Mathilde Chomel
email: mathilde.chomel@manchester.ac.uk

Virginie Baldy

Marie Guittonny

Stephane Greff

Annie Desrochers

Litter leachates have stronger impact than leaf litter on Folsomia candida fitness

Keywords: secondary metabolites, dissolved organic compound, Collembola, mortality, reproduction, metabolomic

Tree litter leachates increased mortality of Folsomia candida in comparison with the litter treatment or the controls, probably due to the solubility of secondary metabolites contained in the leaves and released in a free form in leachates.

days

Litter OR litter leachates (5 or 10%) OR controls (water) 3 species and one mixture 10 individuals of Folsomia candida

Mortality and reproduction

Grass Spruce

Poplar -Spruce Poplar

Introduction

Physical and chemical characteristics of plant leaves exert an important control on litter decomposition processes, mainly through their impact on the activity of litter-feeding invertebrates and microorganisms. For example, previous food choice experiments showed that earthworms or isopods were sensitive to the quality of the litter [START_REF] Hättenschwiler | Isopod effects on decomposition of litter produced under elevated CO2, N deposition and different soil types[END_REF][START_REF] Joy | Food preference and growth rate of porcellio laevis reared on decomposing leaf litter[END_REF][START_REF] Rief | Palatability of selected alpine plant litters for the decomposer Lumbricus rubellus (Lumbricidae)[END_REF]. During the first stages of leaf litter decomposition, an important amount of soluble compounds is lost through leaching, representing up to 30% of total leaf litter carbon, and released into the soil [START_REF] Berg | Plant litter decomposition, humus formation, carbon sequestration[END_REF]. These water-soluble compounds are in a great proportion labile and readily available, and are an important nutrient source for decomposers and microbial communities [START_REF] Joly | Diversity of leaf litter leachates from temperate forest trees and its consequences for soil microbial activity[END_REF][START_REF] Marschner | Controls of bioavailability and biodegradability of dissolved organic matter in soils[END_REF]. However, secondary metabolites can also be leached out from green foliage and decomposing litter, and released into the soil [START_REF] Gallet | Phenolic compounds in natural solutions of a coniferous forest[END_REF][START_REF] Rice | Allelopathy[END_REF]. These compounds are defense compounds to prevent herbivory or parasitism (i.e. terpenes and phenolic compounds), or to resist against inter-and intraspecific competition in allelopathic mechanisms [START_REF] Chomel | Plant secondary metabolites: a key driver of litter decomposition and soil nutrient cycling[END_REF][START_REF] Fernandez | Allelochemicals of Pinus halepensis as drivers of biodiversity in Mediterranean open mosaic habitats during the colonization stage of secondary succession[END_REF][START_REF] Fernandez | Potential allelopathic effect of Pinus halepensis in the secondary succession: an experimental approach[END_REF]. Once released in the environment through leaching or litter decomposition they are in a free form that may resist degradation and can have strong impacts on the growth and activity of decomposers, from macro-arthropods to microorganisms [START_REF] Chomel | Plant secondary metabolites: a key driver of litter decomposition and soil nutrient cycling[END_REF][START_REF] Hättenschwiler | The role of polyphenols in terrestrial ecosystem nutrient cycling[END_REF][START_REF] Kuiters | Role of phenolic substances from decomposing forest litter in plant-soil interactions[END_REF]. Some studies showed that microbial activity is highly responsive to litter leachates input with a change in the soil microbial community [START_REF] Cleveland | Increases in soil respiration following labile carbon additions linked to rapid shifts in soil microbial community composition[END_REF][START_REF] Joly | Diversity of leaf litter leachates from temperate forest trees and its consequences for soil microbial activity[END_REF][START_REF] Wieder | Tropical tree species composition affects the oxidation of dissolved organic matter from litter[END_REF]. However, little is known about their effect on soil fauna communities.

Collembolans are important members of the soil decomposer food web and have important direct or indirect roles on the decomposition process [START_REF] Petersen | A Comparative Analysis of Soil Fauna Populations and Their Role in Decomposition Processes[END_REF]. Some species are microbivores and stimulate or regulate litter fungal colonization by grazing on the fungal hyphae, while others are detritivores and directly participate to organic matter transformation by fragmenting and ingesting the litter; in both cases they derive nutritional benefits from the decomposing litter source [START_REF] Das | Chemical quality impacts of tropical forest tree leaf litters on the growth and fecundity of soil Collembola[END_REF]. Collembolans are selective with their food, and for example they can perceive chemical cues of fungi at a distance of 1 cm -2.5 cm [START_REF] Auclerc | Assessment of movement patterns in Folsomia candida (Hexapoda: Collembola) in the presence of food[END_REF][START_REF] Salmon | Earthworm excreta attract soil springtails: laboratory experiments on Heteromurus nitidus (Collembola : Entomobryidae)[END_REF], prefer some fungal species over others [START_REF] Heděnec | Grazing preference and utilization of soil fungi by Folsomia candida (Isotomidae:Collembola)[END_REF][START_REF] Klironomos | FEEDING PREFERENCES OF THE COLLEMBOLAN FOLSOMIA-CANDIDA IN RELATION TO MICROFUNGAL SUCCESSIONS ON DECAYING LITTER[END_REF][START_REF] Scheu | Growth and reproduction of fungal feeding Collembola as affected by fungal species, melanin and mixed diets[END_REF], and can suffer a reduction in fitness from the ingestion of specific fungal species [START_REF] Klironomos | Reproductive significance of feeding on saprobic and arbuscular mycorrhizal fungi by the collembolan, Folsomia candida[END_REF][START_REF] Scheu | Growth and reproduction of fungal feeding Collembola as affected by fungal species, melanin and mixed diets[END_REF]. Furthermore, collembolans raised on fungal-colonized litter show grazing preferences and reproduction rates that are more affected by litter type than by fungal species [START_REF] Heděnec | Grazing preference and utilization of soil fungi by Folsomia candida (Isotomidae:Collembola)[END_REF], demonstrating the strong influence of litter quality on collembolans activity [START_REF] Das | Chemical quality impacts of tropical forest tree leaf litters on the growth and fecundity of soil Collembola[END_REF]. However, few studies have addressed the direct impact of the chemical composition of litter on the abundance and diversity of soil mesofauna, the physical structure of the litter often being a confounding factor. [START_REF] Das | Chemical quality impacts of tropical forest tree leaf litters on the growth and fecundity of soil Collembola[END_REF] reported that a collembolan species (Cyphoderus javanus Börner) tended to avoid litter with greater amounts of polyphenols and tannins. Furthermore, individuals that remained in contact with litters containing greater quantities of these secondary metabolites exhibited slower growth, lower fecundity and fewer moults [START_REF] Das | Chemical quality impacts of tropical forest tree leaf litters on the growth and fecundity of soil Collembola[END_REF]. [START_REF] Poinsot-Balaguer | Effects of tannin compounds on two species of Collembola[END_REF] found that condensed and hydrolysable tannins extracted from oak leaves were highly toxic for the collembolans [START_REF] Poinsot-Balaguer | Effects of tannin compounds on two species of Collembola[END_REF]. However, these studies related collembolans fitness to the total content of secondary metabolites, but did not consider their solubility in water and their bioavailability in natural environments. Some researchers assert that allelopathic plant-plant interference is most likely due to water-soluble compounds [START_REF] Vyvyan | Allelochemicals as leads for new herbicides and agrochemicals[END_REF]. Following the same idea, these water-soluble secondary metabolites leached from the canopy or the litter could be of greater importance for the activity of soil organisms than the litter content itself.

Previous in-situ studies showed that mixing spruce and poplar in plantations could offer valuable ecosystem services, such as tree productivity and soil carbon storage compared to monospecific plantations [START_REF] Chomel | Non-additive effects of mixing hybrid poplar and white spruce on aboveground and soil carbon storage in boreal plantations[END_REF]. Mixture of different litter species can enhance their respective decomposition rate due to the transfer of nutrient between litter species, the complementarity of diverse resources for the decomposers, or the dilution of secondary metabolites [START_REF] Gartner | Decomposition dynamics in mixed-species leaf litter[END_REF][START_REF] Gessner | Diversity meets decomposition[END_REF][START_REF] Hättenschwiler | Biodiversity and litter decomposition in terrestrial ecosystems[END_REF]. Similar positive results were observed for mixtures of leachates, leading to a stimulation of microbial activity [START_REF] Zheng | Non-additive effects of mixing different sources of dissolved organic matter on its biodegradation[END_REF]. In our in-situ studies, although we did not find higher decomposition in a mixture of poplar and spruce litter, some differences with tree species were observed in the mixed plantations and the collembolan abundance was (i) lower under poplar trees than spruce trees (Chomel et al., 2015a), (ii) greater in grass litter compared to poplar or spruce litter (Chomel et al., 2015b), and (iii) increased when grass litter was added to poplar or spruce litterbags. To understand if these patterns were due to the leaf chemistry of the different species, since poplar, spruce and grass litters have different qualities, controlled experiments are necessary. In this study we used the model collembolan species Folsomia candida Willem (Collembola: Isotomidae), which is among the most intensively studied of all species of Collembola [START_REF] Hopkin | Biology of the Springtails (Insecta: Collembola)[END_REF]. This parthenogenetic species is widely distributed in many environments, including forests [START_REF] Fountain | Folsomia candida (Collembola): A "standard" soil arthropod[END_REF] and is commonly found in the litter layer of coniferous and deciduous forests [START_REF] Christiansen | The Collembola of North America north of the Rio Grande. Part 2. Families Onychiuridae and Isotomidae[END_REF], even under the boreal climate of Canada [START_REF] Skidmore | Checklist of Collembola (Insecta: Apterygota) of Canada and Alaska[END_REF]. Moreover, this species is frequently used in microcosms experiments as it is easy to maintain in laboratory and have a short reproductive cycle [START_REF] Fountain | Folsomia candida (Collembola): A "standard" soil arthropod[END_REF].

The aim of this study was to disentangle the effects of litter water-soluble compounds (released by senescent leaves leaching) from the litter itself on the abundance and activity of the Collembola F. candida. For this purpose, we compared the effect of three litter species and one mixture (hybrid poplar, white spruce, grass and a mixture of poplar -spruce litter) and their respective leachates on F. candida fitness by using the modified ecotoxicological test 'Effects of pollutants on reproduction of Folsomia candida' proposed by the International Standards Organization (ISO 11267, 1999). During the experiment, F. candida were fed with yeast to tease apart nutrient effects associated to the treatments and focus on secondary metabolites effects. We tested the following hypotheses: (i) following results from our in-situ studies (i.e low abundance of collembola under poplar trees and high abundance in grass litter), we expected to find a high concentration of phenolic compounds with allelopathic activities in poplar litter and a low concentration in grass litter; (ii) A gradient of the effect of litter and leachates on the fitness of F. candida should be observed in the following order from positive to negative: grass > spruce > poplar; (iii) litter leachates have a more pronounced impact on F. candida compared to litter since nutrients and secondary metabolites would be directly bioavailable for collembolans; (iv) litter and leachate diversity from the mixture is expected to stimulate F. candida fitness in comparison with monospecific litter and leachates due to complementarity effects induced by mixed species.

Material and methods

Litter sampling and leachates preparation

In late September 2010, three types of senescent leaves (poplar, spruce and grass) were collected from experimental monospecific plantations of hybrid poplar (Populus maximowiczii A. Henry x P. balsamifera L.) and white spruce (Picea glauca [Moench] Voss) located in Amos (48°36'N, 78°04'W) in the boreal region of Abitibi-Temiscamingue, Quebec, Canada. Freshly fallen spruce needles and hybrid poplar leaves were collected by placing a plastic sheet beneath the trees to prevent soil contamination. Aerial parts of grass species (mainly grasses, i.e. Poa sp.) naturally present in the plantation were cut at ground level during the same period. Collected leaf material was homogenized, air-dried and stored at room temperature prior to the experiment. Leachates of each litter type (poplar, spruce, grass and poplar/spruce [50:50]) were prepared by soaking 100 g of litter (air-dried mass) in 1 L of deionized water (10% dry mass) for 24 h in darkness. This concentration is the most commonly used for in vitro allelopathy bioassays [START_REF] Chen | Allelopathic potential and volatile compounds of Rosmarinus officinalis L. against weeds[END_REF][START_REF] Fernandez | Allelochemicals of Pinus halepensis as drivers of biodiversity in Mediterranean open mosaic habitats during the colonization stage of secondary succession[END_REF].

Although they can be more concentrated than in natural conditions, it allows studying the potential allelopathic effect of a species [START_REF] Kil | Effect of pine allelochemicals on selected species in Korea[END_REF]. However, to be more representative to actual field concentrations, a 50% diluted solution was also prepared, producing 5% dry mass leachate.

Folsomia candida cultures

A population of a single clone of F. candida was reared in plastic boxes containing a mixture of permanently water saturated plaster of Paris and activated charcoal in a ratio 9:1 and maintained at 20°C with food (dry yeast pellets) available ad libitum. To synchronize age of the organisms, oviposition was stimulated by imposing a cold period and then placing the adults on a new breeding substrate [START_REF] Fountain | Folsomia candida (Collembola): A "standard" soil arthropod[END_REF]. After oviposition, adults were removed and eggs hatched 3-4 days later. Individuals became sexually mature 16 days after hatching, and to ensure a homogeneous cohort of juveniles, all the young individuals were placed in a large container and fed for the first time at the same time.

Experimental design

To study the effects of litter or leachates on the fitness of F. candida, we set up a total of 65 microcosms. The microcosms were set-up according to 2 interacting factors: the litter form (litter, 5% leachate and 10% leachate) and the litter species (poplar, spruce, grass and poplarspruce mixture) and controls consisted of deionized water without litter. Each combination of factors was replicated five times. The F. candida reproduction test was carried out according to an adaptation of the ISO standard 11267 (ISO 11267, 1999). The microcosms consisted of 120 mL plastic bottles with pierced screw caps containing 32 g of wet artificial soil, made up with 70% quartz sand, 20% kaolinite and 10% peat, ground, dried, and sieved to 0.5 mm with the pH adjusted to 6.0 ± 0.5 by the addition of CaCO 3 . It was moistened to 50% of water holding capacity with distilled water (7 mL). Chemicals to be tested in the ISO norm are generally pollutants dissolved in water at range of concentrations that will give appropriate reduction of reproduction levels (LOEC, CE 50). For this experiment, 7 mL of 5 or 10 % leachates were directly mixed with the soil. For the litter species treatments, 7 mL of distilled water were added to the substrate, and 1 g of coarsely chopped litter was re-humidified and placed on the substrate (corresponding to the amount of litterfall per surface area in natural plantations, [START_REF] Chomel | Non-additive effects of mixing hybrid poplar and white spruce on aboveground and soil carbon storage in boreal plantations[END_REF]. For each microcosm, ten 10/12-days-old juveniles were introduced and the microcosms were randomly placed in a climate chamber at a constant temperature of 20°C and continuous darkness for 30 days. They were opened twice a week for aeration and fed with baker's yeast at the initial time and after 2 weeks. Following the ISO guidelines, the pots were flooded with water and gently stirred to collect and count the animals floating at the surface. The number of surviving adults and the juveniles were recorded using a dissecting microscope.

Chemical composition of litter and leachates

Litter initial content of major nutrients (C, N, P) and phenolics were estimated from 5 subsamples of each litter. The litter samples were finely ground with a ball mill (MM301, Retsch Inc., Newtown, PA) prior to analysis. Carbon (C) and Nitrogen (N) concentrations were analysed in a CHN elemental analyzer (Flash EA 1112 series, ThermoScientific, U.S.A.). Phosphorus (P) was extracted with 20 mL of nitric acid from remaining dry ash after combustion of 0.5 g of subsamples at 500 °C for 5 h in a muffle furnace. The pH was adjusted to 8.5 with a 40 % NaOH solution. A volume of 1 mL of sample, 0.2 mL of mixed reagent (emetic tartar and ammonium molybdate solution), 0.04 mL of ascorbic acid and 0.76 mL of distilled water were placed directly in a microcuvette. After 150 min, the reaction was completed, and phosphorus concentration was measured at 780 nm with a UV/Vis spectrophotometer (Thermo Scientific®, USA). Total phenolics content was measured by the method of Folin-Ciocalteu [START_REF] Folin | A Colorimetric Method for the Determination of Phenols (and Phenol Derivatives) in Urine[END_REF]: 1 g of ground litter was shaken with 20 mL of distilled water for 90 min. A volume of 0.25 mL of the filtered aqueous extract (or of the leachate directly) was mixed with 0.25 mL of Folin-Ciocalteu reagent and 0.5 mL of saturated aqueous Na 2 CO 3 to stabilize the colour reaction, after which 4 mL of distilled water was added to dilute the extract. After 1 hour, the reaction was completed and measured at 765 nm on a UV/Vis spectrophotometer (Thermoscientific, U.S.A.). Quantitative results were expressed with reference to gallic acid.

Litter metabolic fingerprints (untargeted metabolomics)

The metabolomic profile of the litter was analyzed following the method from [START_REF] Hashoum | Biotic interactions in a Mediterranean oak forest: role of allelopathy along phenological development of woody species[END_REF]. A dry mass of 200 mg of sample was suspended in 4 mL of methanol:water (50:50), and subjected to ultrasonication for 5 min at room temperature. Extracts were then filtered using a syringe filter (PTFE 13 mm, 0.22 µm, Restek, USA). Analyses were performed with an UHPLC instrument (Dionex Ultimate 3000 equipped with a RS Pump, an autosampler, a thermostated column compartment and a UV diode array, Thermo Scientific, USA) coupled to an accurate mass spectrometer (qToF) equipped with an ESI source (qToF Impact II, Bruker Daltonics, Germany). UHPLC separation was done on an Acclaim C18 column (150 mm x 2.1 mm, 2.2 µm, Thermo Scientific, USA). Because the negative mode gave a better sensitivity, mass spectra were recorded in this ionization mode in full scan mode from 50 to 1200 amu at 2 Hz. After the dataset normalization [START_REF] Hashoum | Biotic interactions in a Mediterranean oak forest: role of allelopathy along phenological development of woody species[END_REF], around 6000 features were kept before the filtering steps were applied to ensure data quality and remove redundant signals using an in-house script on R [START_REF] Hashoum | Biotic interactions in a Mediterranean oak forest: role of allelopathy along phenological development of woody species[END_REF]. At the end, 3030 ions were kept for data analyses.

Statistical analysis

For all the chemical data, ANOVA were performed to test differences between two interacting factors: Litter species (poplar, spruce, grass and mixture poplar-spruce) and litter form (control, litter, 5% leachate and 10% leachate). Data were ln-transformed before performing statistical tests if the conditions of normality and homoscedasticity of the residuals were not met. After the analyses, multiple comparisons (Tukey contrasts) were done. To have an estimation of the metabolomic diversity of each litter species, the Shannon diversity index was calculated from all the metabolomic data including 3030 ions [START_REF] Quinn | Metabolomics of reef benthic interactions reveals a bioactive lipid involved in coral defence[END_REF][START_REF] Ristok | Plant species richness elicits changes in the metabolome of grassland species via soil biotic legacy[END_REF]. To detect compounds that were specific to each litter species we selected the fifty most discriminating ions (Variable Importance in Projection, VIP) that differentiated the three litter species using a PLS-DA analysis (Fig S1 ) with MetaboAnalystR [START_REF] Chong | MetaboAnalystR: an R package for flexible and reproducible analysis of metabolomics data[END_REF]. The features were annotated with constructor software (Bruker Compass DataAnalysis 5.0, Table S1). The most probable raw formulae and fragmentation patterns (MS 2 spectrum in negative mode) were compared with online databases (Metlin, Massbank of North America, Pubchem using Metfrag, Table S1). Further, we constructed a heatmap with those VIPs to visualise the relative intensities of each of these ions according to each litter type with R. A binomial generalized linear model (GLM) was used to test the difference of collembolans mortality rate in function of the different litter species (poplar, spruce, poplar/spruce mixture and grass litters) and litter forms (control, litter, 5% leachate and 10% leachate). Binomial GLMs are designed to fit proportions or percentages. A negative binomial GLM (i.e., a specific version of a Poisson model that uses an additional parameter to correct for data overdispersion) with a log link function was used to test the difference in reproduction with the different litter species (poplar, spruce, poplar/spruce mixture and grass litters) and litter forms (control, litter, 5% leachate and 10% leachate). Negative binomial GLMs are designed to fit count data (data that usually lacks normality), as it is generally the case when sampling invertebrate taxa. All statistical analyses were done with R v.3.1.0 (R Core Team, 2017), using the package 'stats' for the ANOVA and binomial GLM and the package 'MASS' for the negative binomial GLM.

Results

Chemical composition of litters and leachates

Spruce litter presented greater C concentrations, while grass litter had greater N concentrations compared to the two other litter types (P < 0.01 and P < 0.001 respectively, Table 1). Consequently, grass litter had the lowest and spruce litter the greatest C/N ratios, while poplar had intermediate values (P < 0.001). Phosphorus concentrations in the litters were similar among the different litter species (Table 1). Concerning the total phenolics, grass litter had more than four-fold lower phenolic concentrations compared to the three other litter species (P < 0.001, Fig. 1). For the soluble compounds, however, we observed that grass and spruce leachates contained lower amounts of phenolic compounds compared to poplar and mixed litters leachates (P < 0.005, Fig. 1). The proportion of phenolics in the leachates relative to the litters differed depending on the litter species; these proportions corresponded to 36%, 7%, 31%, and 38% of the total litter concentrations respectively for grass, spruce, poplar-spruce mixture and poplar leachates (Fig. 1). Metabolomic diversity, calculated by the Shannon index of the whole dataset, was lower in spruce and higher in poplar litters, with an intermediate diversity in grass litter (ANOVA, F = 52.9, P < 0.001, Table 1). Qualitative analysis of the 50 most discriminant features of the three litter species is reported in Fig. 2 and shows that the majority of the features were specific to the poplar litter, and were present in greater abundance than in the two other litter species. Among them, we identified caffeic acid derivatives, pinocembrin and several pinocembrin derivatives, salicinin, quercetin and arthromerin A (Table S1). Six features, among which we identified dehydroabietic acid, were specific to the spruce litter, while rare or absent in the poplar litter. Three compounds were found either in the poplar and the grass litter, among them we identified chlorogenic acid and ferulic acid (Table S1).

Effects of litter and leachates on F. candida mortality

Mortality of F. candida differed across combinations of litter species (X² = 63.1, P < 0.001) and forms (X² = 259.7, P < 0.001) with a significant interaction term of the two factors (X² = 57.5, P < 0.001). While the mortality of F. candida with the grass litter or the 10% grass leachate were similar to the mortality observed in the controls (P > 0.05, Fig. 3), a four-fold increase in mortality was observed with the grass 5% leachate compared to the control (P < 0.001, Fig. 3). For the spruce litter, we observed an increase in mortality with all the litter forms compared to the control (Fig. 3). With the litter itself, mortality reached 62%, while it reached 84% and 78% with the 5% and 10% leachates, respectively. Although the mortality with the spruce litter leachates tended to be greater than with the litter, differences were only marginally significant (P = 0.053 and P = 0.058 respectively). While the mortality of F. candida with the mixed litter was similar to the mortality observed in the controls (P > 0.05, Fig. 3), a three-fold increase in mortality was observed with the 5% leachate and the 10% leachate compared to the litter, reaching mortality rates of 93% and 98%, respectively (P < 0.001, Fig. 3). Lastly, for the poplar litter, we observed an increase in mortality with all the litter forms compared to the controls (Fig. 3). Mortality reached 68% with the litter, while it was 94 % and 100 % with the 5 % and 10 % leachates, respectively (P < 0.001, Fig. 3).

Effects of litter and leachates on F. candida reproduction

Reproduction of F. candida differed across combinations of litter species (negative Binomial GLM, X² = 39.4, P <0.001) and forms (Binomial GLM, X² = 70.5, P < 0.001) with a significant interaction term (Binomial GLM, X² = 75.7, P < 0.001). Leachates of spruce, mixed or poplar significantly reduced reproduction in comparison with the litter or the control treatments (P < 0.05, Fig. 4). The grass litter and 10% leachate increased the reproduction of F. candida by 385 % and 70 %, compared to the control, respectively. However, the grass 5% leachate decreased the reproduction by 83% compared to the control (Fig. 4).

Discussion

The aim of this study was to observe the fitness of F. candida in the presence of litter or leachates of different litter species with diverse chemical characteristics. We observed that leachates generally increased F. candida mortality compared to the litters. Additionally, we observed greater mortality rates of collembolans with tree leachates, which contained a greater quantity of phenolic compounds, compared to grass leachates. Our results also showed reproduction rates four times greater with grass litter than for tree litters or controls, in line with our previous in-situ litter decomposition study for which a greater abundance of organisms was observed when grass litter was added to spruce or poplar litter (Chomel et al., 2015a).

Effects of litter species and its quality

While we expected an increase of collembolans fitness with the presence of grass litter, we observed a considerable increase of their reproduction compared to the control and the other litter species, partially confirming our first hypothesis. Nutritional quality of decomposing litter depends on the chemical constituents of leaf tissues [START_REF] Das | Chemical quality impacts of tropical forest tree leaf litters on the growth and fecundity of soil Collembola[END_REF][START_REF] Heděnec | Grazing preference and utilization of soil fungi by Folsomia candida (Isotomidae:Collembola)[END_REF]. Grass litter had relatively low C/N ratios and phenolic compounds contents, indicating greater concentrations of carbohydrates and proteins. This labile organic matter rich in N provides a readily available energy source to decomposers [START_REF] Aber | Predicting long-term patterns of mass loss, nitrogen dynamics, and soil organic matter transformation from initial fine litter chemistry in temperate forest ecosystems[END_REF][START_REF] Aerts | Climate, leaf litter chemistry and leaf litter decomposition in terrestrial ecosystems: A triangular relationship[END_REF], and induced better conditions for feeding and reproduction of collembolans [START_REF] Das | Chemical quality impacts of tropical forest tree leaf litters on the growth and fecundity of soil Collembola[END_REF]. While mortality rate with the grass litter was similar than the control, reproduction rate was much higher. This result confirms the benefit of "high" litter quality to enhance F. candida fecundity [START_REF] Booth | The influence of fungal food quality on the growth and fecundity of folsomia candida (Collembola: Isotomidae)[END_REF][START_REF] Scheu | Growth and reproduction of fungal feeding Collembola as affected by fungal species, melanin and mixed diets[END_REF]) and shorten the period until oviposition starts [START_REF] Scheu | Growth and reproduction of fungal feeding Collembola as affected by fungal species, melanin and mixed diets[END_REF]. Besides C/N ratios, litter phenolic concentrations could also explain F. candida fitness. In comparison with the grass litter, both poplar and spruce litters had more than four-fold greater concentrations of phenolics. Within 'low' quality litters, poplar litter had lower C/N ratios but similar total phenolic concentrations than spruce litter, and both litter types negatively affected the survival and reproduction of F. candida. We observed an increase in mortality of 240 % and 210 % and a decrease in reproduction of 29 % and 18 %, respectively for poplar and spruce litters, compared to the control. We identified several specificities among the tree litter species: spruce litter contained dehydroabietic acid, while pinocembrin and caffeic acid metabolites were found in poplar litter. Pinocembrin is a flavonoid that has been recognized to have antimicrobial activities [START_REF] Rasul | Pinocembrin: A Novel Natural Compound with Versatile Pharmacological and Biological Activities[END_REF], and caffeic acid is also known to have strong allelopathic and antimicrobial activities [START_REF] Batish | Caffeic acid affects early growth, and morphogenetic response of hypocotyl cuttings of mung bean (Phaseolus aureus)[END_REF]. The terpenoid dehydroabietic acid is a defense metabolite abundant in resin, thus common in conifers [START_REF] Phillips | Resin-based defenses in conifers[END_REF]. These three secondary metabolites potentially reduce the colonization of the litter by microorganisms by their antimicrobial activities and can have repulsive effects on detritivores [START_REF] Chomel | Plant secondary metabolites: a key driver of litter decomposition and soil nutrient cycling[END_REF][START_REF] Das | Chemical quality impacts of tropical forest tree leaf litters on the growth and fecundity of soil Collembola[END_REF]. The presence of these secondary metabolites and the high concentration of total phenolic compounds in tree litters compared to grass litter could explain their strong and negative impact on F. candida survival and reproduction. However, when these two species of litter were mixed, mortality rate was similar to the control, confirming our fourth hypothesis. Mixed diets may significantly increase collembolan fitness by providing a balanced nutrient intake from the different litters present in the mixture or by a dilution of toxic compounds contained in one of the species [START_REF] Hättenschwiler | Biodiversity and litter decomposition in terrestrial ecosystems[END_REF][START_REF] Scheu | Growth and reproduction of fungal feeding Collembola as affected by fungal species, melanin and mixed diets[END_REF]. Conversely, although mixing the two litter types increased survival compared to single species litter, it had no effect on the reproduction of F. candida.

Effects of leachates vs litter

In agreement with our third hypothesis, leachates had a greater impact on F. candida fitness compared to the litters. Some of the leached compounds can act as easily-available nutrient sources for decomposers. However secondary metabolites can be toxic and can also have a greater impact on organisms once released in the environment by leaching or decomposition [START_REF] Chomel | Plant secondary metabolites: a key driver of litter decomposition and soil nutrient cycling[END_REF]). It appears that phenolic compounds from the poplar litter were more water-soluble compared to those from spruce litter, as their leachates contained 38 % of the total phenolic compounds of the litter, while spruce leachates only contained 7 % of the total phenolic compounds of the spruce litter. This can be explained by the fact that needles have a thicker epidermis and hypodermis that reduce the leaching capacity of the inner leaf tissues [START_REF] Don | Amounts and degradability of dissolved organic carbon from foliar litter at different decomposition stages[END_REF][START_REF] Joly | Diversity of leaf litter leachates from temperate forest trees and its consequences for soil microbial activity[END_REF]. Microcosms that received 10% leachates of poplar, mixed poplar/spruce or spruce showed 100%, 94 % and 78% of mortality, respectively, whereas grass leachates only induced 32% of mortality and was not different from the control, indicating a strong influence of the compounds contained in the tree leaves on F. candida.

The greater diversity of poplar compounds and their higher solubility could explain why poplar litter leachates induced stronger effects compared to spruce or grass litter leachates. This result is in line with our findings in a previous field experiment where we observed a lower abundance of soil fauna in poplar plantations compared to spruce or mixed plantations (Chomel et al., 2015a). There is some evidence that soil microbial activity is highly dependent on leachates. Indeed, soil respiration has been shown to be quickly responsive to litter leachates input with a change in the soil microbial community [START_REF] Cleveland | Increases in soil respiration following labile carbon additions linked to rapid shifts in soil microbial community composition[END_REF][START_REF] Joly | Diversity of leaf litter leachates from temperate forest trees and its consequences for soil microbial activity[END_REF][START_REF] Wieder | Tropical tree species composition affects the oxidation of dissolved organic matter from litter[END_REF] and soil respiration is significantly explained by the proportion of water-soluble compounds contained in the leaf litter layer covering the soil [START_REF] Fanin | Does variability in litter quality determine soil microbial respiration in an Amazonian rainforest?[END_REF]. The differences in F. candida fitness observed with the different litter species could also be an effect of the different microbial communities that are present in the leachates.

Several studies have shown that leachates can have strong allelopathic activity from one plant species to another [START_REF] Fernandez | Allelochemicals of Pinus halepensis as drivers of biodiversity in Mediterranean open mosaic habitats during the colonization stage of secondary succession[END_REF][START_REF] Fernandez | Potential allelopathic effect of Pinus halepensis in the secondary succession: an experimental approach[END_REF][START_REF] Rice | Allelopathy[END_REF] and can have strong effects on plant community diversity and composition [START_REF] Ma | Effects of Litter Leachate on Plant Community Characteristics of Alpine Grassland in Qinghai Tibetan Plateau[END_REF]; however, studies on the effect of leachates on soil fauna communities are rare. Leaching of leaves and litter is an important process in ecosystems since a large quantity of compounds can quickly be released by a rainfall event, or gradually released during the litter decomposition process. Nonetheless, litter leachates are rarely considered in studies on aboveground and belowground interactions, whereas this study showed that they may contain bioavailable compounds that can be significant drivers of collembolan fitness.

Conclusion

Litter species significantly affected F. candida mortality and reproduction. Grass litter increased survival and reproduction of collembolans, while tree litters reduced survival. This study also showed that leachates had greater impacts on collembolans fitness compared to litter, with stronger negative effects of tree leachates compared to grass leachates. These results indicate a strong influence of the compounds contained in the trees leaves. Phenolic compounds in poplar leaves were more leachable than in spruce leaves and presented a greater diversity, with the presence of several allelopathic compounds as caffeic acid and pinocembrin, which could explain the stronger negative effect of poplar leachates. This study showed that the release of compounds through leaching of leaves or litter could be an important factor for the activity of soil organisms and for aboveground-belowground interactions in natural ecosystems. Other studies should be conducted with more species of collembola to see if similar patterns are observed.

Figure captions Figure 1 .

 captions1 Figure captions

Figure 2 .

 2 Figure 2. Heatmap of the fifty most discriminant ions (Variable Importance in Projection,

Figure 3 .

 3 Figure 3. Mortality of Folsomia candida (mean ± SE) growing in mesocosms with the

Figure 4 .

 4 Figure 4. Reproduction of Folsomia candida (mean ± SE) growing in mesocosms with the

  Figure 1 Click here to download high resolution image

Acknowledgements

The authors thank to Thomas Tully (Ecole Normale Superieure, Paris, France) for providing the initial culture of Folsomia candida. We thank Germain Boungou and Amélie Saunier (IMBE) for the help with the phosphorus and metabolomic analyses. This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC-CRSNG, grant number 381553-09). LC-MS analysis were conducted at the IMBE 'Chemical Ecology and Metabolomics facility' funded by the CNRS, "Sud Provence-Alpes-Côte d'Azur" regional council, TOTAL Foundation and the French National Research Agency (ANR).

Declaration of interests

The authors declare to have no competing interests that could have appeared to influence the work reported in this paper

Supplementary Material

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