and biodiversity of flying predatory insects in crop and non-crop agroecosystems

Keywords: Mediterranean agroecosystem, Organic management, Conservation Biological Control, Entomophagous arthropod, Shelter habitat, Cover crop, Neuroptera, Lacewing

come

Introduction

An agroecosystem, the basic unit of study in agroecology, can be identified as an area which includes crop habitats and non-crop habitats adjacent to the crops [START_REF] You | Non-crop habitat manipulation and integrated pest management in agroecosystems[END_REF]). Intensive agriculture often involves a maximum utilization of the land surface which includes removal of non-crop areas such as surrounding woody vegetation (i.e. hedgerows and shelterbelts), riparian herbaceous vegetation and weeds.

Throughout Europe, the expansion of modern, chemical-intensive agriculture is regarded as the principal cause of the widespread declines in abundance and diversity of predatory insects over the past decades (e.g. [START_REF] Hole | Does organic farming benefit biodiversity?[END_REF][START_REF] Bianchi | Sustainable pest regulation in agricultural landscapes: a review on landscape composition, biodiversity and natural pest control[END_REF]. Sustainable practices such as organic farming, cover cropping and hedgerows preservation may contribute to enhance predatory insects living within agroecosystems [START_REF] Altieri | Vegetation management and biological control in agroecosystems[END_REF][START_REF] Östman | Landscape heterogeneity and farming practice influence biological control[END_REF]. Because arthropod generalist predators (or entomophagous arthropods) must efficiently search for preys, they can be considered ideal models for investigating conservation techniques [START_REF] Panzer | Effectiveness of a vegetation-based approach to insect conservation[END_REF]. Conservation biological control (CBC) is the practice of enhancing natural enemies through modification of the environment or landscape of an agroecosystem [START_REF] Duelli | Population movements of arthropods between natural and cultivated areas[END_REF].The practice of CBC, which involves the manipulation of agricultural habitats to favour the natural enemies, offers the possibility of simultaneously increasing insect and plant biodiversity and reducing pest problems [START_REF] Straub | Are the conservation of natural enemy biodiversity and biological control compatible goals?[END_REF]).

Although higher biodiversity not necessarily means better biological control, conserving natural enemy species richness could increase the chances that the enemy community will contain good enemy species, leading to a positive relationship between natural enemy species richness and biological control [START_REF] Straub | Are the conservation of natural enemy biodiversity and biological control compatible goals?[END_REF]. This has led to growing interest in management practices that promote on-farm biodiversity such us organic farming (e.g. [START_REF] Hole | Does organic farming benefit biodiversity?[END_REF][START_REF] Straub | Are the conservation of natural enemy biodiversity and biological control compatible goals?[END_REF]. In general, organic farming is reported to increase arthropod diversity in agricultural landscapes (e.g. [START_REF] Bengtsson | The effects of organic agriculture on biodiversity and abundance: a meta-analysis[END_REF][START_REF] Hole | Does organic farming benefit biodiversity?[END_REF][START_REF] Smukler | Biodiversity and multiple ecosystem functions in an organic farmscape[END_REF]. Organic farming usually increases arthropod species richness and abundance, having on average 30% higher species richness and 50% more abundance than conventional farming systems [START_REF] Bengtsson | The effects of organic agriculture on biodiversity and abundance: a meta-analysis[END_REF].

When compared with sprayed crops, more natural enemies establish and maintain themselves in organic crops whereas arthropod herbivores usually exhibit an opposite trend [START_REF] Altieri | The dynamics of colonizing arthropod communities at the interface of abandoned, organic and commercial apple orchards and adjacent woodland habitats[END_REF][START_REF] Östman | Landscape heterogeneity and farming practice influence biological control[END_REF].

Non-crop natural or semi-natural habitats inside agroecosystems such as field margins, grassy ground cover, hedgerows and shelterbelts are relatively undisturbed and temporally permanent areas that may provide resources that are critical to sustaining natural enemy population diversity [START_REF] Landis | Habitat management to conserve natural enemies of arthropod pests in agriculture[END_REF][START_REF] Bianchi | Sustainable pest regulation in agricultural landscapes: a review on landscape composition, biodiversity and natural pest control[END_REF]. These patches of vegetation in agricultural landscapes can enhance natural enemies by providing foods (e.g. alternate hosts for parasitoid wasps, preys for predatory insects, honeydew, pollen or nectar), modified 2 3 climate (e.g. windbreaks made by shelterbelts) and/or more niches (e.g. overwintering and nesting habitats) [START_REF] Altieri | Vegetation management and biological control in agroecosystems[END_REF][START_REF] Landis | Habitat management to conserve natural enemies of arthropod pests in agriculture[END_REF][START_REF] Gurr | Multi-function agricultural biodiversity: pest management and other benefits[END_REF][START_REF] Bianchi | Sustainable pest regulation in agricultural landscapes: a review on landscape composition, biodiversity and natural pest control[END_REF]. The abundance and diversity of predatory insects within crop fields and orchards are closely related to the surrounding vegetation [START_REF] Altieri | Vegetation management and biological control in agroecosystems[END_REF]. One of the major limitations to understand how natural enemies are affected by non-crop vegetation is knowing how they move or disperse within their environment throughout the year.

Movement is critical to escape from disturbances and to find resources scattered in space and time. However there is a lack of studies showing year-round movements of predators between croplands and surrounding areas (see [START_REF] Rand | Spillover edge effects: the dispersal of agriculturally subsidized insect natural enemies into adjacent natural habitats[END_REF]) and the knowledge of how highly mobile flying predators overwinter in orchard agroecosystems is even lower. Flying insects are ideal models since they can escape after chemical treatments to surrounding non-crop habitats or stay in the field if they can stand to non-intensive treatments. Since noncrop habitats provide requisites for natural enemies they may act as sinks relative to crop habitats when natural enemies have a year-round preference for these habitats thus reducing the exchange of natural enemies between crop and non-crop habitats [START_REF] Bianchi | Sustainable pest regulation in agricultural landscapes: a review on landscape composition, biodiversity and natural pest control[END_REF]. Appropriate landscape management in agricultural systems requires an understanding of insect seasonal movements and dispersal.

We chose a generalist, highly mobile, flying predator order of insects, Neuroptera, as model to identify the most important habitats for biodiversity conservation in Mediterranean traditional agroecosystems for flying predatory insects. Neuropters have been previously used as standard of value for generalist predators and indicators of the ecological status of rural habitats due to the susceptibility of most species to pesticides and their well-known environmental needs (e.g. [START_REF] Stelzl | Neuroptera in agricultural ecosystems[END_REF][START_REF] Thierry | Typifying ecosystems by using green lacewing assemblages[END_REF]. Neuropters were selected because they occupy the top of the ecological arthropod trophic pyramid, they occur in very diverse habitats all year round, they can easily displace between non-crop and crop habitats, they have different feeding abilities, they need growing vegetation to feed and lay their eggs and they are sensitive indicators of environmental richness, stability and local diversity (see [START_REF] Stelzl | Neuroptera in agricultural ecosystems[END_REF]Villenave et al. 2005). In commercial orchards neuropteran populations develop only when insecticide treatment is not applied in intervals that are too short [START_REF] Pantaleoni | I Neurotteri delle colture agrarie: osservazioni sulle fluttuazioni stagionali di populazione in frutteti[END_REF] and adult specimens can fly the distance between boundary vegetation and fruit trees (some marked Neuroptera species have been collected up to 200 meters away from the marking point) [START_REF] Long | Beneficial insects move from flowering plants to nearby crops[END_REF]).

Here we hypothesize that: a. There will be a reduction in biodiversity and abundance of natural enemies in "conventional" sprayed orchards in relation to organic managed orchards. b. Due to the larger total area and complementary food source all year round of the ground cover vegetation (e.g. pollen from weeds with different flowering periods) it will be the preferred habitat over woodland habitats for most species. c. Flying predators will displace from fruit tree canopies to non-crop habitats to overwinter.

Materials and Methods

Study area

The study was performed in the south of Valencia province (eastern Spain), an area with typical Mediterranean agroecosystems dominated by small fruit orchards of 0.5 -1 ha. The landscape of the study area is composed mostly of clementine mandarins Citrus clementina Tanaka (many of them having grassy ground cover in fruit trees understory) and patches of natural or planted native woody vegetation (shelterbelts) surrounding the orchards. This woody vegetation, which has been historically used to form a natural fence and as windbreaks, is constituted by rows of trees and tall shrubs which can be remnants of existing vegetation from cleared lands, a result of natural plant dispersal, or established via direct plantings by farmers.

We delimited habitat types according to vegetation structure (vegetation strata) and human agricultural activities. The habitat types defined were: (1) fruit tree canopies, (2) ground cover vegetation (grassland), and

(3) shelterbelts of woody vegetation (surrounding fruit orchards).

For the study we selected nine plots constituting nine isolated agroecosystems each of them formed by a citrus orchard with ground cover vegetation growing in fruit trees understory and woody shelterbelts surrounding all sides of the orchards. Plots were separated a maximum of 20 lineal kilometres and about 15-20 lineal kilometres away from the Mediterranean Sea coast.

Citrus trees inside agroecosystems of the study area were cultivated under two forms of farming practice, certified organic management (six orchards) and "conventional" sprayed management (three orchards). Insecticidal treatments for these orchards included phosphorate insecticides, pyrethroids and acaricides which were applied twice a year under integrated pest management strategies. Both organic and sprayed orchards had been under the same farming practice for the previous 7-10 years. We analyzed and compared the biodiversity and abundance of lacewings in both groups.

Shelterbelts of the study area were rather similar and had a mixed composition of several Mediterranean native perennial trees and shrubs species, predominantly Pistacia lentiscus L., Nerium oleander L, Viburnum tinus L. and/or Phillyrea angustifolia. The distance between any of the edges and the central trees of the orchard was always lower than 100 meters (inferior than Neuropters flying capacity).

Ground vegetation was composed of spontaneous or sown herbaceous species (grass), predominantly Cynodon sp., Bromus sp., Amaranthus sp., Sonchus sp., Chenopodium sp., Senecio sp., Calendula sp., Medicago sativa L. and/or Melilotus officinalis L. Natural regeneration of ground vegetation was allowed before mowing (twice a year).

Insect material and sampling methodology

Neuroptera is an insect order of polyphagous predators of many agricultural pests such as mites, scales, whiteflies and aphids [START_REF] Stelzl | Neuroptera in agricultural ecosystems[END_REF]. Almost all feeding behaviours can be found within Neuropteran families, omnivorous (Hemerobiidae and Chrysopidae), carnivorous-glycinophagous (Coniopterygidae); phytophagous (some Chrysopidae) and pollino-glycinophagous (some Chrysopidae can feed on pollen from about 40 trees) and several species are mass reared and released within classical biological control programs [START_REF] Stelzl | Neuroptera in agricultural ecosystems[END_REF][START_REF] Villenave | The pollens consumed by common green lacewings Chrysoperla spp. in cabbage crop environment in western France[END_REF]).

Due to its different feeding preferences and flying abilities and the diverse plant structure and architecture of the three habitats evaluated we choose two very different sampling methods in order to maximize catches of neuropteran insects.

The sampling process was performed fortnightly from May to October and monthly during the cold period for this region, November to April, during three consecutive years (October 2006 -October 2009). In the six organic farming orchards three habitats were sampled: fruit tree crowns, ground cover vegetation and orchards surrounding shelterbelts, whereas in the three conventionally managed orchards only citrus trees were sampled.

Aspiration sampling was carried out with a custom built suction machine composed of a commercial garden two-stroke engine-powered leaf blower (Komatsu Zenoah Co., HBZ2601 model) assembled with a cylindrical plastic suction mouth 30 cm high and 30 cm in diameter. This is a modification of the gasolinedriven vacuum device designed by [START_REF] Dogramaci | A backpack-mounted suction apparatus for collecting arthropods from various habitats and vegetation[END_REF]. The sampling process was standardized for an aspiration of 2 minutes duration (equivalent to 1 -3 trees depending on size) in all cases. All sucked insects were retained in a fine mesh placed at the extreme of the suction tube. The mesh was replaced after each aspiration process and insects captured were conserved inside the mesh which was annotated with a sample number. Sampling with the suction device was randomly performed 3-4 times for citrus tree canopies of each orchard, 3-4 times for shelterbelts and 3-4 times for the ground vegetation (depending of orchard and shelterbelt size).

Yellow traps consist of a 10x25 cm plastic rectangle with sticky surface which were vertically hanged from 2-3 citrus trees and from 2-3 hedge trees or shrubs per orchard. In the ground vegetation 2-3 traps per orchard were placed on wooden posts planted in the soil at constant a height of 50 cm above the ground.

Traps were collected and replaced the same day as the aspiration process was performed (Fig 1).

Trees and ground surface for sampling were randomly selected for both methods. In the laboratory all Neuroptera insects collected or trapped were observed under a stereomicroscope and classified to species or higher taxonomic levels. The several species of the cryptic carnea group were classified into a single category hereby referred to as Chrysoperla carnea Stephens.

A total of 3302 samples were collected with the suction device and 2384 yellow sticky traps from the 6 organic agroecosystems during the 3 years. The citrus trees of sprayed agroecosystems were sampled using the suction device and yellow sticky traps following the same protocol described, with the difference that in this case only the fruit trees were sampled. A total of 346 aspiration samples and 236 yellow sticky traps were collected.

Data analysis

For the statistical analysis of the abundance of captures by the two sampling methods we performed a simple Analysis of Variance (ANOVA) of all the data in all habitats (8 species and 29177 data). For the comparative between D biodiversity indices and between species richness we calculated the montly diversity index and the number of species by summing up the total individuals captured monthly in each agroecosystem followed by ANOVA Multiple Range Test. The comparative between abundance in organic and sprayed agroecosystems was done by simple ANOVA of all data. When we compared fruit trees, ground cover and shelterbelts we performed multifactor ANOVA considering year and habitat as factors for each species.

In order to evaluate the number of species and diversity indices, data were grouped monthly as number of insects or number of species/month for each year and sampling methodology. Two of the most commonly used indices were selected to quantify Neuropteran biodiversity: Taxonomic species richness [S], i.e. the number of species collected and Simpson's diversity index [D].

Results and Discussion

Relative abundance of lacewings

The analysis of the total number of captures of all species by the two sampling methods shows that when using the suction device, lacewings of Chrysopidae family (green lacewings) were the most commonly captured (F = 166.07; df= 7,29 176; P< 0.0001) whereas yellow sticky traps captured, in general, much higher proportion of Coniopterygidae (dusty-wings) (97% of the total lacewings captured with this method; F = 216.64; df= 7, 23 535; P< 0.0001). Chrysoperla carnea was the most frequently captured species with the aspirator whereas Semidalis aleyrodiformis Stephens was the most common Neuroptera on yellow sticky traps (more than 5000 specimens captured). The amount of Hemerobiids (brown lacewings) was small and similar with both methods. This indicates that green lacewings and dusty-wings have different flying or displacement strategies and both capturing methods are complementary in order to evaluate the abundance and diversity of these flying insects.

Out of 14318 adult lacewings collected, we identified ten species (coexisting in both organic and When compared with Neuroptera abundance and species richness found by other authors in agroecosystems composed of arable crops (e.g. [START_REF] Pantaleoni | I Neurotteri delle colture agrarie: osservazioni sulle fluttuazioni stagionali di populazione in frutteti[END_REF] we observed that it was much lower than in these evergreen fruit orchards. Perennial crop systems such as fruit orchards are more stable than annual systems and they are subject to lower levels of disturbance which could explain differences in predator abundance and biodiversity.

Species richness and biodiversity indices

Values reached for species richness and Simpson Diversity Index [D] were higher in fruit orchard agroecosystems conducted under organic management than in sprayed conventional agroecosystems. The mean number of species [S] captured per month was significantly higher (F= 66.13; df = 1, 144; P < 0.0001) in organic orchards (4.68±0.22 species/month) than the number in sprayed orchards (2.33±0.18 species/month). The same pattern was found for the Biodiversity index, with the organic orchards having an average [D] of 2.35± 0.09, significantly higher (F= 20.05; df = 1, 144; P < 0.0002) than the sprayed orchards, 1.31± 0.08 (Fig 2a). These outcomes agree with previous studies in other agroecosystems (see [START_REF] Altieri | The dynamics of colonizing arthropod communities at the interface of abandoned, organic and commercial apple orchards and adjacent woodland habitats[END_REF][START_REF] Bengtsson | The effects of organic agriculture on biodiversity and abundance: a meta-analysis[END_REF].

When comparing the insect biodiversity associated to each habitat type we found that shelterbelts had the highest values for the Biodiversity indices evaluated, followed by the biodiversity associated to fruit tree canopies and lastly by ground vegetation associated insects. Biodiversity indices differences between habitat types were statistically significant (S: F= 41.70; df = 2, 188; P < 0.0001. D: F= 26.61; df = 2, 188; P < 0.0001) (Fig 2b).

Relative abundance in organic and sprayed agroecosystems

When comparing organic and sprayed agroecosystems insect relative abundance (bringing together data from yellow traps and aspirator) we found some significant differences (Table 1). The total mean amount of adult Neuroptera captured in sprayed agroecosystems (3.40±0.54 adults/sample) was significantly higher (F= 16.51; df = 1, 5943; P < 0.001) than in organic agroecosystems (2.32±0.14 adults/sample). This unexpected result was mainly due to the high level of a single dominant species, Ch. carnea, found in sprayed agroecosystems, 0.48±0.07 adults/sample, compared to organic agroecosystems, 0.18±0.01 adults/sample (F= 55.30; df = 1, 5943; P <0.001). The analysis of the population dynamics of Ch. carnea showed that this species was much more abundant in agroecosystems under sprayed management during summer, while their relative abundance in organic agroecosystems remained rather stable all year round. This implies that some species are able to resist chemical treatments up to certain degree when staggered in time. The resistance of Ch. carnea to many pesticides and the sensibility of other Neuropteran species to these pesticides was previously indicated by [START_REF] Stelzl | Neuroptera in agricultural ecosystems[END_REF]. Agricultural pestmanagement practices often lead to altered food web structure and communities dominated by a few common species whereas organic farming methods mitigate this ecological damage by promoting evenness among natural enemies [START_REF] Crowder | Organic agriculture promotes evenness and natural pest control[END_REF]. This could explain why Ch. carnea became the dominant species during summer in sprayed agroecosystems (end of spring was a period for chemical treatments) whereas in ecological agroecosystems the abundance of this species kept rather constant throughout the year.

Among Coniopterygidae no differences were found in both management systems. The more uncommon neuropters, Ch. septempunctata, M. angulatus, W. subnebulosus and Dichochrysa species were significantly more abundant in organic agroecosystems (Table1).

Insect distribution inside organic agroecosystems

Considering together captures with the aspirator and traps within the organic agroecosystems, an average of 2.31±0.21 adult lacewings per sample were captured in citrus tree canopies, 2.51±0.19 lacewings per sample in surrounding trees and shrubs and only 0.11±0.02 lacewings per sample in the ground cover vegetation. Three distribution patterns were observed (Fig 3 ): -Species which were more abundant in citrus tree canopies, represented by C. psociformis (F= 8.77; df= 2, 1288; P< 0.001) and Ch. carnea (F= 12.38; df = 2, 1288; P < 0.001); -Species significantly more abundant in citrus tree canopies and shelterbelts than in the ground vegetation, such as S. aleyrodiformis (F = 12.26; df= 2, 1288; P< 0.001) and Coniopteryx spp. (F= 12.92; df = 2, 1288; P < 0.001); -Species which were present in similar proportion in the three habitat types (F= 0.95; df= 2, 1288; P= 0.3888) such as Chrysopa septempunctata Wesmael.

Unexpectedly, the herbaceous ground cover vegetation showed much lower attractiveness to lacewings than the canopy of shelterbelts and fruit trees. Thus, our first hypothesis, based on the supposition that lacewings would find more complementary food from the many flowering weeds of the ground vegetation, which was supported on previous studies that found high abundance of many predatory insects at ground vegetation level (e.g. [START_REF] Bianchi | Sustainable pest regulation in agricultural landscapes: a review on landscape composition, biodiversity and natural pest control[END_REF][START_REF] Silva | Effect of ground cover vegetation on the abundance and diversity of beneficial arthropods in citrus orchards[END_REF]) was wrong. There may be several reasons explaining this behaviour: the comparative higher disturbance at the ground vegetation level (mowing, fruit picking, etc), the better refuge against wind and extreme temperatures that shelterbelts represent (wooded habitats provide more moderate microclimate [START_REF] Forman | Hedgerows and hedgerow networks in landscape ecology[END_REF]) and/or the higher abundance of non-flying competing predators in the above ground layer (some of them can act also as predators of lacewings). This illustrates the importance of considering plant structure and architecture when evaluating predatory insect habitats. Thus, in terms of economic investment in non-crop habitats enhancing flying predators it is clear that shelterbelts should be considered as the preferable choice when assessing alternative landscape configurations. Nevertheless, it is important to bear in mind that, as demonstrated by [START_REF] Paredes | Effect of non-crop vegetation types on conservation biological control of pests in olive groves[END_REF], in fruit agroecosystems there may be a positive synergistic effect between shelterbelts and ground vegetation in terms of beneficial predator abundance and diversity.

Seasonal population dynamics

We analyzed seasonal dynamics of the most abundant Neuroptera species from data collected with the aspirator device within the organic agroecosystems. Yellow sticky traps were not considered for this purpose since they remain in the field one month during the cold period.

The analysis of the three habitats evaluated allowed separating insect species in several groups (Fig 4 ): -Species that showed seasonal migration from fruit trees to shelterbelts to overwinter: Ch. septempunctata, M. angulatus and W. subnebulosus. Adult populations of these species associated to citrus trees start to decline at the end of summer and beginning of autumn (September-October) at the same time that populations associated with shelterbelts increased. For these species captures of adults during the winter period (equivalent in the case of Valencia to the "non-growing season" from January toMarch) were very scarce or null which mean these species spent winter as egg or larval forms.

-Two species, C. psociformis and S. aleyrodiformis (two dusty-wings), showing an opposite trend to previous group: during winter adult captures of these species were very high in citrus tree canopies while much lower in shelterbelts and very scarce or null at the ground vegetation level. These species had a second similar peak of captures in May but populations declined sharply during summer.

-A species, Ch. carnea, whose captures were very high during summer and autumn both in the citrus trees and in shelterbelts and very scarce during winter and beginning of spring. Seems that the more abundant Chrysopidae and Coniopterygidae share the same resources via temporal niche partitioning since when the former reach the peak of adult captures during summer captures of the later are minimum and the opposite happens during winter.

-All Neuroptera species captured at the ground vegetation level reached the highest peak during the spring period, captures during the rest of the year being very scarce compared to the other habitats, except for Ch. septempunctata which showed a second important peak during august.

The population peaks of lacewings associated with fruit trees and shelterbelts were different in each habitat type which indicates that there was a movement of species between both habitats and shifts in the direction of predator dispersal throughout the year. Furthermore, most Neuroptera species that spent the winter period as egg or larval forms (very low number of adults between January and March) showed the same behaviour: at the end of summer or beginning autumn the adult populations associated to fruit trees declined at the time that populations within shelterbelts increased indicating these species moved there to overwinter. Adults of these species dispersed into the orchards during the following spring as can be deduced by the peaks of captures reached in May or June. These outcomes concur with other predatory insects which were found to show seasonal migration from crops to shelter habitats at different stages of their life cycle and mainly at the beginning of the cold periods to overwinter (e.g. [START_REF] Duelli | Population movements of arthropods between natural and cultivated areas[END_REF][START_REF] Thomas | Aggregation and temporal stability of carabid beetle distributions in field and hedgerow habitats[END_REF][START_REF] Pollard | Arthropods within the woody element of hedgerows and their distribution pattern[END_REF]. However, species whose adults were active during winter, dusty-wings lacewings, highly preferred fruit tree canopies to spend this period. In fact, during most part of the year adult populations of these species were abundant in citrus tree canopies and higher than in the non-crop habitats demonstrating that evergreen fruit crops can harbour predatory insects all year round. Thus, in spite of the intrinsic difficulties associated to any monoculture, some generalist predators are able to find food and shelter within the canopy of fruit trees all year round and persist from year to year within orchards.

The winter-active generalist predators play a key role on the reduction of winter pest populations and the decline of the first seasonal population outbreaks of several agriculture pests since during early spring most natural enemies are still in dormancy or absent. This has been previously stated for other arthropod predator species which were active in fruit crops during winter (see [START_REF] Pekar | Biological control in winter: novel evidence for the importance of generalist predators[END_REF]. This highlights the importance of analyzing natural enemy activity over time rather than take a "snapshot" in one or few samplings which had been the most common in the bulk of the studies considering natural enemy response to agroecosystem complexity (see [START_REF] Chaplin-Kramer | A meta-analysis of crop pest and natural enemy response to landscape complexity[END_REF].

Conclusion

Although biodiversity indices were highest, as expected, within organic orchard agroecosystems, the relatively high year-round predators abundance and diversity inside sprayed tree canopies suggest that when perennial fruit trees are under conventional but not chemical-intensive treatments (such as integrated pest management programs) they can efficiently harbour predatory insects throughout the year.

Our results indicate that strategies for enhancing flying natural enemies that maximize Conservation Biological Control, such as habitat management, should focus more on field margin woody vegetation rather than the ground cover vegetation when considering non-crop habitats. We can say that, under Mediterranean climate conditions, flying seasonal patterns of migration from agricultural lands to non-crop habitats to overwinter depend on species adult winter activity. While dormant insects generally move to shelterbelts to overwinter active insects remain in high proportion on fruit trees canopy. Since many flying predators move from crop towards surrounding shelterbelts to overwinter presence of boundary woody vegetation can avoid their migrations to other areas. Shelterbelts serve to attract flying predators to the farmland and maximize survival possibilities when a perturbation occurs, allowing them to quickly re-colonize the crop afterwards.

More studies to address which particular shrubs or trees species that can be used to build shelterbelts are the more suitable to harbour this and other key predatory insect orders are needed.

  sprayed agroecosystems) belonging to three families. Chrysopidae (green lacewings): Ch. carnea, Chrysopa septempunctata Wesmael and two unidentified Dichochrysa species; Coniopterygidae (dusty-wings): S. aleyrodiformis, Conwentzia psociformis (Curt.) and two unidentified Coniopteryx species; Hemerobiidae (brown lacewings): Micromus angulatus Stephens and Wesmaelius subnebulosus Stephens.

Fig 1 .

 1 Figure Captions:

Fig 2 .

 2 Fig 2. Biodiversity indices (Species richness [S] and Simpson's diversity index [D]) of a) Neuropters living within fruit orchard agroecosystems under organic and sprayed (conventional) management. b) Neuropters living within three habitat types: citrus trees canopy, shelterbelts and ground cover vegetation inside fruit agroecosystems. Samples were collected using a suction device and yellow sticky traps from 9 orchard agroecosystems of eastern Spain during the period 2006 to 2009. Vertical bars indicate standard error (SE) of the means. Values followed by the same letter are not significantly different from each other according to Fisher's LSD multiple range test (p ≤ 0.05).

Fig. 3 .

 3 Fig. 3. Occurrence of the most abundant Neuroptera species within three habitats (fruit trees, shelterbelts and ground covers) belonging to orchard agroecosystems. Samples were collected with a suction device from 2006 to 2009 in six agroecosystems of Valencia Region (eastern Spain) under organic management. Vertical bars indicate standard error (SE) of the means. For each species, values followed by the same letter are not significantly different from each other according to Fisher's LSD multiple range test (p ≤ 0.05).

Fig. 4

 4 Fig. 4. a-f Population dynamics of the most abundant Neuroptera insects in fruit agroecosystems: Chrysoperla carnea, Chrysopa septempunctata, Conwentzia psociformis, Semidalis aleyrodiformis, Coniopteryx sp. and two Hemerobidae species (Micromus angulatus and Wesmaelius subnebulosus). Samples were collected using a suction device and yellow sticky traps from 2006 to 2009 in citrus trees canopy (422 samples), shelterbelts (478 samples) and ground covers (389 samples) within 6 organic agroecosystems of eastern Spain. Vertical bars indicate standard error (SE) of the means.

Fig 1 .

 1 Fig 1. Schematic figure showing the type of habitats evaluated in this study (left side), the sampling methods (centre) and the main species representing the main insect families found in this study (right side). From top to bottom: woody shelterbelts, fruit trees canopy and ground cover vegetation (left side), yellow sticky trap and vacuum device (centre), and adult specimens of Chrysopidae, Coniopterygidae and Hemerobiidae families respectively (right side).

Fig 2 .

 2 Fig 2. Biodiversity indices (Species richness [S] and Simpson's diversity index [D]) of a) Neuropters living within fruit orchard agroecosystems under organic and sprayed (conventional) management. b) Neuropters living within three habitat types: citrus trees canopy, shelterbelts and ground cover vegetation inside fruit agroecosystems. Samples were collected using a suction device and yellow sticky traps from 9 orchard agroecosystems of eastern Spain during the period 2006 to 2009. Vertical bars indicate standard error (SE) of the means. Values followed by the same letter are not significantly different from each other according to Fisher's LSD multiple range test (p ≤ 0.05).

Fig. 3

 3 Fig. 3 Occurrence of the most abundant Neuroptera specieswithin three habitats (fruit trees,shelterbelts, and ground covers) belonging to orchardagroecosystems. Samples were collected with a suction device from 2006 to 2009 in six agroecosystems of Valencia Region (eastern Spain) under organic management. Vertical bars indicate standard error (SE) of the means. For each species, values followed by the same letter are not significantly different from each other according to Fisher's LSD multiple range test (P ≤ 0.05)

Table 1 .

 1 Mean number (M ± SE) of adult Neuroptera captured per sample in fruit agroecosystems under organic and conventional (sprayed) management. Samples were collected with a suction device and yellow sticky traps from 2006 to 2009 in agroecosystems of eastern Spain. Dichochrysa species (Chrysopidae) were not considered due to the low number of captures.Note: Values in rows followed by the same letter are not significantly different from each other according to Fisher's LSD multiple range test (p ≤ 0.05).

	Organic	Conventional
	Agroecosystem	Agroecosystem

Acknowledgements

We thank two anonymous reviewers and the editors for constructive comments on the manuscript.

The authors are very grateful to Rosa Guaita, Pili Mañó, Anna Marco, Altea Calabuig, Bernadette Csőke and Adela Cuesta for their help and collaboration with laboratory and field work and to citrus orchards owners who permitted access for this study. Joan van Baaren and Ferran Garcia-Marí provided valuable comments that improved the manuscript. We thank Lucy Alford and Adrien Bonvin for language corrections.