A H Borsali

T Z Lerch

R Besbes

R Gros

I Laffont-Schwob

J L Boudenne

F Ziarelli

A Pando

A M Farnet-Da Silva

Coastal environments shape chemical and microbial properties of forest litters in circum Mediterranean region

Keywords: extracellular enzyme activities, litter microbial communities, Mediterranean area, water potential stress

This study explores how chemical and microbial properties of litters can be affected by coastal environments across the Mediterranean basin. A litterbag experiment including Pinus halepensis Mill. and Pistacia lentiscus L., collected from both inland and coastal areas was set up in France, Greece and Algeria. Control litterbags were left in their sampling sites and a transfer of litterbags from inland to coastal area was performed to test whether the effect of the specific constraints of coastal environments varies according to the country and the litter type. After 10 months, litter chemical composition (CP/MAS 13 C-NMR) and microbial activities (cellulase, respiration, Biolog) and structure (TRFLP) were analysed. Coastal conditions led to various responses: i) litter aromaticity differed in the coastal zones depending on the country (high in Greek coastal area, low in Algerian coastal zone), ii) less functionally-diversified microbial communities were found in Greek coastal area compared to French and Algerian coasts, iii) genetic diversity and richness were strongly impacted after transfer to the coastal zone whatever the country. The type of litter shaped microbial communities: i) at a local scale i.e. in either coastal or inland area, catabolic profiles and cellulase activities varied with the plant species, ii) at a regional scale, the effect of coastal conditions differed with the plant species (basal respiration, Shannon-Weaver index, catabolic diversity H', cellulases and catabolic profiles). Thus, litter microbial properties differed in coastal environments across the Mediterranean basin and plant litter type plays a major role in microbial properties at large spatial scale.

Introduction

Mediterranean ecosystems are subjected to various environmental pressures: a drastic climate characterized by severe summer drought, soils weakly developed and poor organic matter content [START_REF] Sardans | Experimental drought reduced acid and alkaline phosphatase activity and increased organic extractable P in soil in a Quercus ilex Mediterranean forest[END_REF] and long-standing and intense human activities. Drastic pedoclimatic conditions are more intense in coastal environments where water-potential stress is reinforced by wind regime and combined to an additional stress i.e. osmotic stress due to sea-spray exposure [START_REF] Boukhdoud | Effect of agricultural practices and coastal constraints on soil microbial functional properties in Mediterranean olive orchards[END_REF][START_REF] Qasemian | How do microlocal environmental variations affect microbial activities of a Pinus halepensis litter in a Mediterranean coastal area?[END_REF]. A reduced water availability in soils or litters induces a shift in microbial community diversities [START_REF] Chowdhury | Response of microbial activity and community structure to decreasing soil osmotic and matric potential[END_REF] together with a lower basal respiration and extracellular enzyme production, which hinder organic matter decomposition [START_REF] Yuan | Microbial biomass and activity in salt affected soils under arid conditions[END_REF]. Coastal ecosystems under such particular constraints may be more threatened by climate change that will notably be characterized by higher temperatures and longer periods of summer drought in the Mediterranean area [START_REF] Gibelin | Anthropogenic climate change over the Mediterranean region simulated by a global variable resolution model[END_REF]Déqué, 2003, Sanchez et al., 2004).

Climate in various biomes has been proven to be a major driver structuring decomposer communities and consequently regulating organic matter turn-over [START_REF] Aerts | Climate, Leaf Litter Chemistry and Leaf Litter Decomposition in Terrestrial Ecosystems: A Triangular Relationship[END_REF][START_REF] Wall | Soil Ecology and Ecosystem Services[END_REF], Zhou et al., 2008). However, some studies revealed that, local differences linked to plant species assemblages and thus to organic matter diversity and quality, have a stronger effect on the processes of decomposition than climate variations between continents [START_REF] Cornwell | Plant species traits are the predominant control on litter decomposition rates within biomes worldwide[END_REF]. [START_REF] Zhang | Rates of litter decomposition in terrestrial ecosystems: global patterns and controlling factors[END_REF] found that litter quality is the most important direct regulator of litter properties at the global scale. Their results showed significant relationships between litter decomposition rates and the combination of climatic factors and litter quality (C/N and Total nutrients).

Thus, there is a urgent need to decipher how litter microbial communities may be affected by such climate modifications at various spatial scales and whether it may vary with the chemical quality of litter and pre-exposure to osmotic stress (coastal environments).This study aimed at investigating how microbial functioning in litters from two Mediterranean plant species, commonly found in both Mediterranean coastal and inland areas and exhibiting different functional traits (Pinus halepensis, a coniferous species, and Pistacia lentiscus, an evergreen dicot species) may be driven by the specific conditions of coastal environments. Moreover, we used the contrasted climate conditions across the Mediterranean basin, comparing France, Greece and Algeria environments, to test the effect of a gradient of aridity on litter functioning. Most of litter decomposition experiments used litter from plants exposed artificially to global change factors such as warming, irrigation or elevated CO2 [START_REF] Suseela | Decoupling the direct and indirect effects of climate on plant litter decomposition: Accounting for stress-induced modifications in plant chemistry[END_REF] but very few studies used field-scale transplantation experiments to investigate the in situ impacts of climate variations [START_REF] Berger | Decomposition of beech (Fagus sylvatica) and pine (Pinus nigra) litter along an Alpine elevation gradient: Decay and nutrient release[END_REF][START_REF] Keiser | Climate masks decomposer influence in a cross-site litter decomposition study[END_REF][START_REF] Makkonen | Highly consistent effects of plant litter identity and functional traits on decomposition across a latitudinal gradient[END_REF]. Here, an original experimental design based on litterbag use was performed: litterbags of either P. halepensis or P. lentiscus litters were exposed in inland to coastal areas in the three selected countries to test the effect of a gradient of aridity on litter functioning and, within each country, a pool of litter bags was transferred from inland to coastal area to test the effect of coastal conditions on inland communities. Consequently, we aimed at addressing the following questions: i) are microbial communities and their functions shaped by coastal conditions and do more drastic climate conditions found across the Mediterranean basin strengthen these stresses linked to the coastal conditions? ii) for each country, what are the responses of microbial communities from inland areas when they are subjected to coastal area conditions and their specific stresses and are their responses plantspecies dependent? According to our hypotheses: i) in each country, coastal environmental conditions will play a key role defining a particular microbial functioning in the coastal area and ii) more arid climate conditions will act at a larger scale i.e. the Circum-Mediterranean region and will smoothen the effect of coastal conditions and of litter type on microbial properties. Thus, in fine, this study will provide useful information about environmental factors driving microbial communities and their activities at different spatial scales, more precisely about the vulnerability of coastal areas.

Materials and Methods

Site description and litter sampling

Sampling sites were selected in stands of Pinus halepensis (forest and matorral) in association with Pistacia lentiscus throughout a latitudinal gradient across the Mediterranean basin (Table 1). The inland sites (Table 1) were located in the Massif de l'Etoile in the periurban area of Marseille (France), in the forests of Argoli near Cleones (Peloponese, Greece) and in the forests of Traras near Ain Kebira (Wilaya of Tlemcem, Algeria), respectively. The coastal sites (Table 1) were located in the Parc National des Calanques of Marseille (France), near Monemvassia (Greece) and near Ghazaouet (Algeria). In each country, three sampling sites (400 m 2 ) were chosen in the coastal area (at less than 500 m from the coastal line) and three others in the inland area (at 20 km from the sea). The distance between sites, whether coastal or inland, is 2 km at least, far exceeding the spatial dependence of most microbiological properties in soil [START_REF] Nannipieri | Microbial diversity and soil functions[END_REF]. The selected sites had similar pedoclimatic and topographic features: 25-230 m in elevation, similar exposure (South), slope (10-15%) and soil type (Calcaric Leptosol according to IUSS Working Group WRB, 2006). For each site, the exact location (GPS), maximal and minimal temperatures and the relative humidity of air measured in situ by EL-USB 2.0 probe (Conrad) over a two-year period and annual precipitation, mean of annual temperature, precipitation of driest and wettest months (worldclim, 1970(worldclim, -2010(worldclim, , Hijmans et al., 2005) ) were collected (Table 1). At each site and for each plant species, in May 2014, twenty samples were randomly collected over a 400 m 2 area to obtain a composite sample (2 kg) to prepare the litter bags. The very upper layer of horizon Ol of P. lentiscus and P. halepensis litters was sampled to ensure homogenous characteristics of the litter collected. Contaminating debris (e.g. leaves of other species, including branches and seeds) were removed carefully from each collection. The samples were homogenized and stored at 4°C until litter bag preparation.

Litterbag preparation and experimental set-up 30 g (dry weight, DW) of P. halepensis or P. lentiscus litter were placed per litterbag (3010 cm, 2 mm rigid nylon mesh). A total of 162 litterbags were prepared: 108 control litterbags (3 countries x 2 areas (coastal and inland) x 3 sites x 3 replicates x 2 types of litter) and to test the effect of the coastal zone i.e. 54 litterbags were transferred from inland to coastal areas (3 countries x 3 sites x 3 replicates x 2 types of litter). Litterbags were then placed in 6 sites for litter exposure (3 countries x 2 areas), independent from the sampling sites, in early June 2014: 18 control litterbags in each inland site of the three countries (9 litter bags for each species), 18 control litterbags and 18 'transfer' litterbags in each coastal site of the three countries. After 10 months in the field, litterbags were collected in March 2015. An aliquot of each sample was kept after drying and ground prior to chemical analysis. For microbial analyses, all the experiments started immediately and were performed over one week.

Plant litter physico-chemical properties

The physico-chemical properties of P. halepensis and P. lentiscus litters collected in coastal and inland sites were determined as follows: as a preliminary, litters were characterized for moisture, pH and electrical conductivity (EC). Total Organic Carbon (TOC) and Total Nitrogen (TN) were measured using high temperature catalytic oxidation technique (Multi N/C 2100, Analytik Jena, Germany). The sample was injected (50 µl) into the furnace filled with a Pt preconditioned catalyst. The combustion was realized at 800 °C and the combustion products were carried by high purity oxygen (Linde Gas) allowing detection of CO2 by nondispersive infrared (NDIR) and detection of NO by chemiluminescence (CLD).

After ten months in the field, TOC and TN of litters from litter bags were characterized as described above. Moreover, litter chemical properties were also analysed by solid-state 13 C NMR. Spectra were obtained on a Bruker Avance-400 MHz NMR spectrometer operating at a 13 C resonance frequency of 100.7 MHz and using a commercial Bruker double-bearing probe.

About 400 mg of sample were placed in zirconium dioxide rotors of 4-mm outer diameter and spun at the Magic Angle Spinning (MAS) rate of 10 kHz. The Cross Polarisation (CP) technique was applied with a ramped 1H-pulse starting at 100 % power and decreasing to 50 % during contact time (2 ms) to avoid Hartmann-Hahn mismatches. The experiments were performed at ambient temperature and 20K scans were accumulated using a delay of 2.5s, for an experimental time of 2h. The 13 C chemical shifts were referenced to tetramethylsilane and calibrated with glycine carbonyl signal, set at 176.5 ppm. The 13 C NMR spectra were divided into 7 chemical shift regions according to [START_REF] Dignac | Effect of N content and soil texture on the decomposition of organic matter in forest soils as revealed by solid-state CPMAS NMR spectroscopy[END_REF] (160-190 ppm). Deconvolution of each spectrum was performed on DmFit 2011 to determine the relative intensity of each selected region [START_REF] Massiot | Modelling one-and two-dimensional solid-state NMR spectra[END_REF]. The aromaticity ratio (aromatic C/O alkyl C + alkyl C + aromatic C) was calculated according to [START_REF] Baldock | Assessing the extent of decomposition of natural organic materials using solid-state C-13 NMR spectroscopy[END_REF].

Microbial activities

Cellulase activity was assayed using CarboxyMethylCellulose (CMC) 0.1% (w/v) in 8 mL of sodium acetate buffer (50 mM, pH 5) added to 1 g of litter (dry weight) incubated for 4h at 50°C. Glucose was quantified according to the Somogyi-Nelson method and absorption was read at 870 nm [START_REF] Farnet | A modified method based on arsenomolybdate complex to quantify cellulase activities: application to litters[END_REF]. Enzyme activities were expressed in µmoles of reaction products released per minute (U) per gram of dry soil (U g -1 DS).

Basal respiration was measured to assess the ecophysiological state of litter microbial communities. Three g DW equivalent of fresh litter were placed in 117 ml glass jars. The glass jars were immediately sealed with hermetic rubber septa, and incubated for 4 hours at 25°C. After incubation, 1 ml of air was sampled in the head space with a syringe and injected into a gas chromatograph (Chrompack CHROM 3 -CP 9001) to analyse CO2 production. The gas chromatograph was equipped with a thermal conductivity detector and a packed column (Porapack). The carrier gas helium flow was regulated at 60 ml h -1 . Ambient CO2 concentrations were subtracted from sampled CO2 concentrations and resulting values were adjusted at 22°C according to Ideal Gas Laws using a Q10 = 2. Substrate-induced respiration (SIR) rates were estimated using a procedure from [START_REF] Anderson | A physiological method for quantitative measurement of microbial biomass in soils[END_REF]. Three grams DW equivalent of fresh litter were placed in 117 ml glass jars and amended with powdered glucose (1000 µg C g -1 soil) found to maximize the respiration rate in litter in a preliminary assay (data not shown). One ml of air was sampled in the headspace with a syringe and injected into a gas chromatograph to analyse CO2 production for 1 hour (see above). Substrate-induced respiration was converted into microbial biomass (MB) using the relation established by [START_REF] Beare | A substrate induced respiration (SIR) method for measurement of fungal and bacterial biomass on plant residues[END_REF].

The catabolic diversity of cultivable microbial communities was determined with Biolog EcoPlate™ (BIOLOG Inc., Hayward, CA) using a procedure adapted from [START_REF] Garland | Classification and characterization of heterotrophic microbial communities on the basis of patterns of community-level sole-carbon-source utilization[END_REF]. Briefly, 4 g DW equivalent of sub-sample were added to 50 ml of sterile 0.1 % Na-pyrophosphate solution (pH 7). The mixture was then shaken with an orbital shaker for 20 min and centrifuged at 500 g for 10 min at 4 °C to obtain a microbial suspension. Exactly 1 ml of supernatant was diluted into 99 ml of sterile saline solution (0.85% NaCl), hand-mixed for 30 sec. and left to stand for 10 min. We purposely did not adjust the inoculums in order to obtain uniform cell density, instead considering the total microbial count as an inherent characteristic of the microbial communities of each mesocosm. A 125 µl aliquot of the diluted solution was added to each of 96 wells in a Biolog EcoPlate™. Plates were incubated at 25 °C and colour formation in each well was monitored at 595 nm using a TECAN® spectrophotometer. Measurements were performed three times a day until average well colour development (AWCD) exceeded a value of 1.0 standardized absorbance units (i.e. 3-5 day incubation). Microbial C-use intensity was assessed as the rate of average well colour development (AWCD) calculated as follows: AWCD =Σ ODi/31, where ODi is the optical density for each well in the mid-exponential growth phase (i.e. after 72h). Absorbance values for the wells with C sources were blanked against the control well. The incubation time resulting in an AWCD = 0.5 absorbance unit (T0.5) was calculated for each sample and used to standardise the optical density of each C-containing well [START_REF] Garland | Classification and characterization of heterotrophic microbial communities on the basis of patterns of community-level sole-carbon-source utilization[END_REF].

Microbial genetic structure

DNA was extracted from 0.25g of litter, using NucleoSpin® Soil kit (Macherey-Nagel, Düren, Germany) following the manufacturer instructions. The quality of DNA extractions was checked by electrophoresis on 1% agarose gels stained with GelRed (Molecular Probes, USA) using a Gel Doc image analyser (BioRad, USA). DNA concentration was quantified using Quant-iT TM dsDNA High-Sensitivity Assay Kit (Invitrogen, Canada). Then, all DNA extracts were then diluted to 0.5 ng / μL for subsequent analysis. Bacterial community structures were analyzed using T-RFLP footprinting as described by [START_REF] Blaud | Analysing the effect of soil organic matter on bacterial communities using T-RFLP finger footprinting: different methods, different stories?[END_REF].

Briefly, bacterial 16S rRNA gene was amplified using primers FAM labelled 63F and 1389R. Biorad T100 thermal cycler was used for the amplification with the following programs: initial denaturation at 94 ºC for 2 min, followed by 30 cycles of 94 ºC for 30 s, 57 ºC for 45 s, and 72 ºC for 90 s, followed by a final extension time at 72 ºC for 10 min. Bacterial PCR products (10 µl) were then digested with 10 U of the restriction enzyme AluI and 1× restriction enzyme buffer (Thermo Fisher) in a total volume of 15 µl at 37 ºC for 3 h. After a desalting step, 2µl of PCR products were mixed with formamide containing 0.5% of LIZ500 (T-RFLP) internal size standard (Applied Biosystems,) in a total volume of 12 µl and denatured at 94 ºC for 3 min. Samples were electrophoresed in an ABI 3730 PRISM® capillary DNA sequencer (Applied Biosystems). The T-RFLP profiles obtained with the sequencer were analysed using GeneMapper ® v3.7 software (Applied Biosystems). The fragments between 50 and 500 bp and peaks height ≥ 50 fluorescence units were included in T-RFLP analysis. Fragments having a relative abundance of proportion < 0.5% were removed from the matrices.

Statistical analyses

The catabolic (BIOLOG) and genetic (TRFLP) diversities was calculated using Shannon's index (H'):

where pi is either the ratio of colour development of the i th well to the sum of colour development of all positive wells or the relative abundance of a given restriction fragment for BIOLOG or TRFLP techniques.

The normality and homogeneity of the variances of the data were determined on the residuals from the regression model with the Shapiro-Wilk and Levene tests, respectively. Data were transformed to common logarithms, log10, when necessary to meet the requirements of normality and homogeneity of variance for ANOVA. For control litterbags, three-way ANOVA were used to determine whether, and to what extent, microbial properties (i.e. basal respiration, active microbial biomass via SIR, catabolic diversity H', cellulase activities and bacteria richness and equitability via genetic analysis) and chemical characteristics (using NMR data and C/N ratio) depended upon i) litter species (Pinus halepensis vs Pistacia lentiscus) ii) the area conditions (coastland vs inland), iii) the geographical location (France, Greece and Algeria) and iv) their interactions. When a 2-way interaction was found between geographical location and context (coastal and inland) or plant litter species, we separated data for each modality of geographical location, context, transfer or plant litter species using a one-way ANOVA. Statistically significant (P<0.05) main effects and interactions were analysed further using the Tuckey HSD post hoc tests. Principal component analysis (PCA) was performed on covariance matrices from Biolog® to explore the effects of geographical location, condition and litter type on the catabolic structure of microbial communities and tested by PERMANOVA. PCA provided an ordination of the microbial C-substrate utilization profiles, which were plotted in one and two dimensions respectively, based on the scores of the first two principal components (PC). All statistical analyses when performed using R software (3.2.1, R Development Core team, 2015), levels of significance are indicated as p ≤ 0.05.

Results

Influence of the coastal environmental conditions on litter properties along a gradient of aridity in the Mediterranean basin

Here, we explored whether environmental constraints, which can be enhanced in coastal environments (i.e. differences in salinity exposure, humidity, temperature, etc.), affected litter microbial functioning. Thus, first, it was important to demonstrate how these environmental characteristics actually varied between inland and coastal zones in the three countries. These environmental conditions were indeed highly discriminated with respect to certain parameters and depending on the geographical location. Temperature and precipitation variations were strongly different between both coastal and inland areas. In France and Greece, maximal temperature and annual mean temperature were higher in the area close to the coastline, while precipitations of the driest and wettest months were lower. An opposite trend was observed in Algeria, with more intense arid conditions in the inland site. These contrasted climate conditions are particularly well described using annual precipitation as an index of aridity [START_REF] Stadler | Aridity indexes[END_REF]. The climate gradient between the Northern and the Southern sides of the Mediterranean basin is clearly observed using this parameter and the differences in precipitations between coastal and inland areas are particularly strong in Greece and Algeria with opposite trends as described above. Moreover, values of both conductivity and chloride ion concentrations linked to salinity exposure via sea sprays, were higher in litters situated at the coastal areas in the three countries, showing that salinity stress was stronger in the areas close to the coastline. We also determined the litter chemical properties at t = 0 from the twelve composite samples of litters (2 species x 2 context (coastal vs inland) x 3 countries) used for litter bag preparation and results are shown in Table 2. Several global trends were observed. Whatever the country or the distance from the sea, pH was always lower in Pinus halepensis litter (from 4.7 to 6.24) than in Pistacia lentiscus litter (from 6.22 to 6.55). Moreover, in P. halepensis litter, total Organic Carbon amounts were always higher (from 2.67 to 4.1 g C/L vs 1.55 to 2.22 g C/L) and this trend was also observed for Total N amounts (from 3.49 to 5.12 g C/L vs 2.41 to 3.18 g C/L). 13 C NMR results showed that the proportion of di-O-alkyl C + O-alkyl C was higher in P. halepensis litter and this was also observed for aromatic + phenolic % in most cases. Thus, litter physico-chemical characteristics depended on the type of species and not on the geographical location (coastal vs inland area, Northern vs Southern side of the Mediterranean).

Three way-ANOVA revealed significant interactions between the coastal conditions and the country for most of the microbial markers considered: cellulase activities, catabolic diversity, basal respiration, and microbial biomass (Table 3).

Cellulases varied with the three factors tested (Figure 1 a andc). However, these activities followed a global trend whatever the species and the area (coastal or inland): Greece > France = Algeria.

For microbial biomass (MB), the effects of coastal conditions varied with the plant species and the country (Figure 1b andd): no differences of MB between coastal and inland areas were observed in P. lentiscus litters whatever the country (Fig 1b), while in P. halepensis litters, higher MB was found in Algeria inland area only (coastal x country interaction, F=8.88, p< 0.001, Fig 1d).

Our results also highlighted variations in catabolic diversity H' (ECO) and basal respiration depending on coastal x country interaction (Figure 2a andb). In coastal area, H' (ECO) was lower in Greece where the more drastic climate conditions were found compared to the two other countries and the opposite trend was observed for BR. Thus, microbial communities with less-diversified catabolic potential led to a higher respiration in the Greek coast. In inland area, conversely, H'(ECO) increased with the gradient of aridity (Algeria>Greece>France), while BR was similar whatever the country (Figure 2a). Thus, for RB, MB and H'(ECO), the coastal conditions modified such important microbial markers depending on the plant species and the country across the Mediterranean basin.

These results have to be considered regarding the chemical properties of litters characterised by solid-state NMR of 13 C. Aromatic compounds were negatively correlated with MB (r 2 =-0.26, p<0.05). These results are corroborated by the three-way ANOVA (Table 3): the amount of aromatic compounds was lower in litter where MB was high (Algerian coast) and conversely, aromatics were more abundant in litter where MB was low (Greek coast) (coastal x country interaction F=1.98, p<0.01). O-Alkyl C signal (assigned to polysaccharides) also varied depending on the coastal x country interaction (F=8.08, p<0.001) and higher O-Alkyl C signal in P. halepensis litter was observed together with high MB.

The effects of the country, the area (coastal or inland) and the plant species were also tested on the catabolic structures of litter microbial communities. PERMANOVA showed significant differences of the catabolic structure of litters between the three countries (pseudo-F=5.23, p<0.001) and between areas (pseudo-F=4.40, p<0.001). Moreover, coastal conditions shaped microbial catabolic profiles depending on the country (pseudo-F=3.43, p<0.01). For microbial genetic structure, the first two principal components (PCs) account for 33 % of the variation in the data; PC1 accounts for 20 % and PC2 for 13 %. PCA revealed significant differences between the three countries (pseudo-F=3.39, p<0.001) and between areas (pseudo-F=9.32, p<0.001), showing that geographical conditions shaped microbial genetic structure and that it differed depending on the coastal and inland areas. Figure 3a andb shows the results of the PCA for the four combinations of area (inland or coastal) and litter (based on either genetic or catabolic profiles) plotted on the first two principal components. For catabolic diversity, the first two principal components (PCs) account for 22 % of the variation in the data; PC1 accounts for 12 % and PC2 for 10 %. For the two plant species considered, catabolic and bacterial genetic structures changed depending on coastal and inland areas (Figure 3 a andb) indicating that the effect of coastal context was stronger than the effect of species (which would have homogenized catabolic profiles for one species). On the other hand, in either inland or coastal areas, both catabolic and genetic structures were different depending on the species showing that this is an important environmental driver at a local scale.

It is noteworthy that no significant effect of the factors tested was found on bacterial genetic richness or diversity assessed via H' index (data not shown).

Effect of litter transfer from inland to coastal area on litter properties

In order to understand how environmental conditions of coastal environments may structure microbial functioning, litters were transferred from inland to coastal area to test our initial hypothesis: environmental conditions acting at a regional scale (coastal vs inland) are preponderant over those acting at a local scale (litter chemical signature specific to a plant species).

First, chemical markers from NMR data showed that, for both litters, the Aromaticity ratio was different between transferred litters and litters from the coastal area (Table 4). Thus, the 'chemical environment' of microbial communities was not similar in transferred and coastal litters for both species and whatever the geographical location.

The effects of litter transfer (mainly negative) to the coastal zone observed on different microbial markers (Table 4) varied with the country and the species. For cellulases, variations after transfer to the coastal zone, were only found in Greece and differed with the species: i) in P. halepensis litters, cellulases decreased significantly (to reach a lower level compared to both coastal and inland activities), ii) in P. lentiscus litters, cellulases increased compared to the original area i.e. inland area (Figure 4 a andb). Thus, in Greece, exposure of litter microbial communities to environmental conditions specific to the coastal area (higher temperatures and more intense drought than in the two other countries as shown in Table 1) led to a modification in activities linked to the transformation of the labile fraction of organic matter and the results observed after this exposure to stress differed with the plant species.

Our results also revealed the strong negative effects of the coastal environmental conditions on MB and H'(ECO) depending on the geographical location (Table 4). This is in Algeria where litter transfer to coastal area strongly affected both microbial markers (data not shown), while no variations were observed in France and Greece after transfer for these markers.

PCA from Biolog data revealed a change in catabolic profiles due to litter transfer to the coastal zone (Figure 5a andb). PC1 and PC2 explained 12.7% and 10.7% of variance for P. lentiscus litter (Figure 5a) and 13.5% and 12.5% for P. halepensis litter (Figure 5b).

PERMANOVA indicated effects of transfer to the coastal area on catabolic profiles in Pinus (pseudo-F=2.64, p<0.001) and P. lentiscus (pseudo-F=2.22, p<0.001) litters. These effects were significant for the three countries in the case of P. lentiscus litters and in Algeria only for P. halepensis litter. Interestingly, microbial catabolic structure after litter transfer in Algeria was similar to that from the coastal area for both P. lentiscus and P. halepensis litters (Figure 5a andb).

PCA based on T-RFLP data also revealed a change in genetic profiles due to litter transfer to the coastal zone (Figure 6a andb). PC1 and PC2 explained 9.1% and 4.9% of variance for P. lentiscus litter (Figure 6a) and 11.2% and 5.1% for P. halepensis litter (Figure 6b).

PERMANOVA indicated significant effects of transfer to the coastal area on microbial communities in P. halepensis (pseudo-F=5.64, p<0.01) and P. lentiscus (pseudo-F=7.41, p<0.01) for the three countries.

Discussion

Variations in litter microbial functioning across an aridity gradient in the Mediterranean basin depends on the distance from the sea

Our study revealed that the effects of the coastal environment differed depending on the geographical location: more precisely, in Greece, coastal conditions led to a specific functioning with higher cellulase and basal respiration and a lower catabolic index of diversity. On the other hand, in coastal environments in France and Algeria, more catabolically-diversified communities were found (higher H' (ECO) index). Moreover, all these differences in microbial functioning were accompanied by different genetic structures in coastal and inland areas of the three countries as described by PCA from T-RFLP data. This may be explained by contrasted climate conditions between coastal and inland environments in Northern vs Southern part of Mediterranean basin as described above. Summer drought is indeed more drastic in coastal area in Greece, while higher temperatures are found in the inland area in Algeria. Moreover, additional osmotic stress in the coastal area (as revealed by conductivity and chloride ion concentrations) has probably reinforced the stronger drought constraints found in Greek coastal area. [START_REF] Rath | Salt effects on the soil microbial decomposer community and their role in organic carbon cycling: A review[END_REF] indicated that additional salt stress to drying exerts a stronger effect on microbial growth and moreover a shift in microbial structure (assessed via PLFA) after salt treatment is induced. Our recent study (Kheir et al., 2019) also sustained such findings since osmotic stress, experienced by microbial communities from coastal environments, modified differently catabolic structure of litter microbial communities after drought stress. [START_REF] Rajaniemi | Abiotic conditions and plant cover differentially affect microbial biomass and community composition on dune gradients[END_REF] also showed that microbial community composition and biomass can be strongly modified by abiotic stress such as salinity and provoked a shift towards a higher proportion of Gram-negative bacteria.

Here, our findings also underlined that the type of litter shaped microbial communities: i) at a local scale i.e. in either coastal or inland areas, certain microbial markers (catabolic profiles, cellulase activities) varied with the type of plant species, ii) at a regional scale, the effect of coastal conditions differed with the plant species (variations in BR, H'(ECO), cellulases and catabolic profiles). This goes in line with our previous study [START_REF] Da Silva | Distance from the sea as a driving force of microbial communities under water potential stresses in litters of two typical Mediterranean plant species[END_REF], which demonstrated that catabolic diversity varied with litter type under water potential stresses linked to coastal environments. [START_REF] Sherman | Microbial functional diversity associated with plant litter decomposition along a climatic gradient[END_REF] studied catabolic potential of litter microbial communities across an aridity gradient in Israel, from the Mediterranean shore to the arid zone. They also found a shift in litter microbial catabolic structure explained by plant traits. Moreover, their study revealed that low organic matter input in the arid zone -because of low plant productivity-induced a diversification of resource utilization. These findings are of importance since modification in catabolic potential of microbial communities may induce a shift in decomposition process since transformation of certain compounds such as lignin is sensitive to the structure of microbial communities [START_REF] Mc Guire | Microbial communities their relevance for ecosystem models: decomposition as a case study[END_REF].

Modification of litter microbial functioning when microbial communities from inland area are subjected to coastal environmental conditions

After transfer of litters from inland to coastal area, certain microbial markers, such as cellulase activities, were strongly impacted and the intensity of the effect varied with the plant species. Our initial hypothesis was based on the fact that coastal conditions may have smoothen the effect of litter type leading to homogeneous microbial responses to changes in environmental climate conditions whatever the plant species. Thus, our study underlined the importance of species traits in microbial responses to stress. The quantity and quality of organic matter shape the structure and composition of microbial communities and their resistance to stresses [START_REF] Bradford | Microbiota, fauna, and mesh size interactions in litter decomposition[END_REF]. Mediterranean litters are described as particularly rich in secondary metabolites (terpenes, tannins, VOC …) and lignins [START_REF] Di Castri | Climatographical Comparisons between Chile and the Western Coast of North America. Mediterranean Type Ecosystems Ecological Studies[END_REF][START_REF] Rousk | Soil bacterial and fungal communities across a pH gradient in an arable soil[END_REF][START_REF] Terradas | The influence of summer and winter stress and water relationships on the distribution of Quercus ilex L. Quercus ilex L. Ecosystems: function, dynamics and management Advances in vegetation science[END_REF]. These compounds are known to modify microbial communities and their activities: they can be toxic for microbial cells [START_REF] Popova | Terpenes with antimicrobial activity from Cretan propolis[END_REF][START_REF] Adamczyk | Monoterpenes and higher terpenes may inhibit enzyme activities in boreal forest soil[END_REF], interact with enzymes, leading to adverse effects on their catalytic potential (inhibition/precipitation of enzymes). Some tannins can modify the quantity and the nature of N sources available for microorganisms because of adsorption or enzymatic inhibition (Hättenshwiler and Vitousek, 2000). Thus, the importance of litter chemical signature on microbial community structure and functioning has to be taken into account when studying soil microbial vulnerability under environmental constraints. More particularly, the effect of litter admixture on microbial responses to coastal condition exposure should be valuable to determine since the diversification of nutrient resources is known to enhance microbial diversity and consequently may favour resistance to water potential stress. These subtle variations in organic matter can indeed lead to additive or non-additive (synergistic or antagonistic) effects [START_REF] Brunel | Additive or non-additive effect of mixing oak in pine stands on soil properties depends on the tree species in Mediterranean forests[END_REF] and various plant species exhibiting different functional traits should be included in such research [START_REF] Prescott | Tree species influence on microbial communities in litter and soil: current knowledge and research needs[END_REF].

Previous studies have indeed shown that changes in plant species can actually modify both the biomass and structure of soil microbial communities [START_REF] Laganière | Linking the abundance of aspen with soil faunal communities and rates of belowground processes within single stands of mixed aspenblack spruce[END_REF][START_REF] Matos | Organiccarbon and nitrogen stocks and organic-carbon fractions in soil under mixed pine and oak forest stands of different ages in NE Germany[END_REF] and thus their responses to stress.

Coastal environmental conditions appeared to have induced a shift in microbial catabolic structure of both litters, mainly in Algeria. Interestingly, in P. halepensis litter, the catabolic profile resulting from transfer in the Greek coastal area differed from catabolic profiles of both inland and coastal areas (control and transfer area respectively). These results may partially explain the decrease in cellulase activities after transfer: the shift in catabolic diversity of microbial communities after transfer may have not sustained such activities. On the other hand, in P. lentiscus litter, the potential of cellulose transformation was identical in the transferred litter and in litter from the coastal area. Some studies have attempted to decipher the respective influence of either climate conditions or chemical signature of organic matter, using very contrasted bioclimates. [START_REF] Gholz | Long-term dynamics of pine and hardwood litter in contrasting environments: toward a global model of decomposition[END_REF] and [START_REF] Perez | Home-Field Advantage: A matter of interaction between litter biochemistry and decomposer biota[END_REF] indicated that the effect of 'home field advantage' is particularly pregnant for microbial communities from litters with a high amount of cellulose and hemicellulose. The study of [START_REF] Makkonen | Highly consistent effects of plant litter identity and functional traits on decomposition across a latitudinal gradient[END_REF] revealed utterly different results: transfers of 16 types of litters from four different biomes (subarctic, temperate, Mediterranean and tropical climate) led to a homogeneous dynamics of litter transformation. Moreover, the authors found that the complexity of decomposer communities including micro-, meso and macro fauna (using various mesh size of litterbags), does not accelerate decomposition rate and this is particularly true for the Mediterranean biome. This means that decomposers from a wide range of bioclimates are not specialized in the transformation of 'autochthonous' organic matter. Opposite results were found in the study of [START_REF] Milcu | All size classes of soil fauna and litter quality control the acceleration of litter decay in its home environment[END_REF], which showed that a range of soil fauna size classes contributes to 'home field advantage' effect and that experimental bias (e.g. fine mesh sizes) impacts the contribution of soil fauna. It has to be underlined that our study also involved micro and mesofauna since 2 mm mesh-size litterbags were used. We found that the concept of 'home field advantage' applied to cellulose transformation depended on the litter type. For P. halepensis, this potential clearly depended on the coastal conditions since, when microbial communities from inland areas were transferred to the coastal zones, their potential to hydrolyze cellulose strongly decreased.

Transfer experiment of litters also showed that major microbial markers such as MB and the catabolic index of diversity, H' (ECO), were differently affected depending on the geographical location: after transfer, H' (ECO) decreased in Algeria and not in France or Greece, and MB decreased in Algeria. Thus, this revealed that the contrasted climate conditions across the Mediterranean Basin specialized microbial responses when facing a change in environmental conditions (transfer from inland to coastal area). Piélou Index of diversity based on T-RFLP also underlined that litter transfer led to a decrease in genetic diversity (whatever the geographical location or the litter type). This study indicates that coastal conditions have probably shaped a particular microbial functioning and that microorganisms from other environments have to adapt to these new specific conditions. However, more than the chronicity of the stress linked to sea-spray exposure, the effect of pulses of salinity increase (due to seasonal variations i.e. wind intensity, precipitation…) is probably a more challenging factor for microorganism resistance [START_REF] Schimel | Microbial stress-response physiology and its implications for ecosystem function[END_REF]. Our previous study [START_REF] Qasemian | How do microlocal environmental variations affect microbial activities of a Pinus halepensis litter in a Mediterranean coastal area?[END_REF] indeed revealed variations in conductivity in coastal environments over the year (higher conductivity in summer because of drought). As discussed above, the recent study of [START_REF] Rath | The impact of salinity on the microbial response to drying and rewetting in soil[END_REF] evoked the importance of salinity in enhancing drought exposure impact on soil microorganisms.

Whatever the geographical location and the species, after transfer of litterbags, a shift in the genetic structure was observed and differed from those of both inland and coastal areas. These findings were observed after a 10-month exposure in the new habitat i.e. costal area and this revealed that these variations in environmental conditions are likely to shape differently microbial communities over long periods. This also showed that acclimation of litter microbial communities, in terms of diversity, to other environmental conditions was quite a long-term process. [START_REF] Pesaro | Impact of soil drying-rewetting stress microbial communities and activities and on degradation of two crop protection products[END_REF] found that after a drought stress, functional properties such as respiration, are rapidly recovered, while the initial levels of diversity and biomass are not reached one month after stress. [START_REF] Schimel | Microbial stress-response physiology and its implications for ecosystem function[END_REF] suggested that, while physiological effects of water potential stresses are supposed to drive short-term microbial responses, the modifications in microbial community structure and composition are likely to occur over a longer period. Moreover, these authors assume that microbial diversity in soil should not be considered as a juxtaposition of 'functional groups' but results from the interaction between these functional groups (such as denitrifiers, lignocellulose decomposers…) and from their resistance to variations in environmental conditions and stresses of different types.

This study revealed that litter microbial communities from inland and coastal areas had different genetic and catabolic structures. [START_REF] Crits-Christoph | Colonization patterns of soil microbial communities in the Atacama Desert[END_REF] found that conductivity was one of the major drivers of soil microbial genetic diversity in arid environments. The study of [START_REF] Qasemian | How do microlocal environmental variations affect microbial activities of a Pinus halepensis litter in a Mediterranean coastal area?[END_REF], who tested the effect of coastal conditions on microbial activities at a microlocal scale, also revealed modifications of microbial catabolic structure in litters depending on the distance from seashore. The understanding of area-species diversity relationships is of major importance since it allows prediction of the impact of environmental changes such as more intense climate stresses [START_REF] Decaëns | Macroecological patterns in soil communities[END_REF]. Moreover, the coastal conditions affected major microbial properties across a gradient of aridity in the Mediterranean basin depending on plant litter species, indicating the huge importance of litter chemical signature even at wide spatial scale. Since both microbial diversity and enzyme activities are of major importance in sustaining organic matter turn-over, our results contributed to understand better the relative importance of external filters to shed light on litter microbial functioning in changing environments.

Table 1 Location of the areas where litter bags were left in the three countries, environmental parameters monitored monthly in situ over a two-725 year period and climate variables from worldclim data [START_REF] Hijmans | Very high resolution interpolated climate surfaces for global land areas[END_REF]. 4 Results of the three-way ANOVA to test the effects of the plant species (Pinus halepensis vs Pistacia lentiscus), the transfer (control vs transferred litters), the geographical location and their interactions on the microbial and chemical markers from control and transfer litter bags.

  : i.e. alkyl C (0-45 ppm), methoxyl C (45-60 ppm), O-alkyl C (60-90 ppm), di-O-alkyl C (90-110 ppm), aromatic C (110-140 ppm), phenolic C (140-160 ppm) and carboxyl C

  BR: Basal Respiration, MB, Microbial biomass, H' (ECO): Shannon-Weaver index from catabolic profiles, R (T-RFLP), richness from T-RFLP data, H' (T-RFLP): Shannon-Weaver index from T-RFLP.

Figure 1 a

 1 Figure 1 a and b Cellulase (A and C) and microbial biomass (B and D) from control litter

Figure 2

 2 Figure 2 Shannon-Weaver Index from catabolic diversity (A) and Basal Respiration (B) of

Figure 3 a

 3 Figure 3 a and b PCA from bacteria T-RFLP fingerprints (A) and catabolic profiles from

Figure 4

 4 Figure 4 Cellulase in litter bags from inland control (I), coastal control (C) and transfer from

Figure 6 a

 6 Figure 6 a and b PCA from T-RFLP fingerprints for Pistacia lentiscus (A) and Pinus

Figure

  Figure 1 a and b

Table 2

 2 Chemical characteristics of the composite samples of litters which were used to prepare litter bags for each environmental condition (coastal and inland areas) and geographical location (France, Greece, Algeria).

			France				Greece			Algeria
			coastal	inland		coastal		inland	coastal	inland
		P h	P l	P h	P l	P h	P l	P h	P l	P h	P l	P h	P l
	TOC (mg C/kg)	2851.9	1557.71 2951.67	1855.96 4104.09	1756.91 2647.36	2225.68 2556.62	1694.72 2675.63	1593.12
	Total N (mg N/kg)	88.0	102.4	65.6	69.8	52.0	61.0	55.0	110.0	77.6	97.0	48.2	53.0
	NH4 + (mg N/kg)	0.48	0.63	0.46	0.52	0.26	0.55	0.38	0.85	0.59	0.44	0.27	0.28
	pH	6.16	6.55	6.22	5.76	5.88	6.27	5.7	6.19	5.55	6.46	6.15	6.54
	Conductivity (mS/cm) 1557	2020	590	640	1634	982	701	641	954	830	630	690
	Cl -(%)	2.80	3.62	1.04	1.13	2.92	1.75	1.25	1.40	1.70	1.47	1.11	1.22

Table 3

 3 Results of the three-way ANOVA to test the effects of the plant species (Pinus halepensis vs Pistacia lentiscus), of the environmental condition (coastal vs inland area), the geographical location (France, Greece and Algeria) and their interactions on the microbial and chemical markers from control litter bags. BR: Basal Respiration, MB: Microbial biomass, H' (ECO): Shannon-Weaver index from catabolic profiles, R (T-RFLP), richness from T-RFLP data, H' (T-RFLP): Shannon-Weaver index from T-RFLP.

	Control	Df BR		MB	cellulase	H'	COOH	Aromatic	O-alkyl-C	Alkyl-C
	Species	1	4.82*	1,23	40.51***	0,009	202.39***	3,06	23.01***	17.03***
	Context	1	2,45	5.95*	11.37**	3,62	2,65	1,68	3,27	10.15**
	Geographical location
	(GL)	2	0,39	1,35	108.20*** 13.31***	14.54***	0,8	1,74	2,27
	Species* GL	1	0,02	7.32**	7.73***	1,16	36.42***	33.12***	0,01	0,28
	Species* Context	2	0,4928	4.49*	16.66***	0,67	12.06***	6.43**	7.13**	6.76**

Table

Acknowledgements

This work was supported by EC2CO (CNRS INEE) program MICLIMED. Many thanks to the Labex OT-Med scientific network (n° ANR-11-LABX-0061) for very useful discussions about global change. This work has received funding from Excellence Initiative of Aix-Marseille University -A*MIDEX, a French "Investissements d'Avenir" programme. The authors wish to thank two students of the Université Paris-Est Créteil (UPEC), Parel Makoudie Noubimboo and Marylène Rugard. for their contribution to this project and their enthusiasm in the laboratory. Last but not least: many thanks to Frédérique Peter-Valence, Tiphany Bouriaud, Manuel I. Da Silva Guardado, Dr Sevastianos Roussos and Jean Marc Pentegno for their very useful technical assistance to this project. The Biolog EcoPlate™ (BIOLOG Inc., Hayward, CA) equipment was acquired thanks to a financial support by ANR project MARSECO (ANR-CESA-018-06).

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.


Lucian Florin

Constantin
email: fl.constantin@univ-lille1.fr

Sensing Multifrequency THz-waves with a Photomixer

The nonlinear electrical response from a low temperature grown GaAs photomixer driven with the optical beat of two continuous-wave lasers is exploited for frequency comb generation and phase-coherent THz frequency comb heterodyne detection.

Introduction

The terahertz-waves are useful for several applications including biological imaging, remote sensing, high-speed telecommunications, and security screening [START_REF] Dhillon | The 2017 terahertz science and technology roadmap[END_REF]. The optical frequency down-conversion with a photoconductive antenna (photomixing) was exploited for continuous-wave (cw) THz generation [START_REF] Brown | Photomixing up to 3.8 THz in low-temperature-grown GaAs[END_REF] by driving an antenna with the photocurrent generated by beating two cw lasers in an ultrafast photoconductor. Compact, room-temperature operated, cost-effective and low-power consuming THz photomixing systems with optical frequency control enabled narrow spectral linewidth, wide frequency tunability, high dynamic range and high signal-to-noise ratio. Here, the electrical nonlinear response of a low temperature grown GaAs (LTG-GaAs) photomixer is exploited for frequency comb generation and detection. Multifrequency photonic generation is demonstrated by photomixing with a modulated bias voltage [START_REF] Constantin | Direct-current modulation of a photomixing signal[END_REF]. Alternatively, a THz frequency comb is generated by driving a solid-state electronic frequency multiplier with a pulsed microwave synthesizer [START_REF] Constantin | Photomixing for coherent retrieval of THz waveforms from a frequency multiplier[END_REF]. The heterodyne detection with the photomixer exploits the optical beat as local oscillator to down-convert the THz frequency comb to the microwave domain [START_REF] Constantin | Photomixing for coherent retrieval of THz waveforms from a frequency multiplier[END_REF][START_REF] Constantin | Phase-coherent heterodyne detection in the terahertz regime with a photomixer[END_REF].

Results

The photomixing model developed here is based on uniform unidimensional carrier transport in a thin LTG-GaAs layer. Two spatially superposed coherent optical waves, detuned by an angular frequency ω and carrying the same optical power P enter at normal incidence in the photoconductor and generate a current density expressed as:

( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ⎥ ⎥ ⎦ ⎤ ⎢ ⎢ ⎣ ⎡ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ωτ + ω + ω ωτ + τ + ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ωτ + ω + ω ωτ + τ ϖ η = 2 2 1 1 1 1 2 E t cos t sin E E v E E t cos t sin E E v E Ad P e t j p p p p n n n n h (1)
The laser beams, carrying nearly the same energy , make an optical beat that is focused on a photoconductive area A, and absorbed within a depth d with a quantum efficiency η. The dependences of the carrier lifetimes τ ϖ h n,p (E) with the electric field [START_REF] Zamdmer | Increase in response time of low-temperature-grown GaAs photoconductive switches at high voltage bias[END_REF] are described here with a linear electric field dependence arising from carrier heating :

( ) ( ) [ ] T k eE E B p , n p , n 2 λ 3 1 τ τ 0 0 + ≈ (2)
in function of the Boltzmann constant k B , the electron charge e, the room temperature T=300 K, the low-field carrier lifetimes τ 0n,0p and the distance λ between Coulomb barriers in LTG-GaAs. The carrier drift velocities have a nonlinear dependence on the electrical field [START_REF] Brown | A photoconductive model for superior GaAs THz photomixer[END_REF][START_REF] Brown | Milliwatt output levels and superquadratic bias dependence in a low-temperature-grown GaAs photomixer[END_REF], expressed as :

( ) ( ) [ ] E v v E v p , n sp , sn sp , sn p , n 0 0 µ 1+ = (3)
with the saturation velocities v sn,sp and the low-field mobilities µ 0n,0p of the electrons and holes. The electric field in the photoconductor is expressed in the parallel-plate approximation as an alternative electric field (amplitude E AC =V AC /r, angular frequency ω AC , electrode spacing r) superposed to a bias electric field (E DC =V DC /r ) :

( ) ( ) ( ) (4)
t cos r V r V t E AC AC DC ω × + =
The electric field is applied to the photomixer electrodes either by modulating the bias voltage or by an incoming THz radiation received by the antenna. The electrical nonlinearity arising from v n,p (E) and τ n,p (E) dependences yields a current density with spectral components at angular frequencies ω±nω AC (n integer). The amplitudes and phases of the spectral components of the frequency comb are analytically calculated with eq. (1-4) for a given E DC using a power series expansion in terms of E AC . Particularly, phase-coherent THz frequency comb down-conversion may be performed by heterodyning a multifrequency THz-wave with an optical beat exploited as local oscillator (LO).

The experiment (Fig. 1.A) is based on a LTG-GaAs photomixer with a log-spiral antenna driven by the optical beat of two extended cavity diode lasers emitting at 820 nm. The extremities of the antenna are connected to a coaxial microwave line addressed with a bias-T, which is used for coupling the modulation signal and the bias voltage to the photomixer and to measure its response with a microwave spectrum analyser. A 75-110 GHz electronic frequency multiplier driven by a microwave synthesizer is used to generate a THz-wave which is focused on the photomixer antenna with a silicon lens. On one hand, the modulation of the photomixing current is observed by coupling a radiofrequency signal to the photomixer driven by an optical beat in the microwave domain. The spectrum of the photomixing signal (Fig. 1.B) displays the carrier and sidebands spaced by the modulation frequency. The amplitudes of the spectral components are calculated with eq. (1-4) using the modulation parameters, the LTG-GaAs photoconductor parameters from refs. [START_REF] Zamdmer | Increase in response time of low-temperature-grown GaAs photoconductive switches at high voltage bias[END_REF][START_REF] Brown | A photoconductive model for superior GaAs THz photomixer[END_REF][START_REF] Brown | Milliwatt output levels and superquadratic bias dependence in a low-temperature-grown GaAs photomixer[END_REF], and the photomixer parameters indicated below. The results are multiplied with a common factor in order to match the amplitudes of the most intense spectral components and plotted with bars. The calculated and measured amplitudes agree for ω±2ω AC sidebands but not for the others. The model may be improved by taking into account spatial dependence of the current density, optical power and spatial charge separation effects in the photoconductor. On the other hand, the photomixer is exploited for heterodyne detection of the THz-waves generated with a frequency multiplier driven by a pulse-modulated microwave synthesizer (Fig. 1.C). A monochromatic THz-wave is down-converted to ~1.1 GHz, and recorded with a signal-tonoise ratio of 10 in 1 MHz bandwidth. The responsivity is at the 10 -3 A/W level. The linewidth of the signal is related to the spectral purity of the free-running diode lasers. When the microwave synthesizer is pulse-modulated, the spectral components of the resulting THz frequency comb are down-converted by the heterodyne detection scheme and individually resolved. The heterodyne detection signal is sensitive to the THz-wave amplitude and phase and, via antenna's geometry, to the THz-wave spatial mode and polarization state. The photomixing approach with an ultrafast photoconductor enables integration of the optical beat generation with the electrically-driven optoelectronic frequency conversion in a single photonic circuit for THz frequency comb generation and detection.

Fig. 1 .

 1 Fig. 1. A. The experimental setup. DL1, DL2 diode lasers, SA spectrum analyzer, LF radiofrequency synthesizer, MW microwave synthesizer. B. Spectrum of a modulated photomixing signal. Optical power 2 × 20 mW, optical beat at ω = 2π × 1178 MHz. Modulation voltage V AC =0.5 V at ω AC = 2π × 2 MHz, bias voltage V DC =0.517 V. The calculated amplitudes of the spectral components are plotted with blue bars. Photomixer parameters used in the calculations: r=2 µm, d=2 µm, A=12.6 µm 2 , e ϖ h =1.5 V, η=0.68. RBW=300 kHz, sweep time 4 ms, 2 video averages. C. Spectrum of the heterodyne detection signal of a THz-wave at ω AC = 2π × 98.4 GHz (lower panel, red line). Down-converted THz frequency comb generated with pulse modulation at 6 MHz with 50% duty cycle (upper panel, blue line). Bias voltage V DC =85 mV. Optical beat power 2×20 mW. RBW=1 MHz, sweep time 4 ms, 16 video averages.