Bruno Teste

Jerome Champ

Arturo Londono-Vallejo

Stéphanie Descroix

Laurent Malaquin

Jean-Louis Viovy

Irena Draskovic

Guillaume Mottet
email: guillaume.mottet@pasteur.fr

Lab Chip

Chromatin immunoprecipitation in microfluidic droplets: towards fast and cheap analyses

come   L'archive ouverte pluridisciplinaire

I. Introduction

Chromatin is composed of DNA, RNA and accessory proteins such as histones 1,2 . Chromatin remodelling occurs through epigenetic processes and influences gene regulation. Some epigenetic changes have been strongly associated with human pathologies, including cancer 3 and Huntington's disease 4 , by promoting aberrant gene expression. Altered epigenetic processes may therefore serve as potential biomarkers in medical diagnostics; and furthermore offer new therapeutic targets. These processes are mainly based on DNA methylation and modifications to histone proteins 5,6 including acetylation, methylation, phosphorylation, ubiquitination, sumoylation, deimination, isomerization and poly-ADP-ribosylation. Such modifications will affect protein/DNA interactions, thereby influencing the accessibility of the locus to transcription machinery. Yet how these processes occur to regulate gene expression in the cell is not fully understood. Chromatin immunoprecipitation (ChIP) assays are conventional and widely used techniques to investigate protein-DNA interactions at specific locations in the genome 7 . Briefly, the standard ChIP protocol consists of fixation and lysis of the cells of interest before chromatin fragmentation. Then beads grafted with anti-histone antibodies or other direct DNA-binders are used to specifically extract the target chromatin. Finally, the DNA is released, purified and analysed by quantitative-PCR, hybridization or next generation sequencing. Increased accessibility of the latter contributes in a large part to the renewed interest in developing more efficient ChIP assays 8 . Conventional ChIP assays suffer from three main drawbacks. First, the procedure requires a large number of cells (10 6 -10 7 cells) due to cell loss and inefficiency of chromatin extraction, which is a considerable limitation for studying rare cell populations as in stem cell differentiation, embryo development or oncogenesis. Secondly, the ChIP protocol is both time consuming and technically challenging, and the entire procedure can last from 2 to 7 days. Finally, standard ChIP assays offer only low throughput analyses. There has been substantial efforts in the recent years to improve ChIP performances by reducing the requisite number of cells [9][10][11] , by shortening the procedure 12 and by increasing assay throughput 13 . Nevertheless, it is not yet possible to simultaneously overcome these limitations to obtain a ChIP procedure that may be performed rapidly, easily and on small numbers of cells. Microfluidics-based technologies appeared as a powerful tool to overcome ChIP limitations since they reduce sample consumption and allow the integration, automation and high throughput of multi-step analytical procedures. Microfluidics has been largely developed for proteins 14 , DNA, RNA and cells analyses 15 but very few studies report on ChIP integration at a microfluidic scale. Wu et al. developed an automated microfluidic-based platform to perform 16 assays simultaneously using only 10 000 cells for each experiment, by achieving increased chromatin yield during the immunoprecipitation steps. This integrated approach offers greater sensitivity than conventional procedures 16 . Geng et al. developed an assay that permits the analysis of histone modifications from as few as 50 cells by using magnetic beads in a microfluidic chamber 17 . More recently, Cao et al. combined packed beads and oscillatory washing to effectively remove nonspecific adsorption and obtain high yields of highly enriched DNA 18 . The same team proposed also another chip based on a microchamber integrating an ultrasonic transducer. The internal sonication enables both shearing of chromatin/DNA and mixing/washing of immunoprecipitation beads. This chip allowed sensitive tests starting with 100 cross-linked cells 19 . Although such microfluidic strategies have improved chromatin extraction efficiency from small numbers of cells, the integrated procedure on a chip has been limited to cell lysis and chromatin extraction, and does not include the subsequent steps: reverse cross-linking of DNA from proteins and DNA purification. These latter two steps are particularly time consuming and require organic solvents (chloroform, phenol, alcohol).

In this context, we developed an integrated ChIP procedure, from chromatin immunoprecipitation to DNA purification, using a lab on tube platform that has been previously described (see Fig. 1, Supplementary Fig. 2) [20][21][22][23] . This platform comprises a single 300 µm diameter Teflon tubing and a syringe pump that enables the back and forth movement of droplets. Given that droplets are confined within the tube, internal flows occurring within each droplet improve the internal mixing of reagents. This platform also takes advantage of magnetic beads (MB) and a single pair of magnetic tweezers (MT). MB within the droplets are used as a solid support to transfer the target DNA from droplet to droplet thanks to the activation/deactivation of MT. As such, this platform accomplishes the sequential steps of chromatin extraction, reverse cross-linking and DNA purification. As described above, previously developed microfluidic systems improve cell lysis and chromatin extraction efficiency. While allowing the manipulation of a small number of cells, these approaches remain expensive, complex and limited to the first step of the ChIP procedure. The platform we describe herein offers an automated, cheap and simple analytical procedure, despite not yet Fig. 1. Schematic of the experimental set-up. Teflon tubing was used to generate trains of droplets by sequentially aspirating aqueous (reagents and samples) and oil solutions from a microtiter plate using a syringe pump combined with a pipetting robot. The train of droplets was generated in reverse order based on the syringe pump aspirating mode and then pushed through the tubing toward the magnetic tweezers in order to perform the subsequent steps of the analytical procedure. Activation and deactivation of magnetic tweezers allowed extraction and redispersion of magnetic beads within the droplets. Handling of droplets within the tube was achieved by back and forth movements generated by switching from injection to aspiration mode on the syringe pump.

achieving the high performance of other assays in terms of cell numbers. Moreover, the compartmentalization within nanoliter droplets (i) prevents cross contamination, (ii) quickens reaction kinetics due to internal recirculation flows, (iii) reduces samples and reagents consumption and (iv) simplifies the procedure compared to a conventional ChIP protocol that requires skilled operators. Finally, the combination of tubing, droplets and MB offers multiple trapping-release sequences, which is of great importance in multi-step analytical procedures. In this way, the droplet platform we propose successfully integrates the whole procedure from chromatin extraction to DNA purification, whereas conventional monophasic microfluidic is limited to few steps of the whole analytical procedure.

In this work we investigated the influence of MB, antibodies and initial chromatin quantities on ChIP assay performances. Then we used this novel droplet ChIP assay to analyse histone modifications at the mir-200b gene region from human fibrosarcoma HT1080 cells. Finally, we compared the analytical performances and the results concerning histone modifications from ChIP in droplets with the performances of a conventional ChIP approach.

II. Materials and methods

A Cell culture and Chromatin preparation HT1080, a human fibrosarcoma cell line, was cultured in DMEM GlutaMAX medium (Gibco) supplemented with 10% fetal calf serum (Dako), 1% non-essential amino acids and 1 mM pyruvate (both Gibco). For chromatin preparation cells were seeded in 300 cm 2 flasks at about 40% confluency 24 h prior to the experiment. For chromatin preparations, cells were fixed directly in culture dishes with 1% formaldehyde (Thermo Scientific) for 10 min at 37°C. For reproducible fixation, a freshly prepared crosslinking solution containing 10% formaldehyde, 50 mM Tris pH 8, 100 mM NaCl, 1 mM EDTA and 0.5 mM EGTA was diluted 10-fold in cell culture medium. Fixation was stopped by the addition of 1/20 th of the volume of 2.5 M glycine and subsequent incubation at room temperature for 5 min. Following three washes in ice-cold phosphate buffered saline (PBS) cells were recovered by scraping and washed once in cold PBS supplemented with protease inhibitors (Roche). Nuclei were prepared by homogenization in cold cell lysis buffer (5 mM PIPES pH 8, 85 mM KCl, 0.5% NP-40) and centrifugation at 1800 g for 10 min at 4°C. Finally, nuclei prepared from two 300 cm 2 flasks were lyzed in 1 ml nuclei lysis buffer (50 mM Tris-HCl pH 8, 10 mM EDTA pH 8, 1% SDS) at room temperature and lysates were sonicated with Bioruptor Plus (Diagenode) for 30 cycles of 30 sec on, 30 sec off, at 4°C. Following a centrifugation at 12,000 g for 10 min at 4°C, the cleared supernatants were snapfrozen in liquid nitrogen and stored at -80°C. The shearing efficiency was monitored by agarose gel electrophoresis after decrosslinking and purification of DNA. We routinely obtain chromatin preparations sheared to 250 to 500 bp.

B Antibodies for ChIP

The following antibodies were used (all rabbit): IgG (abcam, ab37415, lot GR135254, 5 mg/ml), anti-H3 (abcam, ab1791, lot GR50122, 1 mg/ml), anti-H3K4me3 (Active motif, 39159, lot 01609004, concentration unspecified), anti-H3K9ac (Millipore, 17-658, lot 2035642, 1 mg/ml), anti-H3K9me3 (abcam, ab8898, lot GF102533, 1 mg/ml), anti-H3K27me3 (Ozyme, 9733S, lot 6, concentration unspecified).

C Conventional ChIP

Preparation of blocked beads: Dynabeads Protein A (Invitrogen, 50% slurry, 30 mg/ml beads) were washed three time with 1X ChIP dilution buffer (150 mM Tris-HCl pH 8, 15 mM NaCl, 1 mM EDTA pH 8, 1% Triton X-100, 0.01% SDS) and blocked in 1X ChIP dilution buffer containing 1 mg/ml of BSA for 2 hours at 4°C on the rotating wheel. After three washes with 1X ChIP dilution buffer, beads were reconstituted back to 50% slurry using this buffer. For chromatin immunoprecipitations, 30 µg of sonicated chromatin was diluted 10 fold in 1.1X ChIP dilution buffer and incubated for 2 hours with 20 µl of blocked beads while rotating at 4°C. After beads were discarded this pre-cleared chromatin was incubated overnight with an antibody of interest (2.5 µg or 10 µl when concentration unspecified). Chromatin-antibody complexes were pulled down using 30 µL of blocked beads (2 hours incubation at 4°C while rotating). The beads were washed for 10 min at 4°C in each of the following washing buffers: Low Salt Buffer (20 mM Tris-HCl pH 8, 150 mM NaCl, 2 mM EDTA pH 8, 1% Triton X-100, 0.1% SDS), High Salt Buffer (20 mM Tris-HCl pH 8, 500 mM NaCl, 2 mM EDTA pH 8, 1% Triton X-100, 0.1% SDS), LiCl Buffer (10 mM Tris-HCl pH 8, 0.25 M LiCl, 1 mM EDTA pH 8, 1% NP-40, 1% Na Deoxycholate) and twice, 5 min each, in TE. ChIPed material was eluted by two 15 min incubations at room temperature with 225 μl elution buffer (0.1 M NaHCO3, 1% SDS). Chromatin was reverse-crosslinked by adding 20 μl of 5 M NaCl and incubated over night at 65°C. DNA was submitted to RNase A digestion (at 0.4 mg/ml for 30 min at 37°C) and proteinase K digestion (at 0.08 mg/ml for 1 h at 42°C). DNA was extracted by UltraPure pheno:chloroform:isoamyl alcohol 25:24:1 (Thermo Fisher) and chloroform:isoamyl alcohol 24:1 (Sigma). DNA was precipitated with sodium acetate (1/10 v/v) and ethanol (2 v/v) over night at -20°C prior to microcentrifugation for 30 min at 16,000 g at 4°C. Precipitated DNA was washed with 70% ethanol and recentrifuged for 10 min under the same conditions. Dried DNA pellets were resuspended in 40 µl water.

D Droplet-based ChIP assay

The analytical procedure within the droplets is depicted on Fig. 2 (see supporting information for a more detailed procedure). Fluid and magnetic beads handling were performed using a previously described platform 20,21 . Briefly, Teflon tubing was inserted in between two magnetic tips made of Fe-Co soft magnetic alloys (AFK502, Imphy Alloys Arcelor Mittal). An electromagnetic coil (33.5 mm diameter, 6 cm long) made of 1000 loops of copper wire (0.8 mm diameter) was used to induce a magnetic excitation in one of the tips by applying current generated from adjustable power supply (0-4 A, 0-25 V). The second magnetic tip was placed in front of the first tip and was magnetized by influence. MB within the droplets were magnetically confined by switching on the current (2A) and released in another droplet by switching off the current. After the release internal recirculation flows in the droplet induce shear forces that favor MB redispersion. Trains of aqueous droplets were generated by sequentially aspirating 100 nL of aqueous suspension (reagents and samples) and 200 nL of fluorinated oil (FC-40, 3M) mixed with 1H,1H,2H,2Hperfluorodecan-1-ol, surfactant (Fluorochem) at 2 % (w/w) in Teflon tubing (300 μm ID and 600 μm OD, Sigma-Aldrich) used as microchannel. Fluid handling was performed using a syringe pump (Nemesys, Cetoni GmbH) with a 1-mL syringe (internal diameter of 0.4 mm). The sequential generation of the droplets train was monitored using a three-axis robot to sample the different reagents and samples (RotAXYs, Cetoni GmbH). Each reagent, sample and oil solutions, were previously stored in separate wells of a microtiter plate. The droplets were injected in the microfluidic device at 5 μL/s and were visualized using a portable USB camera (Dino-lite). A photo of the platform is provided in the supporting information (Supplementary Fig. 2). The procedure was performed in two steps since two different trains of droplets were generated. These trains were generated in reverse order and then pushed within the tube. The first one was generated in the following order: (1) one droplet of proteinase K (20 mg/mL) diluted in citrate buffer at pH 6.5; (2) one droplet of RNase (10 mg/mL); (3) five droplets of washing buffers (see section C and supporting information for more details); (4) one droplet containing a mix of anti-histone antibodies (from 10 -2 to 4 µg), magnetic beads (from 1 to 15 µg) and chromatin sample (from 0.1 to 12 µg), this mix was prepared and incubated for 4h at 4°C before droplets generation. The process optimization was performed with anti-H3 antibodies while histones modification analyses were performed with anti-H3K4me3, anti-H3K9ac, anti-H3K9me3 and anti-H3K27me3 antibodies. Non-specific signal was evaluated using rabbit IgG antibody. All the incubation steps were performed with forth and back moving droplets at a flow rate of 5 µL/s (droplet velocity of ~3x10 -3 m/s) thus allowing internal fluids recirculation and improving kinetics reactions. The first droplet containing the captured target chromatin on MB was confined between the magnetic tweezers and the five droplets of washing buffers passed through the MB cluster. The MB were then released in the RNAse droplet for 5 min. Finally, MB were extracted and transferred into the Proteinase K droplet for 5 min at 70°C. This step allows the release of DNA from the proteins. After these incubations all the droplets except the proteinase K droplet containing the DNA were discarded from the tube. A second train of droplets was generated in the tube containing the proteinase K droplet (with DNA). This 2 nd train of droplet was also generated in reverse order by using the ThermoFisher scientific ChargeSwitch ® gDNA Tissue Kit. The train of droplets was made as follows: one droplet of elution buffer, three droplets of washing buffer and one droplet of charge switch magnetic beads (CSMB). The CSMB were transferred into the proteinase K droplet to capture the DNA for 10 min. Then the CSMB were extracted and washed with the droplet of washing buffer. Finally, the CSMB were released in the elution buffer and after 10 min of incubation, the CSMB were removed and the purified DNA droplet was collected in 5 µL of water in a tube. The technique can be operated manually or automatically 23 .

E Quantitative (q)-PCR assay

Purified DNA recovered by ChIP was analyzed by real-time q-PCR. The DNA quantity was estimated from a qPCR standard curve generated from serial dilution of known DNA quantities. Samples were analyzed using the qPCR mix from GoTaq Promega Kit in the final volume of 25 µl using up to 5 µL of purified sample DNA, 12.5 µL of GoTaq mix and 1 µL of each primer, #989 (ACGCAGAGGGAAGAACCTG) and #990 (GACGGGCTTAATCAATGGTG), at 10 µM. These primers amplify 147 bp from the human miR-200b promoter region about 2.5 kb upstream of the transcription start site (Karina Jouravleva, personal communication). DNA was quantified using a smart cycler (Cepheid) using the following program: 3 min at 95°C (1 cycle) and then 50 cycles at 95°C/15 sec, 60°C/1 min. To calculate the percentage of enrichment at miR200b locus, for each particular mark a fold change of expression relative to IgG is first calculated. Then this fold change is converted into nanograms of DNA based on the input DNA calibration curve set up in each qPCR. Final data are represented as percentages of miR200b loci enriched by various anti-histone antibodies from 30 µg of input DNA for each immunoprecipitation. The efficiency of ChIP depends on the efficiency of the antibody (recognition of the epitopes). All antibodies used in this study were polyclonal, however the number of available epitopes exposed on the surface of each histone is not known. Consistently, the H3 antibody shows a poorer performance than most antibodies against H3 modifications (others have published similar observations, regardless of the anti-H3 antibody used). It only makes sense to compare relative levels of enrichment with individual antibodies between biological samples.

III. Results and discussion

A Principle of ChIP in droplets assay

The proof of concept of the ChIP in droplets assay was demonstrated by generating a sequence of 100 nL confined droplets containing in the first droplet the MB and the chromatin sample, and for the others droplets, reagents needed for all steps of the ChIP procedure. Droplets were used (i) for compartmentalization to prevent cross contaminations, (ii) to reduce sample and reagent consumption and (iii) to increase reaction kinetics. The MB were handled by magnetic tweezers (MT) previously developed by our group. By switching ON the MT, MB in droplets are confined and extracted. The cluster of MB can be resuspended in a new droplet by switching OFF the MT. In that way MB grafted with anti-histone antibodies were used to extract the target chromatin to be analyzed. MB were then transferred from droplets to droplets in order to perform the multi-step ChIP protocol including chromatin binding, washing, elution and purification of DNA. The developed protocol consists of generating two trains of droplets. The first train of droplets was dedicated to chromatin extraction and DNA reverse crosslinking. After cell lysis and chromatin fragmentation, chromatin fragments were specifically captured, based on histone and anti-histone MB interactions, extracted and rinsed through droplets of washing buffers. Then MB were successively resuspended in RNase A and proteinase K droplets to digest RNA and proteins. DNA was released from the digested proteins by thermal elution and MB were discarded from the proteinase K droplet. The second train of droplets was generated for DNA purification by transferring ChargeSwitch MB (CSMB) into the DNA / digested proteins droplet (proteinase K droplet). After DNA capture and washing steps, CSMB were resuspended in the elution droplet in order to obtain a pure DNA solution. Finally, gene-histone associations were determined by qPCR amplification. We first targeted histone H3 and the amount of miR-200b gene was quantified by qPCR in order to investigate and optimize the influence of different parameters on ChIP in droplet sensitivity. Then we studied the efficiency of this new platform to analyze histone modifications (methylation, acetylation) involved in gene activation and repression of transcription processes.

B Influence of the quantity of added magnetic beads on ChIP assay performances

Anti-H3 MB were used as a solid support for the target chromatin capture and extraction. Different quantities of MB ranging from 1 to 15 µg were added and fully extracted after chromatin capture while keeping all others parameters constant. Quantities of chromatin and antibodies were set at 12 and 1 µg, respectively. As seen in Fig. 3.a, the CT (cycle threshold) obtained by qPCR decreases when the quantity of added MB increases, indicating an increased sensitivity. The MB extraction is governed by the magnetic force generated between the MT compared to the interfacial force as previously described 20 . Here the maximum quantity of captured anti-H3 MB is 15 µg. Beyond this value, the produced MB cluster exceed the magnetic confinement volume offered by the MT geometry. Higher current intensity would be required to confine greater amounts of MB quantity, however this approach would also necessitate bulky equipment to maintain the same magnetic force and gradient. In order to demonstrate the specificity of the immunoprecipitation, non-specific IgG antibodies were used as negative control. Using added MB in the 1-15 µg range, the CT values remained in the range of background signal, thus demonstrating that the specificity of the procedure was not affected by MB quantity. For subsequent experiments, we fixed the quantity of MB at 15 µg.

C Influence of anti-histone antibodies quantity on ChIP in droplet assay performances

The sensitivity of the developed procedure is also governed by the quantity of anti-H3 antibody used to specifically extract the target chromatin. In order to highlight the influence of anti H3 quantity on assay sensitivity, the quantity of MB and chromatin were set to 15 µg (1.5x10 6 MB) and 12 µg, respectively, while anti-H3 antibody quantity varied from 0.01 to 4 µg. As observed in Fig. 3. A) Influence of the quantity of added magnetic beads (MB) on the CT signal from the qPCR for the enrichment of H3 at miR-200b promoter. The input of total chromatin and anti-H3 antibody quantity was set to 12 and 1 µg, respectively. B) Influence of the quantity of anti-H3 antibodies on the CT signal from the qPCR. The input of total chromatin and MB quantity was set to 12 and 15 µg, respectively.RSD = 2.5% (N=3) Fig. 3.B there is no signal when the anti-H3 quantity is 0.01 µg. The absence of signal may result from the synergetic effect of the following phenomenon: first the quantity of antibody is too low to efficiently interact with H3 histone, second the MB specific surface is so high that antibodies are instantly captured on the MB thus becoming less available to efficiently interact with H3 compared to free anti H3 antibodies. The effect of these phenomenon decreases when antibody concentration increases. In addition, a loss of antibodies and chromatin may occur after molecules adsorption on tube walls or droplet internal surface. In both cases the surfaces are treated (by the manufacturer for the tubes and using surfactant in our droplet platform) to avoid nonspecific adsorption. For anti-H3 quantities ranging from 0.06 to 1 µg the CT value obtained by qPCR was constant: in this range of values the quantity of anti-H3 is high enough to efficiently capture the specific chromatin while the surface of the MB is not saturated. At a quantity of 4 µg we observed an increase of the CT value, indicating reduced sensitivity. In these conditions, the quantity of anti-H3 is in excess compared to chromatin, therefore introducing competition between free anti-H3 and anti-H3/chromatin complex for the surface of the MB. This condition leads to steric hindrance at the surface of the MB thus explaining the decrease of assay sensitivity.

D Influence of initial chromatin quantity on ChIP in droplet assay performances

One of the main limitations concerning ChIP performances is the requisite use of large numbers of cells, thus prohibiting its application to analyses of rare cells. We found that the lowest quantity of input chromatin required to obtain a significant signal in our system was about 0.1 µg (Fig. 4), which corresponds to the amount of chromatin extracted from about 10,000 cells considering that a diploid mammalian cell contains approximately 10 pg of DNA. This new method therefore permits the reduction of the number of starting cells by two orders of magnitude, compared to conventional ChIP approaches. The overall performances of the ChIP in droplet platform are summarized in the table 1, highlighting the time and reagents consumption for both conventional and droplet approaches. The droplet approach enables a 7-fold reduction in analytical time, as well as a 100-, 20-, and 16-fold reduction in chromatin, MB and antibody consumption, respectively.

E Study of miR-200b expression for HT1080 in droplets microfludic versus conventional method

Based on the results of H3 analysis, we investigated the enrichment of histone active/repressive marks at the miR-200b promoter. This gene belongs to the micro RNA family 200 of non-coding RNAs involved in regulation of cancer progression. miR-200b is deregulated in different cancers 24 and its aberrant down-expression is also known to enhance tumor invasion [25][26][27] . Furthermore, a prognostic value of miR-200b in different cancers has been shown [28][29][30] . Histone modifications, such as methylation or acetylation have a direct influence on gene activation or repression. In this context, we analyzed two active marks (H3K4me3, H3K9ac) and two repressive marks (H3K9me3 and H3K27me3) at the miR-200b promoter in a cancer cell line H1080, which has been previously shown to express low to undetectable levels of miR-200b 31 .

MB quantity

Using the droplet technique, we showed a strong enrichment of repressive marks H3K9me3 and H3K27me3 at the miR-200b promoter (Fig. 5), consistent with the published repressed status of the miR-200b promoter in this cancer cell line 31 . Furthermore, the H3K9ac active mark was poorly represented at the locus. Unexpectedly, we detected a fair enrichment of an active mark H3K4me3, suggesting the bivalent nature of the miR-200b promoter, showing both repressive and active marks.

Since the repressive marks are more prevalent, this gene is repressed in H1080.

To confirm that results obtained using the droplet technique are comparable to those obtained by conventional ChIP methods, we performed a manual ChIP using the same chromatin preparation and antibody lots, but at lower concentrations than the one recommended for the manual ChIP. Very similar results were obtained by both techniques (Fig. 5), validating the use of the proposed droplet approach to study the histone modifications at a particular locus of interest. In additional to significantly reducing the amount of the starting reagents needed, the droplet approach is more efficient since the percentage of captured DNA is higher for all enriched marks tested. The confinement of analytical steps to droplets minimizes DNA loss, which may occur at various steps such as immunoprecipitation, washing, DNA elution and purification steps. When we attempted a conventional ChIP method with the same concentrations of reagents as used in droplets, the ChIP was 10 times less efficient, thus attesting to the benefits of the droplet technique (Fig. 5).

Conclusions

We have demonstrated a new ChIP approach combining droplets, MB and magnetic tweezers. This platform takes advantage of MB used as a capture support to achieve a sequential and multi-step protocol, combined with droplets to compartmentalize individual reactions at a nanoliter scale. The integrated steps of chromatin extraction, DNA elution and purification were completed in shorter analysis time with reduced sample consumption while offering greater sensitivity than a conventional ChIP assay. We demonstrated that the ChIP in-droplet was an efficient technique to evaluate histone modifications at a locus of interest. We described the bivalent nature of the miR-200b locus with a prevalence of repressive marks, suggesting a low or negligible expression of miR-200b in HT1080, as described previously 31 . Finally, we validated the performances of the developed ChIP in-droplet technique by obtaining a similar profile of histone modifications using conventional ChIP methods. This new platform is therefore an efficient tool for ChIP analyses with increased sensitivity, improved processing time and greater cost effectiveness. In addition, since this is a low-cost approach, the platform offers the possibility to incorporate multiple tubes containing trains of droplet sequences, thus opening the way to develop easy multiplex assays. We envision also to combine our

Fig. 2 .

 2 Fig.2. Principle of the droplets microfluidic ChIP platform. Droplets handling within the tube was based on forth and back movements generated by switching from pushing to aspirating mode on the syringe pump. A) Generation of the 1 st train of droplets made of: magnetic beads (MB) + antibodies + chromatin; washing buffer; RNase; Proteinase K. B) MB were confined between the magnetic tweezers (MT), extracted, rinsed in the washing buffer droplets, transferred in the RNase droplet for RNA digestion and extracted after 5 min incubation. C) MB were dispersed in proteinase K droplet for protein digestion and heated for 5 min at 70°C to release DNA from digested proteins. D) MB were discarded and E) a new train of droplets was generated: charge switch magnetic beads (CSMB), washing buffer and elution buffer droplets. F) CSMB were transferred to the droplet containing DNA and digested proteins (yellow droplet) to capture the DNA, G) before it was extracted, washed and eluted in elution buffer. CSMB were discarded before qPCR.

Fig. 4 .

 4 Fig.4. Influence of the initial quantity of chromatin on the CT signal from the qPCR. The anti-H3 antibody and MB quantity was set to 0.25 and 15 µg, respectively. RSD = 2.5% (N=3).

Fig. 5 .

 5 Fig.5. Percentage of enrichment at miR200b for negative control (IgG), positive control (H3), active marks (H3K4me3, H3K9ac) and repressive marks (H3K9me3, H3K27me3) using droplet, conventional techniques or conventional technique with droplet reagents concentration (Droplet experiments average RSD=2.5%, Conventional experiments average RSD=20,7%).

  Due to the lengthy protocol some steps, such as DNA decrosslinking and/or DNA precipitation, are usually performed over night. Comparison of the droplet and conventional ChIP analytical performances.

			Antibody quantity	Chromatin quantity	Chromatin immuno-precipitation	DNA elution	DNA purification	qPCR	Total Time
	Droplet ChIP	15 µg	0,06 µg	0,1µg	4 h	20 min	20 min	2 h	7 h
	Conventional ChIP	300-900 µg 1-5 µg	10-30 µg	24 h	1 h	24 h*	2 h	51 h
	Tab.1.

*

J. Name., 2013, 00, 1-3 | 3

Acknowledgements

This work was supported by European research Council Advanced Grant N°:321107 "CellO", by Investissement d'avenir ANR projects "Digidiag" and IPGG, and by Nadine and Diatools European Projects (FP7). Work in the Telomere and cancer laboratory is supported by a grant from INCa (CINEPIMIR). We thank Karina Jouravleva for communicating results prior publication. The authors also thank C. M. Gillis for proofreading this article.

Notes and references


C Bochaton

S Bailon

I Ineich

M Breuil

A Tresset

S Grouard

From a thriving past to an uncertain future: zooarchaeological evidence of two millennia of human impact on a large emblematic lizard (Iguana delicatissima) on the Guadeloupe Islands (French West Indies)

Keywords: Archaeology, Consumption, Extinction crisis, Morphological drift, Quaternary paleontology, Zooarchaeology

Among the lizards in the Lesser Antillean Islands, iguanas are undoubtedly the most emblematic, especially the endemic species, Iguana delicatissima. However, although much effort is currently made for the conservation of this species as a result of the present biodiversity crisis, nearly

nothing is known of the history of this animal on these islands during the last millennia. Here we present the first data relating to the distribution, morphology, and interaction of past iguanas with human populations in the Lesser Antilles. To do so, we review the archaeological Iguana remains collected over the past 15 years on the Guadeloupe Islands. Our results show that the only Iguana species occurring in pre-Columbian archaeological deposits is Iguana delicatissima. Moreover, we demonstrate that this species occurred on all the islands of Guadeloupe during pre-Columbian times and then suddenly became extinct between 1960 and 1990 on most of these islands. We also confirm the modern introduction of I. iguana to the Guadeloupe Islands. In addition, zooarchaeological research demonstrates that pre-Columbian human populations occasionally used iguanas as a source of food, but with no apparent impact on the native population. However, the first data relating to past size variations of I. delicatissima on the Guadeloupe Islands indicate that archaeological iguanas were much larger than the largest remnant modern specimens and that a marked decrease in body length (more than 20%) occurred in these lizards after contact with European populations. This evidence of widespread extinction and morphological change during modern times is another demonstration of the extensive effects of disturbance and selection induced by modern human societies on endemic insular faunas.

Introduction

The fauna of the Lesser Antilles Islands is rich in lizards and snakes with a high degree of endemism [START_REF] Henderson | Natural History of West Indian Reptiles and Amphibians[END_REF][START_REF] Henderson | Island list of West Indian amphibians and reptiles[END_REF]. However, due to the small size and ecological sensitivity of these islands, squamates are, like many other vertebrate taxa, strongly impacted by the current worldwide biodiversity crisis [START_REF] Barnosky | Has the Earth's sixth mass extinction already arrived?[END_REF]. The islands of Guadeloupe are no exception to this, and both historical [START_REF] Breuil | Histoire naturelle des Amphibiens et Reptiles terrestres de l'archipel Guadeloupéen: Guadeloupe, Saint-Martin, Saint-Bathélemy[END_REF][START_REF] Breuil | Iguana iguana, in: Les Vertébrés Terrestres Introduits En Outre-Mer et Leurs Impacts[END_REF][START_REF] Breuil | Iguana delicatissima. IUCN Red List Threat[END_REF] and fossil data [START_REF] Bailon | New data on Pleistocene and Holocene herpetofauna of Marie Galante (Blanchard Cave, Guadeloupe Islands, French West Indies): Insular faunal turnover and human impact[END_REF]Bochaton et al., 2016a[START_REF] Bochaton | Fossil and subfossil herpetofauna from Cadet 2 Cave (Marie-Galante, Guadeloupe Islands, F. W. I.): Evolution of an insular herpetofauna since the Late Pleistocene[END_REF][START_REF] Boudadi-Maligne | Evidence for historical human-induced extinctions of vertebrate species on La Désirade (French West Indies)[END_REF][START_REF] Grouard | Subsistance, systèmes techniques et gestion territoriale en milieu insulaire antillais précolombien -Exploitation des Vertébrés et des Crustacés aux époques Saladoïdes et Troumassoïdes de Guadeloupe[END_REF][START_REF] Stouvenot | L'abri sous roche Cadet 3 (Marie-Galante): un gisement à accumulations de faune et à vestiges archéologiques[END_REF] show that most of the past endemic squamates of these islands have become extinct, mainly during the recent past.

The most emblematic of the surviving reptiles is probably the genus Iguana Laurenti, 1768, represented by the green iguana (Iguana iguana [Linnaeus, 1758]), and the Lesser Antillean iguana (Iguana delicatissima Laurenti, 1768) [START_REF] Breuil | Histoire naturelle des Amphibiens et Reptiles terrestres de l'archipel Guadeloupéen: Guadeloupe, Saint-Martin, Saint-Bathélemy[END_REF][START_REF] Henderson | Island list of West Indian amphibians and reptiles[END_REF]. These two species diverged around 22.8 Mya ago [START_REF] Hedges | Tree of life reveals clock-like speciation and diversification[END_REF] and are both present in the Lesser Antilles. However, they seem to have undergone different local evolutionary histories, although they can still breed together and produce fertile offspring [START_REF] Vuillaume | Genetic evidence of hybridization between the endangered native species Iguana delicatissima and the invasive Iguana iguana (Reptilia, Iguanidae) in the Lesser Antilles: management implications[END_REF]. I. iguana has a very broad natural distribution area in continental Central and South America [START_REF] Stephen | Geographical structure and cryptic lineages within common green iguanas, Iguana iguana[END_REF] and was recently (accidentally or deliberately) introduced far from its natural distribution area to many places in the world, such as Israel, Hawaii, Florida, the Greater Antilles, and the Lesser Antilles, probably including Guadeloupe [START_REF] Breuil | Caractérisation morphologique de l'iguane commun Iguana iguana (Linnaeus, 1758), de l'iguane des Petites Antilles Iguana delicatissima Laurenti, 1768 et de leurs hybrides[END_REF][START_REF] Falcón | Biology and Impacts of Pacific Island Invasive Species. 10. Iguana iguana, the Green Iguana (Squamata: Iguanidae)[END_REF][START_REF] Henderson | Island list of West Indian amphibians and reptiles[END_REF][START_REF] Krauss | An assessment of control methods for invasive alien iguanas in Saint Lucia[END_REF][START_REF] Pasachnick | Green Iguanas (Iguana iguana) in the Dominican Republic[END_REF]. Endemic populations also seem to occur on some of the Lesser Antillean islands, such as Saba, Montserrat, and Saint Lucia [START_REF] Breuil | Caractérisation morphologique de l'iguane commun Iguana iguana (Linnaeus, 1758), de l'iguane des Petites Antilles Iguana delicatissima Laurenti, 1768 et de leurs hybrides[END_REF][START_REF] Malone | Genetic contributions to caribbean Iguana conservation[END_REF][START_REF] Morton | Management of critical species on Saint Lucia (No. 13)[END_REF][START_REF] Stephen | Geographical structure and cryptic lineages within common green iguanas, Iguana iguana[END_REF].

Conversely, I. delicatissima is endemic to the Lesser Antilles. It is now considered to be an endangered species on the most recent IUCN red list [START_REF] Breuil | Iguana delicatissima. IUCN Red List Threat[END_REF], as it is now extinct on most of the islands in Guadeloupe [START_REF] Breuil | Histoire naturelle des Amphibiens et Reptiles terrestres de l'archipel Guadeloupéen: Guadeloupe, Saint-Martin, Saint-Bathélemy[END_REF][START_REF] Henderson | Island list of West Indian amphibians and reptiles[END_REF], and is currently the focus of an international plan of action [START_REF] Knapp | Lesser antillean Iguana, Iguana delicatissima: conservation action plan, 2014-2016[END_REF]. Recent genetic data show little divergence between the different Lesser Antillean populations and suggest that this animal may have spread to the islands recently, possibly as a result of human activity [START_REF] Martin | Phylogeography of the endangered lesser antillean iguana, Iguana delicatissima: a recent diaspora in an archipelago known for ancient herpetological endemism[END_REF][START_REF] Vuillaume | Genetic evidence of hybridization between the endangered native species Iguana delicatissima and the invasive Iguana iguana (Reptilia, Iguanidae) in the Lesser Antilles: management implications[END_REF]. This hypothesis is congruent with the paleontological results obtained from Pleistocene and Holocene fossil data from the island of Marie-Galante, which show that no Iguana osteological remains occur in layers preceding the arrival of human populations [START_REF] Bochaton | Fossil and subfossil herpetofauna from Cadet 2 Cave (Marie-Galante, Guadeloupe Islands, F. W. I.): Evolution of an insular herpetofauna since the Late Pleistocene[END_REF].

Both of these Iguana species can hybridize when syntopic, which probably played a major role in the extinction of I. delicatissima in Guadeloupe, except for the islands of La Désirade and Îlets de Petite Terre, where there are no I. iguana [START_REF] Breuil | Histoire naturelle des Amphibiens et Reptiles terrestres de l'archipel Guadeloupéen: Guadeloupe, Saint-Martin, Saint-Bathélemy[END_REF]. However, although this genus is absent from the very sparse Pleistocene and early Holocene fossil record in Guadeloupe, historical [START_REF] Breuil | Histoire naturelle des Amphibiens et Reptiles terrestres de l'archipel Guadeloupéen: Guadeloupe, Saint-Martin, Saint-Bathélemy[END_REF][START_REF] Du Tertre | Histoire générale des Antilles habitées par les François[END_REF] and especially archaeological [START_REF] Grouard | Caribbean Archaeozoology[END_REF][START_REF] Grouard | Modes de vie des Précolombiens des Antilles françaises[END_REF][START_REF] Grouard | Subsistance, systèmes techniques et gestion territoriale en milieu insulaire antillais précolombien -Exploitation des Vertébrés et des Crustacés aux époques Saladoïdes et Troumassoïdes de Guadeloupe[END_REF] sources provide evidence of the past occurrence of the genus Iguana on these islands. The latter sources show that the genus Iguana sp. has been present on all the islands of Guadeloupe since at least 1500 BC and was hunted by pre-Columbian human populations more than 2,000 years before the arrival of Europeans. On account of a combination of extensive data concerning the modern representative of this genus and archaeological data, the iguana is an excellent model for studying the temporal evolution of human impact on insular large lizard populations.

Up until recently, few data concerning archaeological iguanas from the Guadeloupe Islands had been recorded. Zooarchaeological studies have, in the Guadeloupe Islands and in other Caribbean islands, mainly focused on the extensive biodiversity of other taxa, especially aquatic animals, which largely compose the meat diet of most of past human populations rather than taxa, like iguanas, that remain scarce in zooarchaeological assemblages [START_REF] Giovas | Though She Be But Little: Resource Resilience, Amerindian Foraging, and Long-Term Adaptive Strategies in the Grenadines, West Indies[END_REF][START_REF] Grouard | Caribbean Archaeozoology[END_REF][START_REF] Keegan | The Role of Small Islands in Marine Subsistence Strategies: Case Studies from the Caribbean[END_REF][START_REF] Wing | Native American use of animals in the Caribbean[END_REF]. In addition, the paucity of research on iguanas, and more generally archaeological lizard remains, has further complicated these studies. As a consequence, the few studies to date of iguanas from archaeological contexts focused on the taxonomic identification of osteological remains [START_REF] Martin | Les Amérindiens Précolombiens des Antilles et la consommation de Reptiles: Le cas des Iguaninae (Anatomie comparée et archéozoologie)[END_REF][START_REF] Martin | The Iguanas of the pre-Columbian Caribbean islands: human subsistence and paleoecology[END_REF]. These studies showed that two iguana species (I. iguana and I. delicatissima) occurred on the Guadeloupe Islands prehistorically [START_REF] Martin | Les Amérindiens Précolombiens des Antilles et la consommation de Reptiles: Le cas des Iguaninae (Anatomie comparée et archéozoologie)[END_REF][START_REF] Martin | The Iguanas of the pre-Columbian Caribbean islands: human subsistence and paleoecology[END_REF]; a finding that contradicts the notion that I. iguana would be a recently introduced species. An early occurrence of I. iguana was also advanced by [START_REF] Lazell | The lizard genus Iguana in the Lesser Antilles[END_REF], who stated that "no human introduction is responsible for the presence of Iguana iguana anywhere in the Lesser Antilles". However, the conclusions proposed by [START_REF] Martin | Les Amérindiens Précolombiens des Antilles et la consommation de Reptiles: Le cas des Iguaninae (Anatomie comparée et archéozoologie)[END_REF] and [START_REF] Martin | The Iguanas of the pre-Columbian Caribbean islands: human subsistence and paleoecology[END_REF] based on archaeological specimens are questionable, since a recent study conducted by Bochaton et al. (2016b), focusing on the osteological differences between the two Iguana species, has shown that most of the osteological characteristics used up until now are unreliable. Consequently, a review of the archaeological material using new reliable identification criteria was necessary in order to analyze variations in the taxonomic composition of Iguana in archaeological assemblages from the Guadeloupe Islands throughout time. In addition, the reconstruction of the past snout-vent length (SVL) of archaeological Iguana [START_REF] Bochaton | Describing archaeological Iguana Laurenti, 1768 (Squamata: Iguanidae) populations: size and skeletal maturity[END_REF] allows for size comparisons with modern populations.

The aim of this paper is to provide a new analysis of osteological Iguana remains from 25 Guadeloupian archaeological sites using new methodological tools developed for the identification (Bochaton et al., 2016b) and zooarchaeological study [START_REF] Bochaton | Describing archaeological Iguana Laurenti, 1768 (Squamata: Iguanidae) populations: size and skeletal maturity[END_REF] of archaeological Iguana remains. These newly obtained zooarchaeological data are then compared with data collected from modern Iguana on the Guadeloupe Islands, as well as textual historical data. Finally we use the results to provide evidence of the occurrence of Iguana species and morphological changes through time, and describe interactions between past humans and iguanas on the Guadeloupe Islands.

Regional setting

The Guadeloupe Islands are situated in the northern Lesser Antilles between Dominica and Montserrat. The archipelago is composed principally of two large islands, Basse Terre (848.1 km²) and Grande-Terre (585.7 km²), separated by a narrow marine channel. Other smaller islands are now clearly separated from the two main islands: La Désirade (21.1 km²) and Îlets de Petite Terre (1.5 km²), Marie-Galante (157.5 km²), and Les Saintes (14.2 km²) (Fig. 1), each with their own geological history. Some of the Guadeloupian islands (Îlets de Petite Terre, Marie-Galante, La Désirade and Grande-Terre) are flat limestone islands, which emerged during the late Calabrian around 800 Ky ago, although La Désirade developed much earlier during the Piacenzian/Gelasian transition at around 2.7 Ma [START_REF] Bouysse | Données sur la structure de l'arc insulaire des Petites Antilles, entre Sainte Lucie et Anguilla[END_REF][START_REF] Münch | Pliocene to Pleistocene carbonate systems of the Guadeloupe archipelago, French Lesser Antilles: a land and sea study (the KaShallow project)[END_REF]. The vegetation of these islands is mainly composed of xerophytic forests and is poorly diversified [START_REF] Lasserre | La Guadeloupe: étude géographique Tome 1: Le milieu naturel et l'héritage du passé[END_REF]. The other islands, Basse Terre and Les Saintes, are volcanic islands which formed between 2.79 and 0.435 My and 4.7 and 0.6 My, respectively [START_REF] Andreieff | Reconnaissance géologique de l'arc insulaire des Petites Antilles. Résultats d'une campagne à la mer de prélèvements de roches entre Sainte-Lucie et Anguilla (Arcante 1)[END_REF][START_REF] Jacques | L'archipel des Saintes (Guadeloupe, Petites Antilles): Géologie et pétrologie[END_REF][START_REF] Samper | Timing of effusive volcanism and collapse events within an oceanic arc island: Basse-Terre, Guadeloupe archipelago (Lesser Antilles Arc)[END_REF]. Basse Terre is mountainous with many peaks above 1,000 m and a culminating point at 1,467 m (la Soufrière). Pluviometry is higher there than on the other islands and the vegetation is far more diversified with xerophilic, mesophilic, and hydrophilic forests [START_REF] Lasserre | La Guadeloupe: étude géographique Tome 1: Le milieu naturel et l'héritage du passé[END_REF][START_REF] Sastre | Plantes, milieux et paysages des Antilles françaises: écologie, biologie, identification, protection et usages[END_REF]. The morphology and the size of the Guadeloupe Islands varied markedly in relation to sea level fluctuations and the islands of Basse Terre, Grande-Terre, Petite Terre and la Désirade formed a single island during the Late Pleistocene. However, during the chronological period covered by our study (mainly the last two millennia), the morphology of the archipelago was very similar to its current configuration.

As regards past human settlement, the oldest archaeological site of these islands is the site of Morel Zero [START_REF] Paulet-Locard | Guadeloupe: Bilan et orientation de la recherche archéologique[END_REF], radiocarbon dated to around 1500 cal. BC (Mesoindian or Archaic Age), but indirect evidence of human occupation in the form of increased charcoal particles, presumably from human-induced fires, suggests that settlement could have been earlier, ca. 3000 cal. BC. [START_REF] Siegel | Paleoenvironmental evidence for first human colonization of the eastern Caribbean[END_REF][START_REF] Stouvenot | L'abri sous roche Cadet 3 (Marie-Galante): un gisement à accumulations de faune et à vestiges archéologiques[END_REF]. Morel I on Grande-Terre is the oldest ceramic archaeological site in Guadeloupe Islands dating to around AD 80 and attributed to the Huecan Saladoid culture (Neo-Indian or Early Ceramic Age) [START_REF] Bérard | The Saladoid[END_REF][START_REF] Clerc | Le peuplement précolombien des Antilles et ses vestiges en Guadeloupe[END_REF][START_REF] Fitzpatrick | The pre-columbian Caribbean: colonization, population dispersal, and island adaptations[END_REF][START_REF] Hofman | New perspectives on a Huecan Saladoïd assemblage on Guadeloupe: the case of Morel I[END_REF][START_REF] Romon | Fouille préventive de la Gare Maritime de Basse-Terre (Rapport final d'opération No. 2005-33)[END_REF]. This early Saladoid component includes Cedrosan Saladoid ceramic traditions, which occurred between AD 300-900 and evolved into the Marmoran Troumassoid tradition that lasted from AD 900-1500 (i.e., the Late Ceramic Age) [START_REF] Bonnissent | Archéologie précolombienne de l'île de Saint-Martin, Petites Antilles (3300 BP -1600 AD)[END_REF][START_REF] Honoré | Evaluation du potentiel archéologique du site de l'Anse du Coq[END_REF][START_REF] Keegan | The Oxford Handbook of Caribbean Archaeology[END_REF].

After Columbus' second trip in 1493 and a period of contact between Amerindian and European populations archaeologically characterized by Cayo complex ceramics, French colonization began around 1635 and led to the disappearance of Amerindian populations [START_REF] Keegan | The Oxford Handbook of Caribbean Archaeology[END_REF][START_REF] Saunders | The peoples of the Caribbean: an encyclopedia of archaeology and traditional culture[END_REF].

Material and Methods

Zooarchaeological material

The osteological material included in this study comes from 25 pre-Columbian archaeological sites situated on Grande-Terre (5), Basse Terre (7), Les Saintes (1), Îlets de Petite Terre (4), La Désirade (3) and Marie-Galante (5) (Fig. 1; Supplementary material S1; Tab. 1). The chronological or cultural attribution of these sites is reported in Supplementary material S1 and Tab. 1. Most of the sites with the Iguana remains studied here had already been investigated by zooarchaeologists who studied the whole bone assemblages recovered in these archaeological deposits [START_REF] De Waal | Pre-Columbian social organisation and interaction interpreted through the study of settlement patterns: An archaeological case-study of the pointe des Châteaux, La Désirade and Les Îles de la Petite-Terre micro-region[END_REF][START_REF] Grouard | Caribbean Archaeozoology[END_REF][START_REF] Grouard | Modes de vie des Précolombiens des Antilles françaises[END_REF][START_REF] Grouard | Subsistance, systèmes techniques et gestion territoriale en milieu insulaire antillais précolombien -Exploitation des Vertébrés et des Crustacés aux époques Saladoïdes et Troumassoïdes de Guadeloupe[END_REF]. For this study, the complete assemblage of skeletal remains collected from each site was observed in order to review previously identified Iguana remains and, when possible, to add newly identified remains. In total, 2,236 archaeological Iguana remains were collected from the different assemblages and investigated as part of this study.

Methodology

Specific identifications followed the osteological characters described by Bochaton et al. (2016b).

The latter study is based on a large sample of modern Iguana (69 skeletons) and proposes a series of 32 identification criteria distributed among 17 different skeletal (both cranial and postcranial) elements allowing for the distinction of I. delicatissima, I. iguana, and for some characters, hybrids of the two species. The authors also provided a percentage of reliability for each criterion in order to allow for the discussion of the results. In the present study, archaeological bones that did not present discriminating characters according to Bochaton et al. (2016b) were left specifically unidentified as Iguana sp.

The Snout-Vent Length (SVL) estimations of archaeological specimens were obtained using the measurements and predictive equations described by [START_REF] Bochaton | Describing archaeological Iguana Laurenti, 1768 (Squamata: Iguanidae) populations: size and skeletal maturity[END_REF] on the different anatomical elements. Skeletal maturity was estimated on the basis of the fully or partially ossified status of each bone and the maturity stages also described by [START_REF] Bochaton | Describing archaeological Iguana Laurenti, 1768 (Squamata: Iguanidae) populations: size and skeletal maturity[END_REF]. Skeletal maturity and size data were then combined to obtain a "Minimal Theoretical Maximal Size" or MTMS, used to estimate the size that the archaeological specimen would have reached if it had lived until the end of its growth. This MTMS is estimated by considering that the largest immature long bone provides an SVL estimation of a maximum of 80% of the maximal size of the specimen [START_REF] Bochaton | Describing archaeological Iguana Laurenti, 1768 (Squamata: Iguanidae) populations: size and skeletal maturity[END_REF][START_REF] Maisano | Terminal fusions of skeletal elements as indicators of maturity in squamates[END_REF]. Data concerning the size of the modern Guadeloupian I. delicatissima were collected by the French Office National de la Chasse et de la Faune Sauvage (ONCFS) on La Désirade in 2012 (funded by the French state and FEDER) and by the NGO Le Gaiac association on Îlets de Petite Terre (Terre-de-Haut and Terre-de-Bas) between 2007 and 2011, and were made available to us for study.

The minimum number of individuals (MNI) was calculated using the most abundant anatomical element for each site [START_REF] Shipman | Life History of a Fossil and Introduction To Taphonomy and Paleoecology[END_REF]. The global MNI is an addition of the MNI obtained from the different sites considering that it is highly unlikely that a specimen would be divided among different deposits. To study the anatomical distribution of bone remains we used the "Proportional Representation of each skeletal part" (PR) [START_REF] Dodson | Taphonomic Investigations of Owl Pellets[END_REF].

Statistical analyses were performed using the free software "R" (version 3.1, https://cran.rproject.org/) and "R Studio" (version 0.98.1102). Data normality was tested using the Shapiro-Wilk test. As data were not normally distributed, comparisons between two populations were performed using the Wilcoxon test and comparisons between more than two populations using the Kruskal-Wallis test. Differences were considered to be statistically significant for P value <0.01.

Finally, in order to observe traces hypothetically left by humans on Iguana bones, all the remains were carefully observed using a stereomicroscope (Nikon SMZ1000). In addition, Scanning Electron Microscope (SEM) photographs of digested bones were taken.

Results

4.1 Specific identification (Figure 2; Tab. 1) A total of 2,236 Iguana remains (MNI=80) were collected from the different archaeological sites studied here. Among these remains, 1,826 (81.7%) did not present any of the anatomical structures taken into account by Bochaton et al. (2016b) and thus could not be identified at the species level. The other 410 remains were identified at the species level. They nearly all (407 remains-18.2%) presented the following I. delicatissima characteristics that differ from I. iguana (see Bochaton et al., 2016b): On the maxilla, the infraorbital foramen is more anteriorly located on the supradental shelf than on I. iguana and can reach the transversal crest, and the exonarinal margin of the facial process forms an obtuse angle with the premaxillary process in lateral view;

On the frontal bone (Fig. 2), the smallest width of the interorbital constriction is superior to 50% of the maximal lateral length of the bone, the posterolateral processes are oriented posterolaterally and articular facets with the parietal are oriented dorsally and are clearly visible in dorsal view, and the bone presents a posterior braincase depression in ventral view; On the parietal bone (Fig. 2), the pineal foramen is not well delimited on the frontal margin of the bone and articular facets with the frontal bone are oriented ventrally and highly visible in ventral view;

On the postorbital bone, the postorbital articular facet with the jugal is medially directed and more visible in medial view than in I. iguana, and the articular facets with the jugal and squamosal form two individualized facets; On the jugal bone, the dorsal view of the jugal suborbital ramus bears a well-developed anteromedial expansion between the palatine and ectopterygoid insertion regions; On the pterygoid bone, only one weakly posteriorly extended dental row occurs, and the lateral area of the bone is flat; On the sphenoid bone, the sphenoid ventral crests joining the basal tubercle to the basipterygoid is sigmoid in ventral view; On the dentary, the intramandibular lamella is short and expands below the first to second dental positions; On the coronoid bone, the coronoid process is higher and more dorsally rounded in lateral view than in I. iguana, this process may also present a small posterior protuberance; On the articular bone, the length of the retroarticular process is inferior to 150% of the length of the articular fossa and the tympanic crest is wider than the angular crest; On the surangular bone (Fig. 2), the surangular coronoid process is pointed; On the axis vertebra, the ratio between the minimal neural arch length and the posterior neural arch breadth comprising the postzygapophysis is inferior to 0.65; All the caudal vertebrae lack caudal autotomy planes; On the scapulocoracoid bone, the small posterolateral protuberance marking the attachment of the scapulohumeral ligament is absent; On the pelvic bone, the ilium dorsal crest is convex and bears a short and curved ventral edge, the posterior section of the ilium is subtriangular, the anterior section of the pubis forms a triangle with an acute apex angle, and the posterior margin of ischium is more concave than in I. iguana; On the humerus (Fig. 2), the foramen of the supracondylar fossa is more open than in I. iguana.

However, three remains (0.1%) presented the following Iguana iguana characteristics: an infraorbital foramen posteriorly located on a maxilla, and an exonarinal margin of the facial process forming a right angle with the premaxillary process in lateral view on the same bone; an intramandibular lamella anteriorly extended under the four last dental positions on a dentary, and a narrow foramen of the supracondylar fossa on a humerus.

Body size characteristics of Iguana archaeological populations

Past Snout-Vent Length (SVL) and skeletal maturity were observed on all the studied Iguana remains. Past Snout-Vent Length (SVL) estimations were obtained from 488 Iguana remains (MNI=48) from all skeletal parts for which size predictive equations were defined by [START_REF] Bochaton | Describing archaeological Iguana Laurenti, 1768 (Squamata: Iguanidae) populations: size and skeletal maturity[END_REF]. These remains come from all of the archaeological sites included in this study and a list of all the obtained estimations can be found in Supplementary material (S2).

The estimated size ranged from around 70 mm SVL for small newborn specimens to very large individuals with SVL of more than 500 mm (Fig. 3, Supplementary material S2). However, all size classes were not equally distributed in our assemblage. Specimens smaller than 220 mm SVL were nearly absent and most fossil Iguana had a SVL between 300 and 400 mm. Specimens smaller than 220 mm SVL (between 70 and 150 mm SVL) only occur in Morne-Rita on Marie-Galante Island (NMI=1), Cathédrale de Basse-Terre on Basse Terre Island (NMI=1) and Anse à la Gourde on Grande-Terre Island (NMI=2) (Fig. 3, Supplementary material S2).

Skeletal maturity data (bone ossification) were recorded on 223 archaeological Iguana remains.

Among them, 65 (29%) were mature bones of which 35 belong to fully-grown specimens (with fully ossified long bones and/or fused sacral vertebrae = stage 4 sensu [START_REF] Bochaton | Describing archaeological Iguana Laurenti, 1768 (Squamata: Iguanidae) populations: size and skeletal maturity[END_REF]. The other 158 remains (71%) correspond to non-fully grown individuals and at least 13 of them belong to non-sexually mature specimens (for which the pelvic girdle and axis are not fully ossified = stage 2 sensu [START_REF] Bochaton | Describing archaeological Iguana Laurenti, 1768 (Squamata: Iguanidae) populations: size and skeletal maturity[END_REF].

Moreover, 114 remains yield a combination of both size and skeletal maturity data and thus allow for the estimation of the MTMS and the size range of the previously defined maturity stages. The detailed observation of archaeological Iguana sizes was performed at the island scale because the material was too scant to obtain significant results for each site. In addition, we noted that archaeological specimens collected from the different Guadeloupe islands present SVL differences (Kruskal-Wallis test, p.val<0.01) (Fig. 3). Size overlap often occurred between the defined maturity stages. This could reflect a size estimation error linked to measurement error, variability in bone maturation timing or could also be the result of sexual size dimorphism.

On Marie-Galante, the Minimal Theoretical Maximal Size (MTMS) of archaeological Iguana was 475 mm, and the estimated SVL mean was 324 mm (Fig. 3-A). A size overlap of a maximum of 40 mm was observed between the maturity stages. The results were similar for the sites from Îlets de Petite Terre (Fig. 3-B), La Désirade (Fig. 3-C), and Grande-Terre (Fig. 3-D): MTMS are respectively 462, 460 and 475 mm with mean estimated SVL of 330, 358 and 310 mm respectively. On Îlets de Petite Terre and Grande-Terre, some limited size overlap (between 19 and 25 mm) was observed between maturity stages. A single archaeological SVL datum (361 mm) was collected from Les Saintes Island.

The situation was more complex for archaeological Iguana from Basse Terre Island, as two groups of very different sizes seem to occur in the same archaeological contexts. Most of the specimens are larger than any other observed specimens, with estimated sizes between 400-450 mm SVL for non-fully mature bones (MNI=1), reaching a maximum of 511 mm SVL for fully mature bones (MNI=2) (Fig 3-E). However, some specimens (MNI=2) present estimated sizes of only 323 and 388 mm SVL for fully mature bones. This situation led to an important body size overlap between maturity stages and made the definition of a single MTMS impossible.

Consequently, we defined two MTMS on the basis of the smallest mature and largest immature bones and obtained MTMS of respectively 403 mm and 556 mm SVL (Fig. 3-E). These two MTMS correspond to two populations presenting different body sizes. Taken as a whole, the estimated SVL of Basse Terre archaeological iguanas (mean= 364 mm) were the highest for the archipelago.

We did not observe any effects of chronological attribution (Saladoid or Troumassoid) on the size of the Iguana remains (Wilcoxon test, p.val=0.34). However, comparisons of SVL data collected from modern I. delicatissima populations from La Désirade (n= 274) and Îlets de Petite Terre (Terre-de-Haut and Terre-de-Bas) (n=435) with all archaeological SVL data obtained from these islands show that archaeological iguanas were far larger than modern specimens (Wilcoxon test, p.val <0.001). Indeed, modern iguanas from three islands do not exceed 390 mm SVL (max. 390 mm on Terre-de-Haut [see also [START_REF] Breuil | Histoire naturelle des Amphibiens et Reptiles terrestres de l'archipel Guadeloupéen: Guadeloupe, Saint-Martin, Saint-Bathélemy[END_REF], 385 mm on Terre-de-Bas and 360 mm on La Désirade [387 mm following [START_REF] Schardt | Iguana delicatissima in Freiland[END_REF], whereas archaeological iguanas from the same islands can be larger than 450 mm SVL. The SVL mean of archaeological iguanas (330-358 mm) was also higher than the SVL mean of modern iguanas (258-274 mm), but as the specimens occurring in archaeological sites and measured in the field are probably subject to very different sampling methods, SVL means are nearly impossible to interpret. A very dry season in 2001 led to high mortality among iguanas from La Désirade and Petite-Terre islands [START_REF] Breuil | Histoire naturelle des Amphibiens et Reptiles terrestres de l'archipel Guadeloupéen: Guadeloupe, Saint-Martin, Saint-Bathélemy[END_REF] and could have contributed to the elimination of large specimens. However, archaeological iguanas from these islands are still larger than any modern I. delicatissima ever measured in the Lesser Antilles, and a reduction in size during the modern period is very likely.

Skeletal distribution, anthropic marks, and surface alterations of the archaeological remains

Due to the scarcity of Iguana remains in archaeological sites, we did not find clear site-scale differences in the data presented in this section. Consequently, all the sites were treated as a whole, but unusual observations encountered at single sites are also mentioned when pertinent.

The overall representation of skeletal parts was low (PR=12.5%), which means that on average only 12% of Iguana skeletons were present in the archaeological sites, but all the anatomical parts were represented. The most represented anatomical parts are the proximal part of the tail (PR=42), femur (PR=32), humerus (PR= 29), middle part of the tail (PR=28), pelvic girdle (PR=25), dorsal vertebrae (PR=24), and tibia (PR=22) (Fig. 4). These anatomical parts are the most important in terms of meat content, especially the proximal part of the tail, the humerus, and the femur. However, these bones are also the biggest and strongest elements, and are the most likely to be preserved and collected. All the other bones, although less represented, were also present in the assemblages (Fig. 4). These bones are smaller elements, sometimes fragile and subject to high breakage rates, and are thus more exposed to collection bias, especially considering that 80% of the studied bones were broken.

Traces of burning, cut marks and digestion marks were rare on the examined material and only occurred on 84 remains (3.7% of the total assemblage). Most of the observed traces (56) were traces of fire, ranging from black burning marks (Stages 1 and 2 from Stiner and Kuhn 1995; N=45) to white calcination marks (Stages 4 and 5 from Stiner and Kuhn 1995; N=11). These marks indicate that bones presenting black marks were exposed to temperatures superior to 300 °C and those presenting white marks to 675 °C [START_REF] Munro | Effects of heating on the carbon and oxygen-isotope compositions of structural carbonate in bioapatite from modern deer bone[END_REF]. These traces of fire occur mostly on skeletal extremities: caudal vertebrae (N=21), the phalanx (N=8) and cranium (N=6) (Fig. 4,5). Cut marks were also observed on 16 remains (Fig. 4,5), but were difficult to observe because of bone alterations such as the occurrence of concretions or surface degradation. These marks only occurred on long bones (N=5), the pelvic girdle (N=3) and vertebrae (N=8) (Fig. 4).

The most recurring location of cut marks was on mid-caudal vertebrae (N=6), where traces occurred all around the vertebrae and clearly reflected the removal of the distal part of the tail, which is very long and poor in meat. Cut marks observed on this area were sometimes combined with traces of fire (Fig. 5). Digestive marks were also observed on 12 remains, but only attributed to small specimens with SVL of around 90 mm, at Anse à la Gourde and in cave deposits (Anse à l'Ecu, Morne-Rita and Cadet 3). We considered some of these bones to be highly digested, as shown by two dentaries (Fig. 6) displaying surface dissolution, enlargement of labial foramina (Fig. 6-A), and the destruction of most of their teeth. Such traces were probably left by humans, carnivores, or diurnal raptors [START_REF] Andrews | Owls, caves and fossils[END_REF][START_REF] Stoetzel | Taphonomic Analysis of Amphibian and Squamate Remains from El Harhoura 2 (Rabat-Témara, Morocco): Contributions to Palaeoecological and Archaeological Interpretations[END_REF].

Discussion

Taxonomic identification of archaeological iguanas

Among the 410 archaeological remains identified to the species level, 407 were attributed to I. delicatissima and only three to I. iguana. However, these I. iguana identifications are highly uncertain. Indeed, two of them (a maxilla and a humerus from the Basse-Terre Gare Maritime site) were identified using characteristics that are not considered to be 100% reliable (Bochaton et al., (2016b). They could represent misidentifications in view of the large number of studied archaeological remains. The third bone presenting an Iguana iguana characteristic is a dentary from the Tourlourous site on Marie-Galante. It was identified based on a 100% reliable character (character I from Bochaton et al., 2016b). However, this attribution is still dubious as the bone is very isolated in an assemblage where 99.7% of the remains were unambiguously attributed to I. delicatissima. Given this context, a single remain bearing an I. iguana character is clearly insufficient to consider the occurrence of that species at the Tourlourous site. The most likely explanation is that the diagnostic characteristic described for the dentary by Bochaton et al., 2016b is not 100% reliable. This would not be surprising considering the important skeletal morphological overlap between I. iguana and I. delicatissima described by previous authors and the limited number of available comparative specimens. Consequently, I. delicatissima seems to be the only Iguana species occurring on the Guadeloupe Islands during pre-Columbian times and there is no clear evidence of the occurrence of I. iguana during that period. In addition, the extreme scarcity of I. iguana elements in our material shows that these I. delicatissima populations bear no traces of past hybridization with I. iguana. These results differ from those obtained by [START_REF] Martin | Les Amérindiens Précolombiens des Antilles et la consommation de Reptiles: Le cas des Iguaninae (Anatomie comparée et archéozoologie)[END_REF] and [START_REF] Martin | The Iguanas of the pre-Columbian Caribbean islands: human subsistence and paleoecology[END_REF], who stated that both I. iguana and I. delicatissima occurred prehistorically on the Guadeloupe Islands. This contradiction is undoubtedly due to the unreliability of the identification characters used by these authors (Bochaton et al., 2016b).

Consequently, previous results should no longer be taken into account and we should currently consider that there is no evidence of past occurrences of I. iguana on the Guadeloupe Islands prior to European contact. This conclusion can be related to the long-standing discussion concerning the introduced or native status of I. iguana in the Lesser Antilles. The introduced status of I. iguana was first suggested by [START_REF] Dunn | Notes on Iguana[END_REF] and then [START_REF] Underwood | Reptiles of the eastern Carribean[END_REF], but subsequently refuted by [START_REF] Lazell | The lizard genus Iguana in the Lesser Antilles[END_REF]. Later, [START_REF] Lescure | Introductions passives et actives de reptiles et d'amphibiens dans les Antilles et les Guyanes[END_REF] suggested an Amerindian role in the dispersal of I. iguana in the Antilles and more recently [START_REF] Breuil | Caractérisation morphologique de l'iguane commun Iguana iguana (Linnaeus, 1758), de l'iguane des Petites Antilles Iguana delicatissima Laurenti, 1768 et de leurs hybrides[END_REF][START_REF] Breuil | Histoire naturelle des Amphibiens et Reptiles terrestres de l'archipel Guadeloupéen: Guadeloupe, Saint-Martin, Saint-Bathélemy[END_REF], [START_REF] Breuil | Iguana delicatissima. IUCN Red List Threat[END_REF] and [START_REF] Vuillaume | Genetic evidence of hybridization between the endangered native species Iguana delicatissima and the invasive Iguana iguana (Reptilia, Iguanidae) in the Lesser Antilles: management implications[END_REF] assumed that I. iguana was introduced to the Guadeloupe Islands and St Maarten during the 19 th and 20 th centuries. Our results concur with the latter hypothesis and should contribute to addressing this long-standing debate.

Size of past iguanas

Moreover, we investigated the past size of I. delicatissima in the Guadeloupe Islands for the first time. In order to achieve this, we observed two estimated size variables; the mean SVL and Minimal Theoretical Maximal Size (MTMS) of past iguanas from each island and found that these variables showed conflicting results. Indeed, although both variables showed inter-island differences, the mean SVL indicated that past iguanas were larger on Basse Terre and La Désirade than on Marie-Galante and Îlets de Petite Terre (Terre-de-Bas), and even smaller on Grande-Terre (Fig. 3). Conversely, the MTMS (between 460 and 475 mm SVL) indicated that iguanas were of similar size on all the islands with the exception of Basse Terre, where they were larger (Fig. 3). The advantage of the MTMS value is that it also takes account of the skeletal maturity of past iguanas and not simply their size. Thus, it allows for the study of the body size of archaeological populations of consumed animals, with often strongly biased size composition: we clearly demonstrated a size selection by human hunters on the studied archaeological iguanas (see below). Thus we consider the mean body size differences observed on archaeological iguanas from the different islands as a bias induced by differential sampling selection by past humans rather than true biological differences.

Consequently, we assume that the past size of I. delicatissima was similar on Marie-Galante, Îlets de Petite Terre (Terre-de-bas), La Désirade and Grande-Terre. However, larger iguanas occurred on Basse Terre Island, as shown by the MTMS (superior to 550 mm). The larger sizes observed on that island could be linked to indirect geographic and environmental effects. On account of its volcanic nature, Basse Terre is more humid and presents more mountainous zones and habitat diversity than the limestone islands [START_REF] Lasserre | La Guadeloupe: étude géographique Tome 1: Le milieu naturel et l'héritage du passé[END_REF][START_REF] Lescure | Le peuplement en reptiles et amphibiens des Petites-Antilles[END_REF]. It is also noteworthy that the biggest modern I. delicatissima ever measured on Guadeloupe Islands (410 mm SVL) originated from that island [START_REF] Breuil | Histoire naturelle des Amphibiens et Reptiles terrestres de l'archipel Guadeloupéen: Guadeloupe, Saint-Martin, Saint-Bathélemy[END_REF]. However, smaller archaeological specimens (MTMS= 403 mm) were also recovered from Basse Terre. Size differences between these small and large iguanas occurring on Basse Terre are far too high to be related to the same sexual dimorphism as in modern I. delicatissima, which does not appear to be as marked according to the ONCFS data collected on La Désirade and Petite Terre (a difference of 20 mm in SVL between the largest measured female and male) and to data collected on Dominica (a difference of 42 mm in SVL between the largest measured female and male) [START_REF] Knapp | Movements and nesting of the lesser antillean iguana (>i>Iguana delicatissima</i>) from Dominica, West Indies: implication for conservation[END_REF], or to a size estimation error.

It is possible that sexual dimorphism may have been more marked in Basse Terre iguanas than in modern iguanas but genetic or isotopic data would be needed to confirm this hypothesis. Another alternative explanation is that these small iguanas could correspond either to an isolated local population subject to unfavorable biotic and/or abiotic conditions or exogenous iguanas translocated by pre-Columbians from another island.

Pre-Columbian population interactions with iguanas

The similarity in size of I. delicatissima indicated by the MTMS on most islands, including Marie-Galante (Fig. 3), where they were probably introduced [START_REF] Bochaton | Fossil and subfossil herpetofauna from Cadet 2 Cave (Marie-Galante, Guadeloupe Islands, F. W. I.): Evolution of an insular herpetofauna since the Late Pleistocene[END_REF], could also point to their introduction to other islands (see also [START_REF] Vuillaume | Genetic evidence of hybridization between the endangered native species Iguana delicatissima and the invasive Iguana iguana (Reptilia, Iguanidae) in the Lesser Antilles: management implications[END_REF], possibly by human populations, probably for feeding purposes. However, this hypothesis lacks theoretical grounds considering that island size only has a slight effect on squamate body size [START_REF] Meiri | Size evolution in island lizards[END_REF] and this similarity in size could also be the consequence of convergent evolution. The occurrence of Iguana delicatissima remains in most of the Guadeloupian pre-Columbian archaeological sites indicates that these large lizards were collected by human pre-Columbian populations for two thousand years (from 500 BC to AD 1500), possibly using specific hunting strategies. Indeed, hunted iguanas were not randomly selected, as suggested by archaeological Iguana size distribution, which does not reflect natural population size ranges. Most of the archaeological assemblages exclusively include large iguanas (mainly between 300 and 400 mm SVL) and young individuals (with SVL of less than 180 mm) are largely absent from archaeological deposits (Fig. 3). Although archaeological collection methods (for instance, the use of sieves and mesh size) could influence the observed size distribution, they cannot account for the lack of medium-sized individuals, as smaller taxa occur at all of the investigated sites. Thus, the observed distribution is likely to reflect human selection of the largest animals, which provide the most meat, and points to the difficulties involved in catching young iguanas or to the lack of interest they present in terms of meat intake. However, this size selection seems to differ between islands. Indeed, the difference between the MTMS of iguanas from each island and the average size observed in the archaeological sites of the same island differs among islands. These differences can be interpreted in terms of selection intensity: lower differences between the mean size and the MTMS point to the more marked selection of large specimens. This difference is especially low on La Désirade and Îlets de Petite Terre (102-132 mm), where selection seems to be strongest. However, it is higher in Marie-Galante and Grande-Terre (151-165 mm), and even higher in Basse Terre (192 mm), where selection appears to be low. The strong selection of large specimens on La Désirade and Îlets de Petite Terre could reflect the high density of Iguana populations, and thus the wide choice available to hunters, whereas conversely, population density would have been lower on Marie-Galante and Grande-Terre. The results from Basse Terre, indicating populations of different body-size, were difficult to assess. In the light of these data it is interesting to observe that Îlets de Petite Terre and La Désirade are the only islands in Guadeloupe still inhabited by large populations of I. delicatissima [START_REF] Breuil | Histoire naturelle des Amphibiens et Reptiles terrestres de l'archipel Guadeloupéen: Guadeloupe, Saint-Martin, Saint-Bathélemy[END_REF]. This could perhaps be explained by their isolation and high population density, preserving them from extirpation.

This clear size selection applied to archaeological specimens reflects the hunter's capacity to select hunted specimens and thus indicates the use of specific hunting techniques. No evidence of injuries was observed on these bones, but historical sources from European authors describing mid-17 th century Amerindians provide useful information about how pre-Columbian iguanas may have been hunted. These authors indicated that iguanas were caught by Carib Indians using a simple noose mounted on a perch that was discreetly passed around the iguanas' necks predators. Thus, for the digested remains found at the open-air archaeological sites in Anse à la Gourde, one of the most plausible hypotheses would be that these small iguanas were hunted by domestic dogs, since dogs occur in archaeological sites and were part of the daily life of pre-Columbian populations in the Guadeloupe Islands [START_REF] Grouard | Subsistance, systèmes techniques et gestion territoriale en milieu insulaire antillais précolombien -Exploitation des Vertébrés et des Crustacés aux époques Saladoïdes et Troumassoïdes de Guadeloupe[END_REF][START_REF] Grouard | Dog burials associated with Human burials in the West Indies during the early pre-Columbian Ceramic Age (500 BC-600 AD)[END_REF].

Alternatively, some of the remains collected in cave deposits (Anse à l'Ecu, Morne-Rita and Cadet 3) may reflect predation by birds of prey.

Size evolution of Iguana delicatissima over time

Several hundreds of years of pre-Columbian hunting do not seem to have impacted I. delicatissima populations, in contrast to the activities of modern human populations. Indeed, according to archaeological evidence, the size of iguanas seems to remain stable throughout the pre-Columbian period. Conversely, comparisons with modern data collected on Îlets de Petite Terre (Terre-de-Haut and Terre-de-Bas) and La Désirade, the only two islands in Guadeloupe still densely inhabited by I. delicatissima, show a possible significant decrease in size between pre-Columbian and modern times, estimated at about 20% of I. delicatissima SVL size. In addition, no present-day I. delicatissima equivalent in size to the largest archaeological iguanas has ever been measured, since the highest SVL ever recorded on a I. delicatissima specimen is only 430 mm SVL (from Dominica; [START_REF] Day | Lesser Antillean iguana: Iguana delicatissima[END_REF], as opposed to a maximum of 550 mm SVL for the largest archaeological specimen. As a precaution we should nevertheless note that it is difficult to assess the reliability of extreme archaeological SVL values, especially for the largest archaeological specimens with a SVL of more than 500 mm. This is because such sizes exceed the size of the modern specimens used by [START_REF] Bochaton | Describing archaeological Iguana Laurenti, 1768 (Squamata: Iguanidae) populations: size and skeletal maturity[END_REF] to construct SVL estimation equations. However, such very high sizes are close to the size of the iguanas described by Du Tertre in Guadeloupe during the 17 th century, when a total length of 160 cm corresponds to an SVL of around 53 cm (Du Tertre, 1667). However, although no equivalent sized I. delicatissima has ever been measured or collected, this does not mean that it never existed. Indeed, [START_REF] Lazell | The lizard genus Iguana in the Lesser Antilles[END_REF] mentioned that the small size of I. delicatissima in museum specimens could be due to sampling bias. He also claimed that he saw large specimens in Les Saintes and Dominica, with an SVL of around 400-450 mm, an observation confirmed on Dominica where the largest modern iguanas attain a maximal SVL size of 428 mm [START_REF] Knapp | Movements and nesting of the lesser antillean iguana (>i>Iguana delicatissima</i>) from Dominica, West Indies: implication for conservation[END_REF]. Still, even if the accuracy of estimated past iguana size is difficult to assess, a significant decrease in the size of this lizard on the Guadeloupe Islands over the past few centuries seems likely. As a consequence, the decrease in squamate size during the Holocene previously highlighted by [START_REF] Pregill | Body size of insular lizards: a pattern of Holocene dwarfism[END_REF] in other West-Indian squamates also seems to apply to I. delicatissima from the Guadeloupe Islands. This constitutes further evidence of the strong impact of human populations after the European colonization of these islands, where I. delicatissima populations were well established and healthy, as suggested by their large size, despite extensive exploitation by pre-Columbian hunters. These results differ from those obtained by [START_REF] Carlson | Resource depletion in the prehistoric northern West Indies[END_REF] on Grand Turk Island (Greater Antilles), where pre-Columbian hunting seemed to result in a size diminution in rock iguanas, probably on account of overexploitation on a relatively small island (only 18 km²). But this phenomenon concerns preceramic populations relying largely on terrestrial fauna for food sources (40% of the MNI of the Grand Turk Island site corresponds to iguanas). Conversely, the archaeological sites concerned by our study concern more recent ceramic cultures that rely mainly on marine resources and squamate remains represent a very minor part of the osteological assemblages (below 6% and often less than 1% of the vertebrate remains collected in each site [START_REF] De Waal | Pre-Columbian social organisation and interaction interpreted through the study of settlement patterns: An archaeological case-study of the pointe des Châteaux, La Désirade and Les Îles de la Petite-Terre micro-region[END_REF][START_REF] Grouard | Caribbean Archaeozoology[END_REF][START_REF] Grouard | Modes de vie des Précolombiens des Antilles françaises[END_REF][START_REF] Grouard | Subsistance, systèmes techniques et gestion territoriale en milieu insulaire antillais précolombien -Exploitation des Vertébrés et des Crustacés aux époques Saladoïdes et Troumassoïdes de Guadeloupe[END_REF]Bochaton, work in progress]). Up until now, few data were available concerning the pre-ceramic cultures and their subsistence strategies on the Guadeloupe Islands, and iguanas from these periods, as well as from pre-anthropic periods, remain unknown to us.

Conclusion

Our data clearly show that populations of Iguana delicatissima, were formerly present on all of the Guadeloupe Islands during pre-Columbian times and that at the same period, the invasive species, I. iguana was absent, confirming that it was introduced to this archipelago during the past few centuries. Iguanas were the object of selection focusing on large specimens by pre-Columbian hunters, as attested by the size reconstruction of archaeological specimens. The meat from these lizards was cooked and consumed by Amerindians, as described in historical sources recounting the daily life of 17 th century Caribbean Amerindians. Hunting by pre-Columbian hunters had no visible effect on past iguana populations. However, comparisons with modern data demonstrate that I. delicatissima is now extinct on most of the Guadeloupe Islands and is 20% smaller on the islands where it still occurs. This fact highlights the massive impact of European and historical populations on these large lizards, which mostly became extinct or underwent a drastic decrease in body size over the past three centuries. Such phenomena impacting iguanas were also observed using archaeological or subfossil data for several other taxa in the Caribbean [START_REF] Macphee | Insulae infortunatae: Establishing a Chronology for Late Quaternary Mammal Extinctions in the West Indies[END_REF][START_REF] Steadman | Vertebrate community on an ice-age Caribbean island[END_REF][START_REF] Stoetzel | Late Quaternary changes in bat palaeobiodiversity and palaeobiogeography under climatic and anthropogenic pressure: new insights from Marie-Galante, Lesser Antilles[END_REF] and in other geographic areas [START_REF] Grayson | The Archaeological Record of Human Impacts on Animal Populations[END_REF][START_REF] Nakamura | Recent cryptic extinction of squamate reptiles on Yoronjima Island of the Ryukyu Archipelago, Japan, inferred from garbage dump remains[END_REF][START_REF] Steadman | Prehistoric Extinctions of Pacific Island Birds: Biodiversity Meets Zooarchaeology[END_REF]. They provide further evidence of the strong impact of modern societies on fauna in the context of the current biodiversity crisis [START_REF] Barnosky | Has the Earth's sixth mass extinction already arrived?[END_REF][START_REF] Burney | Fifty millennia of catastrophic extinctions after human contact[END_REF][START_REF] Ceballos | Accelerated modern human-induced species losses: Entering the sixth mass extinction[END_REF].

Similar studies to ours would allow for the observation of past hitherto hidden or inaccurately recorded phenomena, such as the timing of colonization, spatio-temporal morphological changes throughout time or the natural or human-mediated introduction of exogenous species. They would also lead to the testing of several hypotheses based on modern data. However, with regard to archaeological lizards and snakes, such studies are extremely rare [START_REF] De Buffrénil | Harvest of the Nile Monitor, Varanus niloticus, in Sahelian Africa. Part 1: Impact of intensive harvest on local stocks[END_REF][START_REF] Klemens | Reptiles as a food resource[END_REF][START_REF] Monchot | Archaeozoological evidence for traditional consumption of spiny-tailed lizard (Uromastyx aegyptia) in Saudi Arabia[END_REF] and much work remains to be done in order to elucidate recent squamate history and interactions of these animals with past human populations all over the world.

  Number of Iguana remains (NR) collected from each archaeological site along with their attribution to I. delicatissima, I. iguana or Iguana sp. and Minimal Number of Individuals (MNI). Cultural attribution of each deposit is also indicated: Archaic= 2000 -500 BC, Saladoid = AD 80 -900, Troumassoid = AD 900 -1490, Modern=1493present.

Figure 1 :

 1 Figure 1: Map of the Lesser Antilles and focus on the Guadeloupe Islands with isobaths (-200m)

Figure 2 :

 2 Figure 2: Examples of Iguana bone remains from the Gare Maritime site (Basse Terre),

Figure 3 :

 3 Figure 3: Distribution of estimated SVL size (in millimeters) obtained on archaeological Iguana

Figure 4 :

 4 Figure 4: Anatomical distribution of Iguana remains from the 25 sites with PR value (Dodson

Figure 5 :

 5 Figure 5: A) Half-burnt (black) left jugal bone of I. delicatissima from Caille à Bélasse site

Acknowledgements

We are grateful to Le Gaïac NGO (F. Guiougou and B. Angin) and the French Office National de la Chasse et de la Faune Sauvage [ONCFS] (J.-P. Poly, B. Guillemot, E. Curot-Lodeon, and C. Rodrigues for providing modern data on I. delicatissima SVL). The collection of ONCFS data was funded by Le Fonds Européen de DÉveloppement Régional (FEDER) and the French government. We also thank the Service Régional de l'Archéologie de Guadeloupe (D. Bonissent, C. Stouvenot and E. Cousin) and the Edgar Clerc Museum (S. Guimaraes) for authorizing and facilitating access to the archaeological collections and administrative documentation. We are also grateful to M. Lemoine and to the UMR 7209 archaeobotanical platform for the SEM pictures. We wish to thank all the excavation directors of the studied sites: D. Bonnissent, M. Boudadi-Maligne, F. Casagrande, R. Chenorkian, E. Clerc, C. Colas, A. Delpuech, P. Courtaud, C. Etrich,

 [START_REF] Rochefort | Histoire naturelle et morale des Antilles de l'Amérique[END_REF][START_REF] Du Tertre | Histoire générale des Antilles habitées par les François[END_REF]Maurile de Saint Michel (ca 1615-1669)in Grunberg, 2013).

The same technique was still used by Guadeloupe and La Martinique inhabitants long after the disappearance of pre-Columbian hunters [START_REF] Labat | Nouveau voyage aux isles de l'Amerique T[END_REF][START_REF] Pinchon | D'autres aspects de la nature aux Antilles[END_REF]. After being caught, the authors also explained that the animals were carried to the site and kept alive, sometimes for several weeks [START_REF] Du Tertre | Histoire générale des Antilles habitées par les François[END_REF]Grunberg, 2013b;[START_REF] Rochefort | Histoire naturelle et morale des Antilles de l'Amérique[END_REF], as suggested by the occurrence of all the anatomical pieces in the bone remains from the sites (Fig. 4). But iguanas were also cooked, as shown by the rare traces of fire and cut marks observed on archaeological remains, which can also be correlated to historical literature sources. Indeed, the anonymous author from Carpentras who described 17 th century Amerindians, explained that after being brought back to the village, the iguanas were burned to kill them and remove their skin before being dismembered (Anonyme de Carpentras (1618-1620) in Grunberg, 2013b). We observed rare fire marks on Iguana bones on the thinnest anatomical parts most exposed to fire (skull and extremities including sectioned tails). These marks could reflect the burning practices described by the anonymous Carpentras author (Anonyme de Carpentras (1618-1620) in Grunberg, 2013b).

The rarity of such marks could also correspond to the practice of boiling described by the same author (Anonyme de Carpentras (1618-1620) in Grunberg, 2013b), or to drying or smoking the carcass, as often practiced by Amerindian populations. However, there is no mention of removing the tail; a practice clearly established on archaeological iguanas and observed by one of us (S.

G.), along with the removal of the skin and extremities of the carcass just before cooking.

The rare digestion traces observed on archaeological remains are intense, easily visible, and only occur on small specimens, highlighting the particularity of these small remains, which clearly differ from the main size distribution. These remains could reflect the occasional human consumption of young iguanas but such small animals may also have been eaten by non-human P. Fouéré, J. Gagnepain, E. Gassies, C. L. Hofman, M. l. P. Hoogland, A. Lenoble, T. Romon, N.

Sellier-Segard, N. Serrand, C. Stouvenot, M. Van den Bell and M. de Waal.

Finally, we wish to thank Scott Fitzpatrick and an anonymous reviewer for helping us to improve the quality of this paper. traces (white arrows) and cut marks (black arrows) showing the removal of the tail from Anse à la Gourde site (year 1997-Z64 S93 C1 lvl 4).