Cosmin Saveanu
email: cosmin.saveanu@pasteur.fr

Minimal perturbation analysis of mRNA degradation rates with Tet-off and RT-qPCR

Keywords: RNA degradation, yeast, Saccharomyces cerevisiae, repressible promoter, RT-qPCR, exponential decay

Messenger RNA stability is an important variable in gene expression and its dynamics. High stability ensures a constant level of synthesized protein, whereas mRNA instability can be critical for regulatory processes in which protein production needs to be stopped, such as development, inflammation or adaptation to stress. Accurate measurements of RNA degradation rates are important for understanding how RNA features and RNA binding proteins affect the post-transcriptional life of an mRNA. As an alternative to global transcriptional inhibition methods, the use of a Tet-off repressible promoter has the advantage that cells are minimally perturbed by the addition of doxycyclin during the assay. We illustrate the use of a reporter mRNA expressed from a plasmid in Saccharomyces cerevisiae cells, but similar methods can be applied to other regulated promoters, on plasmids or by genome editing, and in other organisms. RNA levels are measured by reverse transcription followed by quantitative PCR. An exponential decay law is then used to estimate how well the measurements follow this expected trend for the simplest possible mechanism of RNA degradation, where the decay is proportional to the amount of RNA present at any given time.

Introduction

The stability of an mRNA has a profound impact on its steady-state level at a given transcription rate. Therefore, methods for estimating how long an mRNA is present in a cell and identifying the mechanisms responsible for both stability and degradation of mRNAs, are important for understanding gene expression. Many methods can be used to estimate mRNA degradation rates, but the one presented here requires relatively simple equipment and is also inexpensive. Its main disadvantages are the fact that it can only estimate bulk mRNA degradation, the 5' untranslated region of the mRNA of interest is not the native one, and it can only be applied to individual reporters. By comparison, other methods, such as high-resolution microscopy can track individual mRNA molecules in specific subcellular compartments. For example, labeling of the nascent protein and of the 3' untranslated region of a reporter mRNA can estimate the dynamics of RNA degradation during nonsense-mediated mRNA decay [START_REF] Hoek | Single-molecule imaging uncovers rules governing nonsense-mediated mRNA decay[END_REF]. Single-molecule tracking is not without caveats, as it requires specific RNA elements that modify the mRNA of interest and may also affect its stability or translation. In addition, these elements may also be resistant to degradation of the mRNA fragments generated from the reporters, leading to erroneous estimates of half-life values. Recently, technical solutions have been proposed to prevent such artefacts [START_REF] Tutucci | An improved MS2 system for accurate reporting of the mRNA life cycle[END_REF][START_REF] Li | An improved imaging system that corrects MS2-induced RNA destabilization[END_REF].

To avoid altering the mRNA sequence, metabolic labeling methods have the advantage of estimating mRNA stability for thousands of transcripts in parallel. The level of incorporation of such nucleotides indicates the balance between mRNA synthesis and degradation. Initially based on a relatively challenging and error-prone purification of modified RNA molecules [START_REF] Cleary | Biosynthetic labeling of RNA with uracil phosphoribosyltransferase allows cell-specific microarray analysis of mRNA synthesis and decay[END_REF][START_REF] Dölken | High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay[END_REF], variants of pulse-chase labeling of mRNA use now chemical modification of incorporated nucleotides that can be detected by short reads sequencing [START_REF] Herzog | Thiol-linked alkylation of RNA to assess expression dynamics[END_REF].

Between microscopy and metabolic labeling, the most widely used method for estimating mRNA degradation rates involves the inhibition of its synthesis and monitoring mRNA levels at different time points. Global inhibition of transcription, which is achieved in mutants that affect the function of RNA polymerase II, or chemical inhibition of transcription with drugs such as ortho-phenantrolin or thiolutin, can have various secondary effects because a stress response can be induced by such treatments (e.g. Zencir et al, 2020).

Inhibiting transcription of a single gene by using a specific promoter inserted upstream of the coding region minimally disrupts the cellular metabolism, including

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general translation and RNA degradation factors. A widely used method for rapid repression of gene expression uses a chimeric transcription factor consisting of the bacterial tet repressor fused to the transcription activating domain of the herpes simplex virus [START_REF] Gossen | Tight control of gene expression in mammalian cells by tetracycline-responsive promoters[END_REF]. However, the promoter region and the corresponding 5' untranslated part of mRNA can affect the translation and degradation of the mRNA under investigation. This can be partially avoided by careful analysis of the promoter region to insert the tet operator sequences upstream potential TATA box, for example [START_REF] Baudrimont | Multiplexed gene control reveals rapid mRNA turnover[END_REF]. The decrease in mRNA levels can also depend on its nuclear to cytoplasmic distribution, especially if export from the nucleus is slow. Another caveat of using reverse transcription followed by qPCR, as described here, is that the signal observed may not correspond to full length molecules, but, for example to abnormally stable mRNA fragments. An alternative way of looking at mRNA degradation that does not have this problem is Northern blotting, as described elsewhere in this volume. For a comprehensive evaluation of methods allowing the evaluation of mRNA stability, see the review by Wada and Becskei (Wada & Becskei, 2017).

Here, I present the protocol that we use for Tet-off-based mRNA degradation rate estimation using a low copy (centromeric) plasmid expressing the mRNA of interest from the pCM189 backbone [START_REF] Garí | A set of vectors with a tetracyclineregulatable promoter system for modulated gene expression in Saccharomyces cerevisiae[END_REF] in yeast. The levels of the target mRNA are measured over time using reverse transcription and quantitative PCR and the results are fitted to an exponential decay model to calculate the mRNA degradation rate and its associated half-life.

Materials

2.1 Yeast growth media 1. YPD medium: 2% D-glucose, 1% yeast extract, 1% Bactopeptone. Autoclave at 110°C for 20 minutes.

2. SD-URA medium: 2% D-glucose, 6.7g/L yeast nitrogen base containing ammonium sulfate, without amino acids, 1.92 g/L drop-out mix without uracil. Autoclave at 110°C for 20 minutes. Alternatively, filter sterilize.

Cell lysis and RNA extraction

1. Lysis buffer: 10 mM Tris-HCl, pH 7.5, 10 mM EDTA, 0.5% SDS. Stored at room temperature.

2. Phenol-chloroform-isoamyl alcohol (25:24:1), pH 5.2. Store at 4°C.

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3. Ethanol-Ammonium acetate mix (6 volumes of ethanol for 1 volume of 7.5M ammonium acetate. Store at -20°C.

Ehanol 70% (vol/vol

). Store at room temperature.

4. Thermomixer of dry bath that can hold 65°C for 1.5 ml Eppendorf tubes. 5. Optimized DNase I and its buffer containg Mg2+ and Ca2+ ions (e.g. Turbo Dnase).

6. Spectrophotometer for small volumes working at 260 nm (e.g. Nanodrop).

7. Cell culture incubator with shaking (30° C).

8. Doxycyclin 1000x stock solution at 10 mg/ml in ethanol (store at -20°C). 9. Refrigerated centrifuge for 50 ml and 1.5 ml tubes.

Reverse transcription and qPCR

1. Thermal cycler. 2. qPCR system for microplates and its associated software.

Analysis of results and half-life estimation

1. Computer system with R, Rstudio and MASS package installed (reshape2 is optional).

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3 Methods

Cell culture and recovery

1. Grow cells in synthetic SD-URA medium, if the reporter mRNA is expressed from a URA3 marker containing plasmid. Dilute cells grown in selective medium overnight diluted in 300 ml YPD medium at a 600 nm to an initial absorbance of 0.15.

2. Incubate the cells with agitation at 30°C for about 4 hours, until the 600 nm absorbance reaches 0.5. 7. Transfer the cells using 1 ml of ice cold water in an Eppendorf tube. Centrifuge 30 seconds at 10 000 g. Remove supernatant and store the tubes at -80° C or process immediately.

Total RNA extract preparation with hot phenol

1. Suspend the pellet by pipetting up and down 400 μl lysis buffer. Add 400 ul phenol/choloroform/isoamyl alcohol mix. Vortex for 10 seconds.

2. Transfer the tubes to a dry block at 65°C and incubate for 30 minutes with occasional mixing.

3. Move the tubes on ice and incubate for 5 minutes.

4. Separate phases by centrifugation at 14 000 g for 5 min., 4° C. 5. Recover the aqueous supernatant and perform two additional phenol-chloroform extractions.

6. Final aqueous RNA extract is mixed in a 1 to 3.5 volumetric ratio with chilled ethanol/ammonium acetate mix. Incubate at 4°C for 10 minutes.

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7. Recover precipitated RNA by centrifugation at 14 000 g for 15 min in a refrigerated centrifuge (4° C).

8. Wash the pellet with 800 μl ethanol (70%) twice, using 5 minutes of centrifugation to recover the pellet at each wash. 9. Remove all the ethanol from the wash step and leave the pellet to air dry until their appearance becomes translucent. 10. Add 50 μl of water and resuspend the pellet by vigorous pipetting. Optionally, incubate the tubes at 65°C for 5 minutes to ensure that all the RNA has been solubilized.

11. Measure absorbance at 260 nm to estimate the amount of recovered RNA. Adjust the concentration of all tubes to 2 μg/μl. Store the RNA samples at -80°C.

Reverse transcription and qPCR

1. Use 10 μg of total RNA for DNase treatment in a 15 μl total volume, together with DNase buffer and modified DNase I (2 units). Incubate 30 min. at 37°C 2. Add 85 μl water, to increase the working volume and perform a phenol-chloroform extraction.

3. To the recovered aqueous phase, add 8 μl linear polyacrylamide solution (40 μg) and 3.5 volumes ethanol/ammonium acetate mix. Incubate on ice for 10 minutes.

4. Recover the DNase treated RNA by centrifugation for 20 min. at 14 000g at 4°C. 5. Wash once with 0.8 ml 70% ethanol. After drying the pellet, suspend in 10 μl cold water.

6. Use 2 μl of DNase treated RNA to set up the annealing mix for the reverse transcription reaction. The 5 μl total reaction volume, in a 0.2 μl PCR tube, should contain 1 μl 5X RT buffer and specific oligonucleotides for the reaction, 0.5 μl of a 5 μM solution for each NOTE2 . A control reaction, without oligonucleotides, is optional but recommended. An additional control, without RT, can also be performed.

7. Prepare a 2X mix containing the reverse transcriptase. For 5 μl of mix, use 1 μl 5X RT buffer, 1 μl 0.1M DTT, 0.3 μl of a 25 mM dNTP mix (each nucleotide at 25 mM), 0.05 μl Actinomycin D (10 mg/ml), 2.3 μl water, 0.35 μl reverse transcriptase ( e.g. Superscript III or IV at 200 U/μl). Keep on ice.

8. Use a thermocycler to incubate the annealing mix for 5 min. at 85°C, followed by 20 min. at 65°C and 10 min. at 42°C.

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9. While the tubes are in the thermocycler, add 5 μl of reverse transcriptase mix and mix by pipetting up and down. 10. Incubate for 30 min. at 42°C and inactivate the enzyme by an incubation for 10 min. at 70°C. Recover the tubes and place them on ice. The RT reaction can be stored at -20°C. 11. Quantitative PCR reactions are done on serial dilutions of the cDNA obtained in the previous step. An example of serial dilution starts with 5 μl of cDNA in 75 μl of water, followed by dilutions of 10 μl of this initial solution with 70 μl of water, three times. The obtained dilutions differ by a factor of 8, leading to differences in the number of cycles required to get to a threshold fluorescence of about 3.

12. Mix 5 μl cDNA dilution with 20 μl of SYBR Green mix, which contains 12.5 μl of 2X commercial 2X dNTP, enzyme and SYBR Green, 1.25 μl of 10 μM solution for each of the two PCR primers and 5 μl H 2 0.

13. Perform the qPCR reaction. Typical conditions are 95°C for 5 min. followed by 40 cycles of 20 sec. at 95°C and 1 min. at 60°C, with a fluorescence reading at the end of each cycle. An optional melting curve can be also programmed. 4. Use the nls function included in R to estimate the first order kinetic constant for an exponential decay model NOTE5 . This function uses a named vector for the initial estimates of the estimated parameter, that can be expressed using the following commands: initialestimate <-c(initial_k); names(initialestimate) <-c("k"). Starting with an initial_k of 0.1 is reasonable for relatively fast RNA degradation experiments (initial_k <-0.1 before setting the initialestimate variable).

Data analysis and RNA half-life estimation

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5. To enter the nls formula, it is useful assign x to the time values and y to the fraction of RNA remaining at each time point: x <-mydf$time; y <-mydf$fraction. The formula is: m <-nls(formula = y ~ exp-(k*x), start=initialestimate). The result noted "m" contains the fitted parameter. The constant "k" can be recovered from the fitted model through k_estimated <-coef(summary(m))[1].

6. For a visual estimation of how well the results fit the observations, compute the theoretical expectation for the fraction of remaining RNA and overlay the curve on the data points, as illustrated in Fig. 1B. A times variable, from 1 to 30, is generated with: times <-seq(from=0, to=30, by=1), and is combined with computed fraction of remaining RNA: estfraction <-exp(-times*k_estimated). predicteddf <data.frame(time=times, predicted=estfraction). The results are overlayed on a plot of the experimental observations, either as fraction or percentage of value at time 0.

7. Especially if several replicates have been done, but even for a single experiment, obtain an idea of how precise the estimate for k is, by using the confint function of the MASS package. confm <-confint(m, level=0.95). The two value stored in confm correspond to the lower and upper estimate for k, with a 95% confidence. 8. Finally, computing an estimated half-life from the decay constant is straightforward, as they are linked through the formula: half-life=ln(2)/k NOTE6 .

Notes

1. The design of the oligonucleotides for qPCR can be done using specialized tools, such as the one provided by the Potsdam University [START_REF] Arvidsson | QuantPrimea flexible tool for reliable high-throughput primer design for quantitative PCR[END_REF]: https://quantprime.mpimp-golm.mpg.de. For spliced transcripts, oligonucleotides that cover exon-exon junction might be preferred, as they will not amplify genomic DNA contamination. The PCR fragments amplified for qPCR are generally short, in the 60 to 100 nucleotides range.

2.

While random hexamers of heptameric oligonucleotides can be used for reverse transcription, specific oligonucleotides help for a better sensitivity. If random hexamers are used (1 μl of a 100 μM solution for each reaction), the annealing reaction conditions should be adjusted, by incubating the annealing mix for 5 minutes at 65° C and switching to ice for 2 minutes before the addition of the reverse transcriptase.

3.

Quantitative PCR results can be analyzed using an efficiency and threshold cycle difference method described by Michael Pfaffl [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF], although I prefer to transform dilutions using base 2 logarithm, rather than base 10, as it is closer to the 8/12 exponential nature of the PCR reaction. Messenger RNA used for normalization of the amount of RNA in the qPCR reaction are, for yeast two of the following: PGK1, ACT1 or RIM1.

4.

For users of spreadsheet software, such as Microsoft Excel or LibreOffice Calc, the results are most often displayed in the so-called "wide" format. For multiple replicate experiments, it is usual to list their results in separate columns labeled, "exp1", "exp2", and so on. In R, it is often easier to use a "long" format for data analysis. In such a format, each column corresponds to a type of variable. For example, there will be only one column for the type of experiment and each entry will be one of "exp1", "exp2", etc. The advantage of this format is that all values of interest are found in specific columns. Moreover, graphics packages such as ggplot2 can use the specific information from a column to assign colors to points in a graph as a function of the discrete values present in that column ("exp1", "exp2", etc). Switching from a wide to long format for a data frame in R is accomplished with the melt function of the reshape2 package.

5. An exponential decay function might not always correspond to the observations. For example, there might be a lag between the addition of doxycyclin and the transcription inhibition. In this case, one can use time intervals from which a "lag" period has been subtracted. To take into account the possibility that some basal transcription still occurs even in the presence of the drug, leading to an apparent stabilization of the mRNA levels, one can use an additional constant factor in the equation [START_REF] Baudrimont | Multiplexed gene control reveals rapid mRNA turnover[END_REF]. However, please keep in mind that, in general, the least number of factors we need to guess by nonlinear least squares fitting, the more likely it is that these factors can be given a biological meaning.

6.

To establish a confidence interval for the estimated half-life, a possibility is to take the minimal and maximal estimates of the decay constant k and compute the corresponding half-life values. While not formally correct, such computations give a rough idea about how good the fit was and how confident one can be about the reported half-life value.

  3. qPCR pre-mix, containing SYBR Green interacalating fluorophore, thermostable DNA polymerase, dNTPs and the manufacturer buffer. Usually these pre-mixed solutions are provided as 2X stock. Store at -20°C. 4. Oligonucleotides specific for the transcript of interest NOTE1 . Store at 100 μM in H 2 0 at -20°C. 5. Reverse transcriptase derived from murine leukemia virus enzyme, with decreased Rnase H and DNA endonuclease activities and increased thermal resistance (e.g. SuperScript III or SuperScript IV). 6. Reverse transcriptase buffer 5X stock (250 mM Tris-HCl pH 8.3, 375 mM KCl, 15 mM MgCl 2 ). Store at -20°C. 7. Actinomycin D, 10 mg/ml. Store at -20°C. 8. Dithiothreitol (DTT) 0.1 M stock. Store at -20°C. 9. dNTP mix, each deoxy-nucleotide triphosphate (dATP, dGTP, dCTP, dTTP) at 25 mM. Store at -20°C. 10. Linear polyacrylamide 5 mg/ml stock (store at -20° C).

  3. Split the culture in 5 flasks marked for incubation with doxycyclin for 0, 5, 10, 15, 20 and 30 minutes. Continue incubation with agitation at 30°C. 4. Add 50 μl doxycycline stock to the flask labeled "30 min.". Add doxycyclin 10 minutes later to the flask "20 min.". Follow the indications of Fig. 1A for the addition of doxycyclin in the other flasks. Adapt the duration for more stable transcripts. 5. At the end of the incubation period, move all the flasks to an ice-water mix for rapid chilling of the media and cells. Transfer cells to refrigerated 50 mL Falcon tubes. 6. Recover cells by centrifugation at 2000g, 4°C, for 10 minutes.

1.

  Use the qPCR software associated with the qPCR thermocycler to measure cycle threshold values and verify that the differences in cycle values correspond to the dilutions NOTE3 . 2. Once normalized, express each of the amounts of the transcript of interest as a fraction of the amount of that transcript in the sample that received no doxycyclin treatment. It is expected that these fractions are lower than 1. 3. Load the results in R in a data frame called mydf. The simplest version of such a data frame will have two columns: time and fraction. If several replicate experiments have been performed, use a three column data frame, with one of the column corresponding to a description of which replicate experiment it represents NOTE4 .

Figure 1 .

 1 Figure 1. Typical workflow and result for the half-life estimation using RT-qPCR with a Tet-off reporter. a) Schematics of the culture conditions and a way to synchronize the recovered samples and ensure equal treatment for the different time points. All the time points are recovered together, but with different periods of doxycyclin treatment. b) Example of obtained results (Dehecq et al, 2018), with a relatively unstable mRNA. The experiment was done three times independently and the results were fitted to an exponential decay function (indicated). "k" is a kinetic first order constant for the degradation of the mRNA. (The flask image was generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license).

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Acknowledgments

I thank Laurence Decourty for results used to illustrate this protocol and exceptional technical assistance and my colleagues for fruitful discussions and criticism. This work was funded by the DEFineNMD grant ANR-18-CE11-0003.


Juan Manuel López-García

Hugues-Alexandre Blain
email: hablain@iphes.cat

Christian Sánchez-Bandera
email: csanchezbandera@gmail.com.

Jessica Cohen
email: jessica.co@outlook.fr

Loïc Lebreton

Sophie Montuire
email: sophie.montuire@u-bourgogne.fr.

John R Stewart
email: jstewart@bournemouth.ac.uk.

Emmanuel Desclaux
email: emmanuel.desclaux@gmail.com.

Christian Sánchez- Bandera

Multi-method approach using small vertebrate assemblages to reconstruct the Marine Isotope Stage 6

Keywords: Mutual Ecogeographic Range, Bioclimatic Model, Quantified Ecology, Taxonomic Habitat Index, Climatograms, Simpson Diversity Index 1

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

Introduction

The end of the Middle Pleistocene is marked by the penultimate glacial period, Marine Isotope Stage 6 (MIS 6 -ca. 185-135 ka), which corresponds with the Saalian glaciation in Europe (e.g. [START_REF] Roucoux | Vegetation history of the penultimate glacial period (Marine Isotope Stage 6) at Ioannina, north-west Greece[END_REF]Magari et al. 2014;[START_REF] Railsback | An optimized scheme of lettered marine isotope substages for the last 1.0 million years, and the climatostratigraphic nature of isotope stages and substages[END_REF].

MIS 6 was a glacial period where generally drier and cooler conditions have been detected in the Mediterranean region of Europe as compared to interglacial periods (e.g. [START_REF] Hodge | Effective precipitation in southern Spain (similar to 266 to 46 ka) based on a speleothem stable carbon isotope record[END_REF][START_REF] Wainer | Millennial climatic instability during penultimate glacial period recorded on a southwestern France speleothem[END_REF]. During the second half of MIS 6 (from 150 ka) the global sea level was 100 m below that of today and the sea surface temperatures (STT) reached 5 ºC lower with respect to current ones (e.g. [START_REF] Elderfield | Evolution of ocean temperature and ice-volume through the Mid-Pleistocene Climate Transition[END_REF][START_REF] Margari | Land-ocean changes on orbital and millennial timescales and the penultimate glaciation[END_REF].

Speleothem data (in Italy, France and Spain) and long pollen sequences (in Greece, France and from a marine core off the Portuguese coast) are available for western Europe (Fig. 1) showing the climatic and environmental conditions during the entirety of MIS 6 (e.g. [START_REF] Margari | Land-ocean changes on orbital and millennial timescales and the penultimate glaciation[END_REF][START_REF] Tzedakis | Comparison of changes in vegetation in northeast Greece with records of climate variability on orbital and suborbital frequencies over the last 450,000 years[END_REF][START_REF] Roucoux | Vegetation history of the penultimate glacial period (Marine Isotope Stage 6) at Ioannina, north-west Greece[END_REF][START_REF] Wilson | Limnetic and terrestrial responses to climate change during the onset of the penultimate glacial stage in NW Greece[END_REF][START_REF] Bard | Hydrological conditions over the western Mediterranean basin during the deposition of the cold Sapropel 6 (ca. 175 kyr BP)[END_REF][START_REF] Hodge | Effective precipitation in southern Spain (similar to 266 to 46 ka) based on a speleothem stable carbon isotope record[END_REF]Guiot el al. 1993;[START_REF] Ponel | Rissian, Eemian and Wurmian Coleoptera assemblages from La Grande Pile (Vosges, France)[END_REF]. For the early part of this glacial period, long pollen sequence data suggest a fluctuating tree abundance in Europe, while extreme conditions with tree loss have been detected for the latest part of MIS 6 (e.g. [START_REF] Roucoux | Vegetation history of the penultimate glacial period (Marine Isotope Stage 6) at Ioannina, north-west Greece[END_REF], with a high amplitude climatic oscillations detected in the diatom species record (e.g. [START_REF] Wilson | Persistent millennial-scale climate variability in[END_REF]. In contrast, archaeological sites in western Europe dated to MIS 6 with small vertebrate studies are scarce. Existing ones include three Spanish sites, Sala de los Huesos in Maltravieso cave [START_REF] Hanquet | Évolution des paléoenvironnements et des paléoclimats au Pléistocène moyen, en Europe méridionale, d'après les faunes de micromammifères[END_REF], Estanque de Tormentas de Butarque H-02 [START_REF] Blain | A new middle Pleistocene (Marine Oxygen Isotope Stage 6) cold herpetofaunal assemblage from the central Iberian Peninsula (Manzanares Valley, Madrid)[END_REF][START_REF] Laplana | Biocronología de la Terraza Compleja de Butarque del río Manzanares en el Estanque de Tormentas al sur de Madrid (España)[END_REF] and Lezetxiki II (García-Ibaibarriaga et al. 2018), four sites in France, Baume Moula-Guercy [START_REF] Desclaux | Étude préliminaire des micromammifères de la Baume Moula-Guercy à Soyons (Ardèche, France). Systématique, biostratigraphie et paléoécologie[END_REF], Grotte des Cèdres (Defleur and Crégut-Bonnoure, 1995), Romain-la-Roche [START_REF] Guérin | Le gisement Pléistocène Moyen récent de Romain-la-Roche (Doubs, France): Synthèse biostratigraphique et paléoécologique[END_REF] and Coudoulous 1 [START_REF] Jaubert | Coudoulous I (Tour-de-Faure, Lot), site du Pléistocène moyen en Quercy[END_REF] and one site in Italy Poggetti Vecchi [START_REF] Benvenuti | Paleoenvironmental context of the early Neanderthals of Poggetti Vecchi for the late middle Pleistocene of central Italy[END_REF]) (Fig. 1). The aim of the present study is to characterize the environment and climate of the MIS 6 archeological sequence of Lazaret cave (Nice, France) using small mammals (mainly rodents) and herpetofaunal assemblages. Applying several paleoclimatic and paleoenvironmental tools to obtain a multi-method approach, which it is hoped allows a better understanding of conditions in Western Europe during the penultimate glacial.

Lazaret Cave

Lazaret Cave is located in southern France on the Mediterranean coast of the city of Nice (43º 41' 25'' N, 7º 17'42'' E) (Fig. 1). Lazaret cave is formed in lower Jurassic dolomite limestone and is 40 m long and 15 m wide with a celling of ca. 15 m high. The current entrance faces the southwest at 26 m a.s.l. and is ca. 100 m from the coast. The lower part of the cave sequence (Fig. 2) is composed by two marine transgressive phases.

The first phase, Complex A or the Lower Marine Beach, is without fossils and is ascribed to Marine Isotope Stage 9 (MIS 9) [START_REF] Lumley | Le sol d'occupation acheuléen de l'unité archéostratigraphique UA 25 de la grotte du Lazaret[END_REF]). The second phase, Complex B or the Upper Marine Beach, is rich in coral and marine mollusks dating to around ca. 230 ka, corresponding to Marine Isotope Stage 7 (MIS 7) [START_REF] Bahain | Datation par résonance de spin électronique (ESR) de carbonates et d'émail dentaire quaternaires: potentiel et problèmes[END_REF][START_REF] Michel | Méthodes U-Th/ESR en Préhistoire et problèmes d'altération des matériaux: exemple de la datation des carbonates et apatites de la grotte du Lazaret[END_REF]. These marine deposits rest under a continental fill (Complex C) 6meters in thickness (Fig. 2), mainly composed of clays and gravels, which are capped by a stalagmitic layer (Complex E) dated between ca. 108-45 Ka [START_REF] Shen | Datation des planchers stalagmitiques de sites acheuléens en Europe par les méthodes des déséquilibres des familles de l'uranium et contribution méthodologique[END_REF][START_REF] Michel | ESR dating of dental enamel from Middle Palaeolithic levels at Lazaret Cave, France[END_REF]). Complex C is divided into three stratigraphic sub complexes, CI, CII and CIII, which correspond to archaeological levels and date between 220-130 Ka [START_REF] Michel | Étude des influences des processus de fossilisation sur le fondement de la datation radiométrique. Application à la datation par U-Th et ESR de mâchoires (os, dent) de Cervus elaphus des niveaux archéologiques de la grotte du Lazaret[END_REF][START_REF] Michel | ESR dating of dental enamel from Middle Palaeolithic levels at Lazaret Cave, France[END_REF]. The bottom of the continental deposits, sub complex CI, is not well known as yet, but the CII and CIII sub complexes contain at least 29 archaeostratigraphic units (UA), CIIinf. (UA 29-26), CIIsup. (UA 25-13) and CIII (UA 12-1). These subcomplexes show a succession of hominin occupations, where humans and numerous faunal (vertebrates and invertebrates) remains have been recovered, together with abundant lithic bifacial tools attributed to the final Acheulean (e.g. [START_REF] Lumley | Le sol d'occupation acheuléen de l'unité archéostratigraphique UA 25 de la grotte du Lazaret[END_REF][START_REF] Hanquet | Caractérisation du climat et de la biodiversité au Pléistocène moyen final, d'après les faunes de vertébrés de la grotte du Lazaret[END_REF][START_REF] Valensi | Cadre climatique et environnemental des acheuléens de la grotte du Lazaret, à Nice. Données paléontologiques, biochimiques et radiométriques établies sur les faunes de vertébrés et d'invertébrés[END_REF], among others).

Material and methods

Small vertebrate sorting, taxonomic and taphonomic study

The small-vertebrate fossil remains used for this study come from the sub-complex CII inf., CII sup. and CIII from Lazaret cave, in which 29 archaeo-stratigraphic units (UA) of anthropic occupation have been identified (Fig. 2). Most of the small vertebrate remains have been recovered by water-screening from the excavation sediments, using two superimposed screens of 5 mm and 0.8 mm mesh sizes. The fossils were processed, sorted and classified at the "Laboratoire de Préhistoire du Lazaret" (Nice, France). The rodents from the sub-complexes CII sup. and CIII have been identified and partially published by [START_REF] Hanquet | Caractérisation du climat et de la biodiversité au Pléistocène moyen final, d'après les faunes de vertébrés de la grotte du Lazaret[END_REF], [START_REF] Desclaux | Evolution des communautés de mammifères dans les Alpes-Maritimes au cours du Pléistocène[END_REF] and [START_REF] Lumley | Les restes humains fossiles de la grotte du Lazaret (Nice, Alpes-Maritimes, France)[END_REF], while the association pf CII inf. is presented here for the first time. The amphibians and reptiles from sub-complexes CII inf, CII sup and CIII have been published by [START_REF] Bailon | Amphibiens et reptiles du Pliocène et du Pléistocène de France etd'Espagne : mise en place et évolution des faunes[END_REF], [START_REF] Lumley | Le sol d'occupation acheuléen de l'unité archéostratigraphique UA 25 de la grotte du Lazaret[END_REF], [START_REF] Hanquet | Caractérisation du climat et de la biodiversité au Pléistocène moyen final, d'après les faunes de vertébrés de la grotte du Lazaret[END_REF], [START_REF] Manzano | Les amphibiens et les reptiles des sites Pléistocène moyen et supérieur de la France méditerranéenne[END_REF] and [START_REF] Lumley | Les restes humains fossiles de la grotte du Lazaret (Nice, Alpes-Maritimes, France)[END_REF].

The rodent assemblages used for this study correspond to minimum number of 5428 individuals, representing at least 16 taxa (Table 1: Fig. 3). The rodent remains were identified using systematic paleontological methods. Specific identification of this material rests principally on the best diagnostic elements: first lower molars for the subfamily Arvicolinae; and isolated teeth for the subfamilies Cricetinae, Murinae and Glirinae. The fossils were quantified using the minimum number of individuals (MNI) method for each sample (i.e. from each level) and determined by counting the diagnostic elements. A previous preliminary taphonomic study of sub-complex CII (UA25-27) has been published by in [START_REF] Hanquet | Évolution des paléoenvironnements et des paléoclimats au Pléistocène moyen, en Europe méridionale, d'après les faunes de micromammifères[END_REF] and [START_REF] Desclaux | Origine(s) des accumulations de micromammifères dans quelques sites préhistoriques du Pléistocène moyen et supérieur d'Europe méridionale[END_REF] using the criteria proposed by [START_REF] Andrews | Owls, Caves and Fossils. Predation, Preservation and Accumulation of Small Mammal Bones in Caves, with an Analysis of the Pleistocene Cave Faunas from Westbury-sub-Mendip[END_REF] and subsequently updated with the work of [START_REF] Fernández-Jalvo | Taphonomy for taxonomists: implication in predation in small mammal studies[END_REF] for alterations caused by digestion present in rodent incisors and molars.

Marmota marmota is not included in the environmental and climatic data analysis for the following reasons: 1) is not possible to use with all the methods because theclimatic distributions data is not available for the species(e.g. [START_REF] Royer | New bioclimatic models for the quaternary palaearctic based on insectivore and rodent communities[END_REF]; 2)

Due to it large size, the accumulation of M. marmota is not commonly caused by small bird of prey or small carnivore predatory activities (e.g. [START_REF] Armitage | Behavioral response of yellow-bellied marmots to birds and mammals[END_REF], like the other rodent species (e.g. [START_REF] Fernández-Jalvo | Taphonomy for taxonomists: implication in predation in small mammal studies[END_REF]). In addition, its presence in archaeological sites may also be the result of anthropic activities (e.g. [START_REF] Romandini | Marmota marmota, the most common prey species at Grotta del Clusantin: Insights from and unusual case-study in the Italian Alps[END_REF]. In order to complement rodent reconstructions, the faunal lists of amphibians and reptiles published by [START_REF] Manzano | Les amphibiens et les reptiles des sites Pléistocène moyen et supérieur de la France méditerranéenne[END_REF] have been used. The amphibian and reptile assemblages used for this study correspond to at least 20 taxa (Table 2). As no detailed taphonomical study of the remains has been done for sub-complexes CII sup and CIII and we used only the presence-absence of taxa for the amphibians and reptiles and no quantificationhas been included.

Palaeonvironmental reconstruction

In order to reconstruct the environment, we used two main methods to analyse the rodent assemblages, the Taxonomic Habitat Index (THI) and the Climatogram methods.

The THI was developed by [START_REF] Andrews | Small Mammal Bone Accumulations Produced by Mammalian Carnivores[END_REF], a method that considers the assemblage of rodent species identified, taking into account the diversity of the habitats where these species live, and regardless of their relative proportions. The representation of species by habitat has been done for Lazaret cave (Table 3) according to the current distribution of the rodent species in France [START_REF] Le Louran | Les rongueurs de France: Faunistique et Biologie[END_REF], as well as the distribution of the fossil rodent species in Pleistocene of France and western Europe, according to [START_REF] Andrews | Owls, Caves and Fossils. Predation, Preservation and Accumulation of Small Mammal Bones in Caves, with an Analysis of the Pleistocene Cave Faunas from Westbury-sub-Mendip[END_REF], [START_REF] Chaline | Les rongeurs au pilori ? Analyse critique des reconstitutions paléoécologiques en microthériodentologie[END_REF][START_REF] Chaline | Les rongeurs, les paléoenvironnements et les climats du Plésitocène moyen de Vergranne (Doubs)[END_REF], [START_REF] Marquet | Paléoennvironment et chronologie des sites du domaine atlantique français d'âge Plèistocéne moyen et supérieur d'après l'etude des rongeurs[END_REF] and [START_REF] Desclaux | Le contexte paléoenvironmental des occupations humaines. L'apport de la microfaune, des restes aviaries et de la malacofaune. Le site de payre-occupations humaines dand la vallée du Rhône à ña fin du Pléistocène moyen et au début du Pléistocène supérieur[END_REF]. The Climatogram quantitative method applied to rodent assemblages was developed by [START_REF] Chaline | Les rongeurs au pilori ? Analyse critique des reconstitutions paléoécologiques en microthériodentologie[END_REF] and applied to numerous case studies (e.g. [START_REF] Marquet | Paléoennvironment et chronologie des sites du domaine atlantique français d'âge Plèistocéne moyen et supérieur d'après l'etude des rongeurs[END_REF][START_REF] Desclaux | Étude préliminaire des micromammifères de la Baume Moula-Guercy à Soyons (Ardèche, France). Systématique, biostratigraphie et paléoécologie[END_REF][START_REF] Desclaux | Origine(s) des accumulations de micromammifères dans quelques sites préhistoriques du Pléistocène moyen et supérieur d'Europe méridionale[END_REF][START_REF] Foury | Evolution des faunes de rongeurs en moyenne vallée du Rhône (rive droite, Ardèche, France) au cours du pléistocène moyen final et du pléistocène supérieur ancien[END_REF]Leberton et al. 2016[START_REF] Crégut-Bonnoure | New Insights into the LGM and LG in Southern France (Vaucluse): The Mustelids, Micromammals and Horses from Coulet des Roches[END_REF]. This method allows the climatic and environmental variation represented by a stratigraphic sequence to be revealed. In this way the rodent associations of Lazaret cave have been grouped into six climate-ecology categories (Table 4).

Palaeodiversity reconstruction

Among the large number of indices proposed for evaluating the biodiversity of a sample, species evenness is fundamental for assessing the homogeneity of an environment [START_REF] Magurran | Biological Diversity: Frontiers in Measurements and Assessment[END_REF]. Evenness is a diversity index that is used to quantify how equal the various communities are numerically. The evenness of a community can be represented by the Simpson index of diversity, which is also equivalent to the probability of interspecific encounter [START_REF] Simpson | Measurement of diversity[END_REF][START_REF] Blois | Small mammal diversity loss in response to late Pleistocene climatic change[END_REF][START_REF] López-García | Small mammal diversity in Spain during the Late Pleistocene to early Holocene: climate, landscape and human impact[END_REF]: i.e., the Simpson index of diversity = 1 -Σ (pi 2 ), where pi is the proportion of total individual samples belonging to the ith species. The evenness index is constrained between 0 and 1. The lower the variation in species within a community, the higher the value of the evenness index. The index will be close to 0 if there is a single dominant species. To avoid the statistical problems of high sample-size dependence, we have standardized the absolute values by dividing them by the total sample abundances, expressed as percentages of the MNI. This standardization allows the taxon evenness to be compared in samples of different size. This index of evenness was obtained using the Paleontological Statistics Program (PAST) [START_REF] Hammer | PAST: Paleontological statistical software for education and data analysis[END_REF].

Palaeoclimatic reconstructions

In order to assess the palaeoclimatic data from Lazaret cave, we used the Bioclimatic Model (BM) and the Quantified Ecology (QE) methods on small mammal assemblages and the Mutual Ecogeographic Range (MER) and the Quantified Ecology (QE) methods on the herpetofaunal assemblages. Restriction Index (CRIi = 1/n, where "n" is the number of climatic zones where the species are represented and "i" is the climatic zone where the species appears) (Table 5)

and was assigned to the following zones: IV Subtropical with winter rains and summer droughts; VI Typical temperate; VII Arid temperate; VIII Cold-temperate (boreal) and IX Polar. After obtaining this distribution, the Bioclimatic Component (BC;

representation by level of each of the three available climates) was calculated using the following formula: BCi = (Σ CRIi) × 100/S, where S is the number of species per subcomplex at Lazaret cave. From the BC, a mathematical model was elaborated using a multiple linear regression [START_REF] Hernández-Fernández | Quantitative palaeoclimatic inference based on mammal faunas[END_REF]; by means of a series of functions, this allows the estimation of mean annual temperature (MAT) and mean annual precipitation (MAP).

The QE was developed by [START_REF] Jeannet | L'ecologie quantifiée. Essai de description de l'environnement continental à l'aide des microvertébrès[END_REF]. This method is based on the combination of geographic and climatic distributions of small vertebrate species today, in this case we use the small mammal and herpetofaunal assemblages separately (Table 6), calculated from the sum of the climatic parameters (mean temperature and mean precipitation) by species divided by the total of species represented in each sub-complex of Lazaret cave.

On the basis of these methods, two climatic factors are estimated: the mean annual temperature (MAT) and the mean annual precipitation (MAP).

The MER method [START_REF] Blain | Long-term climate record inferred from early-middle Pleistocene amphibian and squamate reptile assemblages at the Gran Dolina cave, Atapuerca, Spain[END_REF][START_REF] Blain | Refining upon the climatic background of the Early Pleistocene hominid settlement in western Europe: Barranco León and Fuente Nueva-3 (Guadix-Baza basin, SE Spain)[END_REF] has been used to estimate the temperature and precipitation, for which we used the current geographical ranges of the herpetofaunal species present in the Lazaret cave assemblages. To determine the current spatial distributions of European amphibians and reptiles, we used the data from [START_REF] Sillero | Updated distribution and biogeography of amphibians and reptiles of Europe[END_REF], represented in a geographic coordinate system (datum WGS 84) with a 50

× 50 km grid. Once the common overlapping area of the species from the assemblage was identified, approaching a more precise common species distribution area, the UDA-ODA discrimination methodology [START_REF] Fagoaga | Improving the accuracy of small vertebrate-based palaeoclimatic reconstructions derived from the Mutual Ecogeographic Range. A case study using geographic information systems and UDA-ODA discrimination methodology[END_REF] was applied. This procedure, partially derived from the MER method, sharpens each species distribution creating the real Occupied Distribution Areas (ODA) that will be used as current analogue associations from where climatic parameters will be extrapolated to past fossil assemblages. For example, the upper elevation limits of Pelobates cultripes (1770 meters) and Pelodytes punctatus (2000 meters) [START_REF] Cejudo | Nueva altitud máxima para Pelobates cultripes[END_REF][START_REF] Guixé | Rècord altitudinal de Pelodytes punctacus (Daudin, 1802) a Catalunya i a la península Ibèrica[END_REF] were used to obtain more precise areas within the overlapping area in which all the species could coincide.

The climate data are from WorldClim 2.0 (Ficks and Hijmans, 20176), also codified in a geographic coordinate system (datum WGS 84). Both data sets were processed using the ArcGIS 10.5 application [START_REF] Redlands | ArcGIS desktop: Realeas10[END_REF]. The bioclimatic information used are the bioclimatic variables BIO1 (annual mean temperature) and BIO12 (mean annual precipitation) from [START_REF] Hijmans | Very high resolution interpolated climate surfaces for global land areas[END_REF]. The mean and standard deviations were calculated using the statistical program IBM SPSS Statistics 22.

The same climatic parameters were calculated for Lazaret today , choosing an area of 3,5 km 2 surrounding the site (i.e, in a radius of 1 km). MAT have been estimated to be 15.0 ± 0.17ºC and MAP to be 811.70 ± 3.8 mm.

Results

Taphonomic remarks

The results obtained in the previous taphonomic studies by [START_REF] Hanquet | Évolution des paléoenvironnements et des paléoclimats au Pléistocène moyen, en Europe méridionale, d'après les faunes de micromammifères[END_REF] and [START_REF] Desclaux | Origine(s) des accumulations de micromammifères dans quelques sites préhistoriques du Pléistocène moyen et supérieur d'Europe méridionale[END_REF] from units UA 27-25 (CII inf. and CII sup.) show that most of the small mammal accumulations were the result of predation by nocturnal birds of prey. The signs of digestion in the small mammal bones and teeth suggest that four nocturnal raptors (Bubo bubo, Asio otus, Asio flammeus and Strix aluco) may have been responsible for the accumulations at Lazaret cave [START_REF] Hanquet | Évolution des paléoenvironnements et des paléoclimats au Pléistocène moyen, en Europe méridionale, d'après les faunes de micromammifères[END_REF][START_REF] Desclaux | Origine(s) des accumulations de micromammifères dans quelques sites préhistoriques du Pléistocène moyen et supérieur d'Europe méridionale[END_REF]). These kinds of predators do not generally display specific prey consumption patterns [START_REF] Andrews | Owls, Caves and Fossils. Predation, Preservation and Accumulation of Small Mammal Bones in Caves, with an Analysis of the Pleistocene Cave Faunas from Westbury-sub-Mendip[END_REF].

Therefore, palaeoecological interpretations based on the relative abundance of the rodent taxa from Lazaret cave are likely to be reliable indicators of the habitat in which the predators hunted.

In the case of the amphibians and reptiles, a taphonomic study has been carried out by [START_REF] Manzano | Les amphibiens et les reptiles des sites Pléistocène moyen et supérieur de la France méditerranéenne[END_REF] for units UA 27-26 (CII inf.). It suggested that the bone accumulation is in situ because the bone surfaces lack polish and that no skeletal elements are missing.

The skeletal remains show a high level of fragmentation, mostly with angular and irregular fractures attributable to diagenetic processes in the cave (Pinto-Llona and [START_REF] Pinto-Llona | Amphibian taphonomy and its application to the fossil record of Dolina (middle Pleistocene, Atapuerca, Spain)[END_REF]Manzano,2015). The signs of digestion in amphibian and reptile bones are rare and can hardly be recognized. Traces of digestion have been observed in some bones of Rana temporaria, but the intensity of this digestion is very low (category 1, type

Tyto alba; sensu Pinto-Llona and Andrews, 1999).

Small vertebrate assemblages

The rodent assemblage of complex C (including CII inf., CII sup. and CIII) of Lazaret cave is dominated by two species, the field vole (Microtus (Agricola) agrestis, more than 700 individuals) and the wood mouse (Apodemus (Sylvaemus) sylvaticus, more than 400 individuals). These two taxa represent more than 75% of the sub-complex (4209 individuals) in relation to the total number of individuals (5428 individuals) (Table 1).

Both species are also present in other MIS 6 sites with small mammal studies in France, such as Coudoulous I in Lot [START_REF] Jaubert | Coudoulous I (Tour-de-Faure, Lot), site du Pléistocène moyen en Quercy[END_REF], Romain-la-Roche in Doubs [START_REF] Guérin | Le gisement Pléistocène Moyen récent de Romain-la-Roche (Doubs, France): Synthèse biostratigraphique et paléoécologique[END_REF] and Moula Guercy in the Ardèche [START_REF] Desclaux | Étude préliminaire des micromammifères de la Baume Moula-Guercy à Soyons (Ardèche, France). Systématique, biostratigraphie et paléoécologie[END_REF]

Palaeoenvironmental and Paleoclimatic approach

In comparison with current climatic data (Table 7), the bioclimatic model and the quantified ecology methods applied to the small mammals of the Lazaret cave sub complexes suggested a generally colder climate (ΔMATCII = -6.7 °C and -6. 175-130 Kya), these could correspond to MIS 6 substages 6d to 6a (Fig. 4). These data are also concordant with the percentage representation in both sub complexes, CII and CIII, of the species associated with climate categories VI (typical temperate, related with a temperate deciduous forest), VII (Arid temperate, related with a steppe and cold desert), VIII (Cold-temperate, related to a boreal coniferous forest-taiga) and IX (Polar, related with Tundra) which represents more than 70% of the Bioclimatic Component of the bioclimatic model (Table 8). It is also consistent with the relatively high representation (10 of 17) of small mammal species that require mean temperatures below 10ºC (Table 5), according to the Quantified Ecology method.

On the other hand, both methods (THI and Climatograms) used in the environmental reconstruction at Lazaret cave with rodent assemblages indicate a landscape dominated by deciduous temperate forest and humid meadows with a significant representation of shrublands (Fig. 4). The percentage of deciduous forest obtained with the THI has values around ca. 35% in all the sub-complexes (Table 9), mainly represented by the most abundant species M. (A.) agrestis and A. (S.) sylvaticus and to a lesser extent by Eliomys querciuns, Muscardinus avellanarius, Glis glis and Clethrionomys glareolus. These five last species are also those indicative of a temperate forest using the Climatogram method, with percentages ranging between ca. 21-35% (Fig. 4; Table 10). Following the environmental category Mediterranean, Shrubland and Humid habitats are the most abundant, between 16-21 %, according to the THI method (Fig. 4; Table 9). The Mediterranean category is mainly represented by the species E. 10).

Finally, the Simpson index (1-D) indicates a relatively high diversity (>0.5) with similar values in the three studied sub complexes (1-DCIII-CIIsup. = 0.6 and 0.67) from

Lazaret cave (Fig. 5). This is a signal of a relatively heterogeneous vegetation (mainly related to deciduous forest, shrubland and mountain habitats) and rodent community (relation between number of species and individual's distribution in each sub complex) (Fig. 5). These most abundant rodents are one mouse (A. (S.) sylvaticus) and five vole species (M. (A.) agrestis, M. (T.) multiplex, A. mosbachensis and Pliomys sp. nov.) which represent more than 50 individuals in each sub complex (Fig. 5).

Discussion

Comparison with other Lazaret environmental proxies

The environmental studies done using other proxies from Lazaret cave include the published works on herpetofauna (amphibians and reptiles), birds, marine gastropods, and large mammals from subcomplexes CIII and CII sup [START_REF] Hanquet | Caractérisation du climat et de la biodiversité au Pléistocène moyen final, d'après les faunes de vertébrés de la grotte du Lazaret[END_REF][START_REF] Valensi | Cadre climatique et environnemental des acheuléens de la grotte du Lazaret, à Nice. Données paléontologiques, biochimiques et radiométriques établies sur les faunes de vertébrés et d'invertébrés[END_REF]. In general, all the studied proxies from complex C of Lazaret cave indicate a cooler climate and a more humid environment than today [START_REF] Hanquet | Caractérisation du climat et de la biodiversité au Pléistocène moyen final, d'après les faunes de vertébrés de la grotte du Lazaret[END_REF][START_REF] Valensi | Cadre climatique et environnemental des acheuléens de la grotte du Lazaret, à Nice. Données paléontologiques, biochimiques et radiométriques établies sur les faunes de vertébrés et d'invertébrés[END_REF]Valensi and Abbassi, 1998). Former quantification of climate based on herpetofauna at the level of the UAs [START_REF] Manzano | Les amphibiens et les reptiles des sites Pléistocène moyen et supérieur de la France méditerranéenne[END_REF] using the MER method at a regional level (200 km around the site) and QE for the sequence of Lazaret suggested an alternance of temperate and cold phases during a globally colder than present MIS 6. Cold periods (CII inf. and CIII) were characterized by MAT as being between 12 and 9.6ºC (i.e. -5.4ºC to -3ºC in relation to the present climate). CII sup., however, shows a general improvement in climatic conditions with MAT indicating temperatures between 12 and 14ºC (i.e. -3ºC to -1ºC). Reconstructed MAP suggested somewhat lower levels of rainfall than today for temperate periods (around 700 mm; i.e. 100 mm lower than present level) in opposition to much higher values obtained for cold periods with MAT reaching peaks above 1000 mm (i.e. +200 mm in comparison with the present amount) [START_REF] Manzano | Les amphibiens et les reptiles des sites Pléistocène moyen et supérieur de la France méditerranéenne[END_REF].

Some of the UAs (UA27, 18, 15 and 3) were said to correspond to non-analogue herpetofaunal communities [START_REF] Manzano | Les amphibiens et les reptiles des sites Pléistocène moyen et supérieur de la France méditerranéenne[END_REF], but even with a larger stratigraphical consideration (i.e., at level of complexes) we found no non-analogue assemblages when

MER was used at a European scale. If climate reconstructions are quite similar concerning anomalies (Δ) between [START_REF] Manzano | Les amphibiens et les reptiles des sites Pléistocène moyen et supérieur de la France méditerranéenne[END_REF] and the present reconstructions, some differences can be seen according to the methods applied. According to the MER, CII sup. would be slightly warmer than the two other complexes, whereas, according to QE, CII sup. is slightly warmer than CIII and CII inf. (Table 7). However, the differences fall within one standard deviation and are therefore not significant. CIIsup. and CIII sub complexes [START_REF] Hanquet | Caractérisation du climat et de la biodiversité au Pléistocène moyen final, d'après les faunes de vertébrés de la grotte du Lazaret[END_REF]. Finally, the marine gastropods assemblage highlights the presence of Melarhaphe neritoides, a Mediterranean Littornidae species, together with Nordic Littornidae, Littorina saxatilis and Littorina fabalis. The association of these gasteropods indicates a cooling of the sea in sub complex CIII respect to CII [START_REF] Valensi | Cadre climatique et environnemental des acheuléens de la grotte du Lazaret, à Nice. Données paléontologiques, biochimiques et radiométriques établies sur les faunes de vertébrés et d'invertébrés[END_REF]. In general, the data obtained from the different methods applied to the rodent assemblages are in concordance with the trend shown by the other proxies, with a relatively cold climate and a humid environment in relation to today (Fig. 4). When considered according to sub-complexes within the cave sequence, the results obtained from the rodent assemblages agree with the herpetofaunal data in that there is a similar association in sub-complexes CII sup. and CIII (Table 1). However, the cooling and/or closure detected in the other proxies from CII sup. to CIII are not observed with the rodent results (Fig. 4). Nevertheless, relative colder conditions have been detected with the rodent assemblage in sub complex CII inf., in relation to the overlying complexes (Fig. 4). This is mainly indicated by the presence of Lasipodomys gregalis (narrow-headed vole) in sub complex CII inf., a species that currently inhabits tundra and forest tundra from the White Sea to the Kolyma River in Russia and in the steppes of Kazakhstan, Kyrgizia, SW Siberia, Yakutia, Mongolia and Northern China [START_REF] Batsaikhan | Microtus gregalis. The IUCN Red List of Threatened Species[END_REF].

Comparison with other MIS 6 sites

As noted above, there are few archaeological sites in western Europe dating to MIS 6 with small vertebrate studies. Together with Lazaret cave, four sites have been studied

in France, Baume Moula-Guercy [START_REF] Desclaux | Evolution des communautés de mammifères dans les Alpes-Maritimes au cours du Pléistocène[END_REF]Defleur, 1997, Defleur et al.,1998;[START_REF] Defleur | Présentation paléoenvironnementale du remplissage de la Baume Moula-Guercy à Soyons (Ardèche) : implications paléoclimatiques et chronologiques[END_REF][START_REF] Defleur | Impact of the last interglacial climate change on ecosystems and Neanderthals behavior at Baume Moula-Guercy, Ardèche, France[END_REF][START_REF] Defleur | The Eemian: Global warming, ecosystem upheaval, demographic collapse and cannibalism at Moula-Guercy. A reply to Slimak and Nicholson[END_REF], Grotte des Cèdres (Defleur and Crégut-Bonnoure, 1995), Romain-la-Roche [START_REF] Guérin | Le gisement Pléistocène Moyen récent de Romain-la-Roche (Doubs, France): Synthèse biostratigraphique et paléoécologique[END_REF] and Coudoulous 1 [START_REF] Jaubert | Coudoulous I (Tour-de-Faure, Lot), site du Pléistocène moyen en Quercy[END_REF], three in the Iberian Peninsula, Sala de los Huesos in Maltravieso cave [START_REF] Hanquet | Évolution des paléoenvironnements et des paléoclimats au Pléistocène moyen, en Europe méridionale, d'après les faunes de micromammifères[END_REF], Estanque de Tormentas de Butarque H-02 [START_REF] Blain | A new middle Pleistocene (Marine Oxygen Isotope Stage 6) cold herpetofaunal assemblage from the central Iberian Peninsula (Manzanares Valley, Madrid)[END_REF][START_REF] Laplana | Biocronología de la Terraza Compleja de Butarque del río Manzanares en el Estanque de Tormentas al sur de Madrid (España)[END_REF] and Lezetxiki II (García-Ibaibarriaga et al. 2018) and one in Italy, Poggetti Vecchi [START_REF] Benvenuti | Paleoenvironmental context of the early Neanderthals of Poggetti Vecchi for the late middle Pleistocene of central Italy[END_REF]. Regarding the expansion of the continental glaciers in western Europe during MIS 6 [START_REF] Batchelor | The configuration of Northern Hemisphere ice sheets through the Quaternary[END_REF], the composition of the rodent assemblages of the aforementioned sites may be influenced by the position of the localities (Fig. 6).

Strictly cold species, such as Dicrostonyx torquatus (Artic lemming), Lasiopodomys gregalis and Lemmus lemmus (Norway lemming) are only represented in French sites, while in Iberian and Italian sites these species are not present, and the assemblages are composed mainly of temperate taxa.

Indeed, if we take the example of Baume Moula-Guercy, located in the Ardèche In Poggetti Vecchi Unit 2 the rodent assemblage is composed of Arvicola amphibius, Microtus (Terricola) sp. and Microtus cf. M. arvalis [START_REF] Benvenuti | Paleoenvironmental context of the early Neanderthals of Poggetti Vecchi for the late middle Pleistocene of central Italy[END_REF].

In H-02 the rodent assemblage is composed of Allocricetus bursae, Arvicola cf. sapidus.

Microtus (Microtus) arvalis, Microtus (Iberomys) brecciensis and A.gr. sylvaticusflavicollis [START_REF] Laplana | Biocronología de la Terraza Compleja de Butarque del río Manzanares en el Estanque de Tormentas al sur de Madrid (España)[END_REF]. In Maltravieso-SH the rodent assemblage is composed by Eliomys quercinus, M. (Ibeormys) brecciensis, Micortus (Terricola) cf.

duodecimcostatus, Allocricetus bursae and A.(Sylvaemus) sylvaticus [START_REF] Hanquet | Évolution des paléoenvironnements et des paléoclimats au Pléistocène moyen, en Europe méridionale, d'après les faunes de micromammifères[END_REF]. In the lower layers Lezetxiki II, the more abundant rodent species are A. amphibius, M.

(Agricola) agrestis and M. (Terricola) sp. (García-Ibaibarriaga et al. 2018). During MIS 6 Lazaret combines, the presence of strictly Mediterranean species, such as M. (I.)

brecciensis and M. (T.) savii with strictly cold species, such as L. gregalis. This phenomenon could be related to the position of the site, as previously suggested by [START_REF] Valensi | Cadre climatique et environnemental des acheuléens de la grotte du Lazaret, à Nice. Données paléontologiques, biochimiques et radiométriques établies sur les faunes de vertébrés et d'invertébrés[END_REF] and [START_REF] Desclaux | Evolution des communautés de mammifères dans les Alpes-Maritimes au cours du Pléistocène[END_REF], showing that the glacial expansion in the Maritime-Alps during MIS 6, caused changes in ecology and the area forms a transitional zone between the steppe identified in the southern limits of permafrost in France and the forest and grass identified in in the Italian peninsula (Fig. 6). The area therefore may have acted as a refugial leading edge for some more temperate species.

The nature of the Maritime-Alps coastal areas during MIS 6 was not the same during the cold stages of the Late Pleistocene. For example, during MIS 4, Mammuthus primigenius is observed in level 6 of Grotte du Prince and Foyer III of Grotte du Cavillon, in Ventimiglia, at the French-Italian border [START_REF] Moussous | Identification de l'ivoire de proboscidiens des grotte des Balzi Rossi (Ligurie, Italie) à partir de la méthode des lignes de Schreger[END_REF]. Furthermore, during MIS 2, the cold climate was also sufficiently marked to allow populations of Mammoths (Mammuthus primigenius) to reach the Mediterranean shores of Liguria, as evidenced for example by the observations of [START_REF] Braun | Mammuthus primigenius in the cave and portable art: an overview with a short account on the elephant fossil record in southern Europe during the last glacial[END_REF] in the Arene Candide cave in Finale Ligure, [START_REF] Onoratini | The Barma Grande cave (Grimaldi, Vintimiglia, Italy): From Neandertal, hunter of « Elephas antiquus » to Sapiens with ornaments of mammoth ivory[END_REF] in the Barma Grande, in Ventimiglia. It is possible that the occurrence of cold adapted taxa in Mediterranean coastal areas only took place during the Late Pleistocene and that during the Middle Pleistocene the glacial stadials did not have the same intensity, at least in this region of Europe.

Comparison with other environmental and climatic proxies in western Europe

Studies of marine cores, terrestrial pollen sequences and speleothems have been done in western Europe containing the MIS 6 fluctuations [START_REF] Margari | The nature of millennial-scale climate variability during the past two glacial periods[END_REF]2014[START_REF] Roucoux | Vegetation history of the penultimate glacial period (Marine Isotope Stage 6) at Ioannina, north-west Greece[END_REF][START_REF] Wilson | Limnetic and terrestrial responses to climate change during the onset of the penultimate glacial stage in NW Greece[END_REF][START_REF] Tzedakis | Comparison of changes in vegetation in northeast Greece with records of climate variability on orbital and suborbital frequencies over the last 450,000 years[END_REF][START_REF] Guiot | A 140,000-year continental climate reconstruction from two European pollen records[END_REF]1993[START_REF] Ponel | Rissian, Eemian and Wurmian Coleoptera assemblages from La Grande Pile (Vosges, France)[END_REF][START_REF] Bard | Hydrological conditions over the western Mediterranean basin during the deposition of the cold Sapropel 6 (ca. 175 kyr BP)[END_REF][START_REF] Hodge | Effective precipitation in southern Spain (similar to 266 to 46 ka) based on a speleothem stable carbon isotope record[END_REF][START_REF] Wainer | Millennial climatic instability during penultimate glacial period recorded on a southwestern France speleothem[END_REF].

The analysis of pollen from the marine core MD01-2444 off the Portuguese coast divided the penultimate glacial, on basis of the amplitude of the millennial-scale variability in two main periods (one between 185 to 160 Kya and the other one between 150-135 Kya), with a transitional period (between 160-150 Kya). The early period is mainly characterized by a prominent oscillation in foraminiferal isotopes and tree pollen values while the last period is mainly characterized by mild temperature swings in the Antarctic and minimum tree pollen values. This suggests that MIS 6 is characterized by cool climatic conditions with an open forest environment [START_REF] Margari | The nature of millennial-scale climate variability during the past two glacial periods[END_REF]2014).

The terrestrial pollen sequences of Ioannina and Tenaghi Philippon in Greece suggest generally cool and wet conditions for the MIS 6 [START_REF] Roucoux | Vegetation history of the penultimate glacial period (Marine Isotope Stage 6) at Ioannina, north-west Greece[END_REF][START_REF] Wilson | Limnetic and terrestrial responses to climate change during the onset of the penultimate glacial stage in NW Greece[END_REF][START_REF] Tzedakis | Comparison of changes in vegetation in northeast Greece with records of climate variability on orbital and suborbital frequencies over the last 450,000 years[END_REF]. Similarly, the French pollen sequences of Les Echets and Grande Pile, have mean temperatures and precipitation estimated suggesting MAT between 12 ºC and 4 ºC and MAP between 200 mm and 800 mm lower than at present [START_REF] Guiot | A 140,000-year continental climate reconstruction from two European pollen records[END_REF]1993[START_REF] Ponel | Rissian, Eemian and Wurmian Coleoptera assemblages from La Grande Pile (Vosges, France)[END_REF].

The speleothem data coming from the Italian Argentarola cave, the Spanish Gitana cave and French Villara cave, show lower rainfall values during glacial periods and specifically characterize MIS 6 by cool and humid climatic conditions [START_REF] Bard | Hydrological conditions over the western Mediterranean basin during the deposition of the cold Sapropel 6 (ca. 175 kyr BP)[END_REF][START_REF] Hodge | Effective precipitation in southern Spain (similar to 266 to 46 ka) based on a speleothem stable carbon isotope record[END_REF][START_REF] Wainer | Millennial climatic instability during penultimate glacial period recorded on a southwestern France speleothem[END_REF].

These data are consistent with the results obtained here where different methods were applied to the rodent assemblages of Lazaret cave. On one side, the methods applied to estimate climatic parameters show a generally cold conditions with relatively low rainfall for all subcomplexes analyzed. On the other hand, the methods applied to investigate the vegetational landscape show a habitat dominated by deciduous temperate forest with humid meadows including relatively high proportions of scrublands (Fig. 4; 5).

The apparent difference in results between the climatic parameter method and the landscape representation method could be due to a difference in rainfall regime predicted.

Even if the rainfall is lower, however, the way in which precipitation is distributed throughout the years may have more impact on the vegetation. Unfortunately, it is currently not possible to reconstruct this variable.

Conclusions

On basis of a multi-method approach using the small-vertebrate assemblages of Lazaret cave MIS 6 sequence to reconstruct the past climate and environment, our analysis enables the following conclusions to be drawn:

1) The set of the used methods applied to the small-vertebrate associations (mainly rodents and herpetofauna) from the MIS 6 Lazaret cave sequence suggest a cold climate with a relatively humid environment dominated by deciduous temperate forest.

2) The comparison with other proxies (large mammals, birds and marine gastropods) identified at Lazaret cave shows, in general, the same environmental and climatic trend (cold climate and relatively humid environmental conditions) as identified using the small vertebrate assemblages.

3) [START_REF] Desclaux | Étude préliminaire des micromammifères de la Baume Moula-Guercy à Soyons (Ardèche, France). Systématique, biostratigraphie et paléoécologie[END_REF], Cèdres (Defleur and Crégut-Bonnoure, 1995), Romain-la-Roche [START_REF] Guérin | Le gisement Pléistocène Moyen récent de Romain-la-Roche (Doubs, France): Synthèse biostratigraphique et paléoécologique[END_REF], Codoulous [START_REF] Jaubert | Coudoulous I (Tour-de-Faure, Lot), site du Pléistocène moyen en Quercy[END_REF], Poggetti Vecchi [START_REF] Benvenuti | Paleoenvironmental context of the early Neanderthals of Poggetti Vecchi for the late middle Pleistocene of central Italy[END_REF]. Pollen sequences: MD01-2444 [START_REF] Margari | Land-ocean changes on orbital and millennial timescales and the penultimate glaciation[END_REF], Les Echets [START_REF] Guiot | The climate in western Europe during the last glacial/interglacial cycle derived from pollen and insect remains[END_REF], La Grande Pile [START_REF] Ponel | Rissian, Eemian and Wurmian Coleoptera assemblages from La Grande Pile (Vosges, France)[END_REF], Ioannina [START_REF] Roucoux | Vegetation history of the penultimate glacial period (Marine Isotope Stage 6) at Ioannina, north-west Greece[END_REF], Tenaghi Philippon [START_REF] Tzedakis | Comparison of changes in vegetation in northeast Greece with records of climate variability on orbital and suborbital frequencies over the last 450,000 years[END_REF]. Speleothem data : Gitana cave [START_REF] Hodge | Effective precipitation in southern Spain (similar to 266 to 46 ka) based on a speleothem stable carbon isotope record[END_REF], Villars cave [START_REF] Wainer | Millennial climatic instability during penultimate glacial period recorded on a southwestern France speleothem[END_REF], Argentola cave [START_REF] Bard | Hydrological conditions over the western Mediterranean basin during the deposition of the cold Sapropel 6 (ca. 175 kyr BP)[END_REF]. Poggetti Vecchi; 7. Lezetxiki II; 8. Maltravieso-Sala de los Huseos; 9. Estanque de Tormentas Butarque (H-02)

Table 1. Minimum number of individuals (MNI) and percentage of minimum number of individuals (% MNI) of the rodent's assemblage from the different sub-complexes of Lazaret cave.

The

  BM was established byHernández-Fernández (2001a and b) and we use the recently updated R script PalBER published in[START_REF] Royer | New bioclimatic models for the quaternary palaearctic based on insectivore and rodent communities[END_REF]. This method is based on the hypothesis that a significant correlation exists between climate and mammal communities. According toHernández-Fernández (2001a and b);[START_REF] Hernández-Fernández | Quantitative palaeoclimatic inference based on mammal faunas[END_REF] and[START_REF] Hernández-Fernández | Bioclimatic analysis of rodent palaeofaunas reveals severe climatic changes in Southwestern Europe during the Plio-Pleistocene[END_REF], mammal assemblages can be assigned to ten climatic types, five of which are represented by the rodent assemblages of Lazaret cave. These assemblages were analyzed using the Climatic

  2 ºC; ΔMATCIII= -6.35 ºC and -6.2 ºC) and with relatively lower rainfall (ΔMAPCII = -294 mm and -201 mm; ΔMAPCIII = -243 mm and -210 mm). The herpetofaunal assemblages also suggest cold climate conditions with somewhat less harsh temperatures than reconstructed by the rodents (ΔMATCIIsup = -4.4°C and -3.8ºC; ΔMATCIIinf = -3.8°C and -4.5ºC; ΔMATCIII= -4,0ºC and -4.1ºC). They also indicated different results for rainfall.The MER reconstructed MAP suggests slightly more humid conditions (ΔMAPCIIsup = +70.4 mm; ΔMAPCIIinf = +15.9 mm; ΔMAPCIII = +18 mm), whereas the QE reconstructed rainfall suggests a dryer climate (ΔMAPCIIsup = -123.7 mm; ΔMAPCIIinf = -104.6 mm; ΔMAPCIII = -149.2 mm) than reconstructed using the small mammals. Taking into account the chronological placement of these sub complexes, CII and CIII (between ca.

  querciuns, G. glis, M. avellanarius, Microtus (Iberomys) brecciencis, A. mosbachensis and Pliomys sp. nov.. The Shrubland category is mainly represented by G. glis, Cricetus cricetus, Microtus (M.) arvalis, M. (I.) brecciensis and Pliomys sp. nov.. Finally, the humid habitat is mainly represented by C. cricetus and M. (A.) agrestis. M. (A.) agrestis is responsible for the percentage representation of humid meadows, between 48-58%, obtained with the Climatogram method (Fig. 4; Table

(

  France), along the Rhône corridor rodent species characteristic of open environments and cold climates, such as Dicrostonyx torquatus, Allocricetus bursae, Lasiopodomys gregalis and Sicista betulina (northern birch mice) are represented in levels assigned to MIS 6 (XIX to XVI). These small mammals are associated with large mammals such Rangifer tarandus (reindeer) and Mammuthus primigenius (woolly mammoth). Further south, in Provence, in the Grotte des Cèdres, these cold species are however absent in the levels dating from MIS 6 (Defleur and Crégut-Bonnoure, 1995). Rodent associations are present at the Grotte des Cèdres with a more temperate character (such as M. (I.) brecciensis, M. (A.) agrestis, Eliomys quercinus, M. (T.) duodecimcostatus and A. (S.) sylvaticus), similar to what is observed in the CIII and CII complexes of the Lazaret cave. It would therefore indicate that a latitudinal gradient was relatively well pronounced in south-eastern France during the late middle Pleistocene (MIS 6).

Figure 2 .

 2 Figure 2. Stratigraphic section of Lazaret cave. ESR ages taken from Michel et al. (2009).

Figure 3 .

 3 Figure 3. Some rodent's teeth identified from Lazaret cave. 1-4. m1 left Microtus (Agricola) agrestis; 5-7 m1, two left-one right Microtus (Terricola) multiplex; 8-11. m1, two left-two right Arvicola mosbachensis; 12-13. M11, one right-one left Pliomyssp. nov.; 14-15. M1 and M2 right and M1 left Apodemus (Sylvaemus) sylvaticus; 16. m1 right Cricetus cricetus. All teeth are in occlusal view.

Figure 4 .

 4 Figure 4. Comparison of the climate and landscape values obtained with the rodent's assemblages from the different complex and sub-complex of Lazaret Cave with the 18 O isotope curve values (modified from Railsback et al. 2015) for the MIS 6. BM: Bioclimatic Model; QE: Quantified Ecology; MAT: Mean Annual Temperature; MAP: Mean Annual Precipitation; TF: Temperate forest; RF: Riparian Forest; HM; Humid Meadow; DF: Deciduous Forest; HH: Humid Habitat; Sh: Shrubland; Med: Mediterranean; THI: Taxonomic Habitat Index.

Figure 5 .

 5 Figure 5. Values obtained of the Simpson diversity index (1-D) for the sub-complexes of Lazaret cave. MNI: Minimum Number of Individuals

  as well as sites in the Iberian Peninsula such as Lezetxki II in the Basque Country (Garcia-Ibaibarriaga et al. 2018). Both species are relatively abundant later on in western Europe during the late Pleistocene, with records in the Italian and Iberian Peninsula as well as in southernFrance (e.g.[START_REF] Royer | Investigating the Influence of climate changes on rodent communities at a regional-scale (MIS 1-3, southwestern France)[END_REF][START_REF] Berto | Changes in the Late Pleistocene smallmammal distribution in the Italian Peninsula[END_REF][START_REF] López-García | Los micromamíferos del Pleistoceno Superior de la Península Ibérica. Evolución de la diversidad taxonómica y cambios paleo-ambientales[END_REF]. On the other hand, while A. (S.) sylvaticus is currently present across the whole of France, M. Hyla sp. are only recorded from CII sup. and Pelobates cultripes is absent from CIII. For squamate reptiles, CII inf. is the least diverse assemblage with an absence of Timon Podarcis is absent from CII sup. and the snakes Hierophis viridiflavus and cf. Zamenis longissimus are not recorded in CIII (Table2).

(A.)

agrestis is probably absent in the Maritime-Alps region (https://inpn.mnhn.fr). Moreover, while A. (S.) sylvaticus is an adaptable species, inhabiting a wide variety habitats including all types of woodland

[START_REF] Torre | Ratón de campo (Apodemus sylvaticus Linnaeua 1758)[END_REF]

, M. (A.) agrestis mainly inhabits wet meadow areas and forests with dense herbaceous understories

(Krystufek et al. 2016)

, suggesting that humid and open woodland landscapes dominated at the time the accumulation of rodents in sub-complexes CII inf., CII sup. and CIII of the Lazaret cave were being formed.

Among the herpetofauna, five taxa are the most abundant in the Lazaret assemblage, including Bufo bufo, Rana temporaria, Angis fragilis, Coronella austriaca and Vipera spp. Their presence in the Mediterranean region usually indicates a colder climate with a humid environment in a landscape composed of mountain and valley habitats

[START_REF] Valensi | Cadre climatique et environnemental des acheuléens de la grotte du Lazaret, à Nice. Données paléontologiques, biochimiques et radiométriques établies sur les faunes de vertébrés et d'invertébrés[END_REF]

). The distribution of herpetofaunal species among the sub-complexes is relatively homogeneous. However, urodeles (Ichtyosauria alpestris and Salamandridae indet.) and lepidus, Malpolon monspessulanus, Natrix gr. natrix, Coronella cf. girondica and Zamenis scalaris which are present in the other sub-complexes. The absence of such Mediterranean species may suggest that CII inf. would be colder than the other sub-complexes. However,

  The same issue is observed concerning precipitation. MER suggests a globally more humid climate whereas QE a slightly dryer climate, but again most of the differences observed falls within the standard deviation. In conclusion, climate indications obtained from the QE method on herpetofauna is more in accordance with the occurrence of thermophilus, typically Tetrao tetrix[START_REF] Hanquet | Caractérisation du climat et de la biodiversité au Pléistocène moyen final, d'après les faunes de vertébrés de la grotte du Lazaret[END_REF]. In general, the avifaunal assemblage shows a predominance of species from cold climates and open environments, together with species related to temperate climates and open environments as well as rock and mountain habitats, with a remarkable closing of the vegetational landscape between

Mediterranean, species (T. lepidus, M. monspessulanus and Z. scalaris) in the different sub-complexes and in line with the results obtained by

[START_REF] Manzano | Les amphibiens et les reptiles des sites Pléistocène moyen et supérieur de la France méditerranéenne[END_REF]

at a regional level.

The large mammal assemblage has a faunal composition dominated by Cervus elaphus and Capra ibex. More specifically the sub-complex CIIsup. is composed of more temperate species, with the abundance of C. elaphus, the presence of Bos primigenius and Capreolus capreolus and a lower representation of C. ibex, than sub complex CIII, where Rangifer tarandus and Bison priscus are well represented and C. ibex is more abundant

[START_REF] Valensi | Cadre climatique et environnemental des acheuléens de la grotte du Lazaret, à Nice. Données paléontologiques, biochimiques et radiométriques établies sur les faunes de vertébrés et d'invertébrés[END_REF]

). The avifaunal assemblage of sub-complexes CIIsup. and CIII has sedentary species associated with cold climates such as Aegolius funereus, Bubo scandiaca and

  The comparison with other MIS 6 sites in western Europe that include small vertebrate studies, shows that, although Lazaret cave is more similar to Mediterranean sites, than to sites in central France, it combines the presence of Mediterranean species, such as M. (I.) brecciensis or M. (T.) savii with strictly cold species, like L. gregalis. This could be related to the geographical position of the cave, that provides for this period, with the glacial expansion in the Maritime-Alps, a transitional zone between the French cold steppe and the warmer

	Southern Europe during Marine Isotope Stage 6. Quaternary Science Advances 3,
	Italian forest-grassland. 100016.
	4) Finally, the comparison with other climatic and environmental proxies (marine
	cores, terrestrial pollen sequences and speleothems) in western Europe
	demonstrates similar environmental (open forest landscape) and climatic
	conditions (cool and relatively wet climate with a relatively low rainfall values)
	as those detected using the small-vertebrate assemblages from Lazaret cave during
	MIS 6.
	Figure 1. Location of the Lazaret cave and the other MIS 6 sites mentioned in the
	manuscript. Archaeological sites: MTV-SH (Hanquet, 2011), H-02 (ETB) (Blain et al.
	2017; Laplana et al. 2015), Lezetxiki II (García-Ibaibarriaga et al. 2018), Moula-Guercy

Table 2 .

 2 Herpetofaunal presence/absence faunal list from the different sub-complexes of Lazaret cave

Table 3 .

 3 Repartition of the rodent species according to the Taxonomic Habitat Index

	(THI)

Table 4 .

 4 Repartition of the rodent's species according to the Climatogram method

Table 5 .

 5 Repartition of rodent's species in climates types according to the Bioclimatic Model. IV Subtropical with winter rains and summer droughts; VI Typical temperate; VII Arid temperate; VIII Cold-temperate (boreal) and IX Polar.

Table 6 .

 6 Repartition of small-vertebrate species by mean temperature and mean precipitation according to the Quantified Ecology method.

Table 7 .

 7 Comparison of the data obtained by means of the various climatic methods used applied to the small vertebrate assemblage's sub-complexes of Lazaret cave with the current mean temperature and precipitation. MAT: Mean Annual Temperature; MAP: Mean Annual Precipitation; SD: Standard deviation of the obtained values; Δ: difference with the nowadays values.

Table 8 .

 8 Repartition of rodent's species by climate types to obtain the Bioclimatic component (BC) values. CRI: Climatic Restriction Index; S: total number of the species; IV Subtropical with winter rains and summer droughts; VI Typical temperate; VII Arid temperate; VIII Cold-temperate (boreal) and IX Polar.

Table 9 .

 9 Repartition of the percentages obtained with the Taxonomic Habitat Index (THI) applied to the Lazaret cave rodent's assemblages

Table 10 .

 10 Repartition of the percentages obtained with the Climatogram method applied to the Lazaret cave rodent's assemblages

Acknowledgments

This research was supported by the 2017-SGR-836 and 2017-SGR-859 projects of the Catalonian Government. J.M.L.-G. was supported by a Ramon y Cajal contract (RYC-2016-19386), with financial sponsorship of the Spanish Ministry of Science and Innovation. This publication is part of the collective research project named "Paleoecology of the Lazaret cave: human-environment interactions on the coast of the meridional Alps during the late Middle Pleistocene (MIS6)", granted by the DRAC PACA (French Ministry of Culture). We want also to thank the editors Prof. Thomas Algeo and Dr. Howard Falcon-Lang and the two anonymous reviewers for their suggestions and comments that improved the final version of the manuscript.


Youness Ezziani

Mounsif Pooyan Manoochehrnia

Ech-Cherif El-Kettani

Damien Leduc

Pierre Marechal

Mathieu Ducousso

Nicolas Cuvillier

Characterization of Multilayer Structures by Acoustic Microscopy

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

DISCUSSION / CONCLUSION METHODS AND MATERIALS Introduction

Characterization of Multilayer Structures by Acoustic Microscopy

Youness EZZIANI (1) , Pooyan MANOOCHEHRNIA (1) , Mounsif ECH-CHERIF EL-KETTANI, (1) Damien LEDUC (1) , Pierre MARECHAL (1) , Mathieu DUCOUSSO (2) , Nicolas CUVILLIER ( 2

g T T R R R                            2 0 1 2 1 0 0 ( ) cos 2

  ) , (1) Laboratory of Waves and Complex Media (LOMC UMR 6294), Le Havre Normandy University, Le Havre, France (2) Safran Tech, 78772 Magny-les-Hameaux, France Contact RESULTS for the Bilayer Trilayer structure: epoxy film thickness and adhesion qualityEpoxy/Composite), 50 MHz plane transducer -Quantification with X-scan imaging Bilayer structure: Estimation of local material geometric & elastic parameters?? -85µm thick Resin-Epoxy -50 MHz plane transducer -Model: Debye Series Model (DSM) formalism: of the thickness of the adhesive for two samples with A-scan and X-scan Imaging (135 µm for Sample 1, 69 µm for sample 2) -Sample 1 (135 µm epoxy): High background Epoxy amplitude, and composite is less visible and on few windows of the X-scan. -Sample 2 (69 µm epoxy): low background Epoxy amplitude, and composite clearly visible on several windows of the X-scan. the background echo amplitude of the epoxy film depends on the epoxy-composite interface and, consequently, on the adhesion quality. We can conclude that the adhesion quality of sample 2 is better than that of sample 1. Bilayer: -Estimation of elastic and geometric parameters of thin layer by analysis of specular insonation of thin layer using SAM -Inverse problem algorithm used for estimation of best match between parameters 𝒆 𝑬𝒑𝒐𝒙𝒚 = 𝒄 𝑳 𝑬𝒑𝒐𝒙𝒚 × 𝜟𝒕 𝒄𝒐𝒓𝒓 𝟐 ≅ 𝟎. 𝟏𝟑𝟓𝟕 𝒎𝒎 𝒆 𝑬𝒑𝒐𝒙𝒚 = 𝒄 𝑳 𝑬𝒑𝒐𝒙𝒚 × 𝜟𝒕 𝒄𝒐𝒓𝒓 𝟐 ≅ 𝟎. 𝟎𝟔𝟗 𝒎𝒎 Size of the X-scan gate = 20 ns  d(Epoxy)=23 µm , d(Composite) = 29.2 µm Epoxy medium : 3 Gates (C-scan 1 => C-scan 3)  epoxy = 3*23 = 69 µm Mounsif ECH-CHERIF EL-KETTANI, Pr Head of Acoustic of Materials and Structures team(AMS), LOMC UMR 6294 75 RUE BELLOT, 76600 LE HAVRE echcherm@univ-lehavre.fr +33649283051 TA6V Epoxy Composite -Ultrasonic acoustic methods have the advantage of being non-intrusive with respect to the materials tested, rapid and inexpensive implementation.-Acoustic microscopy: characterization and imaging with high resolution (10ns-100ds MHz) for a wide range of materials and structures .

  =2500 [m/s] • ρ=1300 [kg/m 3 ] • Th=85 [µm] • δ=1.5e -5 • α=3.5e -8[Np/(m.Hz)] Matched model parameters:• C L =2500*1.07 [m/s] • ρ=1300*0.75 [kg/m 3 ] • Th=85*0.85*0.93 [µm] • δ=1.5e -2 • α=3.5e -5[Np/(m.Hz)]