Mami Inoue

Shinsuke Kamiguchi

Katto Ugawa

Shaima Hkiri

Dr Jules Bouffard

David Sémeril
email: dsemeril@unistra.fr

Tetsuo Iwasawa
email: iwasawa@rins.ryukoku.ac.jp

Evaluation of the Catalytic Capability of cis -and trans -Diquinoxaline Spanned Cavitands

Keywords: cavitands, Diquinoxaline-extended resorcin[4]arene, Gold and Rhodium catalysis

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

Introduction

Resorcin [4]arene-cavitand, able to carry out a catalytic reaction, is an emerging supramolecule that mimics enzymatic catalysis and achieves marvelous chemical transformations from the viewpoint of green chemistry. 1 The cavitands have strong resemblances in two points to natural enzymes: one, cavitands are partly open, guest substrates readily fill the space, enter and leave; the second, cavitands are endowed with gently curved concave large enough to accommodate catalytic centers. Despite the relevant role played by catalytic cavitands, such a class of cavitands is underrepresented owing to synthetic difficulties. 3,4 To make supramolecular cavitands those weigh over MW ca. 1000 preparatively in pure form is a basically embarrassing synthesis. When we attempt to introduce reactive centers inside the hydrophobic pockets, isomeric production of in-and outwardly oriented cavitands towards the pockets often occurs. Hence, we chemists are now always struggling to understand even basic aspect of cavitand catalysis. It might be quite a challenge for us to imitate enzymatic virtue that Mother Nature has meticulously created for more than long, long four billion years, because synthetic chemistry has been in just one or two hundred-yearperiod history since Friedrich Wöhler synthesized urea from ammonium cyanate. 5,6 Recently, we have developed for catalytic applications trans-diquinoxaline-spanned resorcin[4]arenes. 7 An introverted bis-gold cavitand was found to be efficient in the dimerization of two different terminal alkynes. 8 A second type of gold cavitand, in which a lewis acid gold cation was associated to a lewis basic P=O moiety, displayed high regioselectivities in addition of H 2 O to unsymmetrical triple bonds; for example, 3-octanone was obtained in 91 % yield starting from 3-octyne. 9 Although the advantage of cavitand catalysis in selective transformations are obvious, but systematic studies of structure-activity relationship remains rare. Particularly, from the viewpoint of basic research, comprehension of mechanistic aspects awaits upgrading, enhancement, and accomplishment. Herein we report a structureactivity relationship of phosphinated cavitands in transition metal-Figure 1. Cis-1-3 and trans-4-6 di-quinoxaline-spanned resorcin[4]arenes. catalyzed reactions. We prepared new cis-type P(III) (1 and 2) and mixed P(III)/P(V) (3) extended cavitands, which are structurally similar to previously reported trans-type 4, 5, and 6 derivatives (Figure 1): those are used as ligands in gold-and rhodiumcatalysis. The question we pursue here is "How does the difference between these trans-and cis-arrangements affect the catalytic activity and selectivity?"

Results and Discussion

The synthesis of cis-1 starts with a reaction between resorcin[4]arene and 2,3-dichloroquinoxaline (Scheme 1(a)). Preparation of cis-positioned di-quinoxaline-spanned resorcin [4]arene was laborious 10 and required the use of DABCO (1,4-diazabicyclo[2.2.2]octane) and pyridine improved the chemical yield to 22%. The obtained cis-positioned tetra-ol cavitand reacted, in the presence of a base in refluxing toluene, with P(OCH 3 ) 3 to give two of the three possible isomers ("outout", "in-out", and "in-in"). The two compounds were carefully separated by silica-gel column chromatography and were isolated in 44% and 22% yield for the be "out-out" 1 and "in-out" iso-1, respectively (Scheme 1(b)). [START_REF]isomer was observed in the 1 H NMR spectrum of the crude reaction mixture[END_REF] Orientation of the lone pair of the phosphorus atoms were deduced from their 1 H NMR spectra. In the case of 1, its 1 H NMR spectrum reveals one doublet located at 3.87 ppm ( 3 J PH = 8.6 Hz) for the two "out-out" POCH 3 ; in the spectrum of iso-1, two doublets at 3.87 ppm ( 3 J PH = 8.6 Hz) and at 3.20 ppm ( 3 J PH = 12.6 Hz), which can be attributed to the two "inout" POCH 3 protons (Figure 2 Di-phosphoramidite derivative cis-2 was obtained by reaction between the tetra-ol resorcinarene and P[N(CH 3 ) 2 ] 3 in the presence of NEt 3 at 75°C in toluene. Although, after 1 hour, TLC displayed mostly a single spot, due to decomposition degradation during silica-gel column chromatography, compound cis-2 was isolated in only 44% yield (Scheme 1(a)). Its 1 H NMR spectrum exhibits one doublet located at 2.76 ppm ( 3 J PH = 10.5 Hz), which could be attributed to the two outwardly directed PN(CH 3 ) 2 moieties. A similar signal at 2.85 ppm ( 3 J PH = 11.05 Hz) was previously observed in the cavitand trans-5 (Figure 2 (e) and (f)). 13 Scheme 1. (a) Synthesis of cis-1 and cis-2; (b) iso-1 bearing both inwardand outward POCH3 groups; (c) synthesis of cis-3 (mCPBA = metachloroperbenzoic acid). The conformational flexibility of tetra-quinoxaline-spanned resorcin [4]arenes is well known and can fluctuate between vase (close) and kite (open) conformations. The conformation of the macrocycle could be deduced from its 1 H NMR spectrum and more particularly from the chemical shift of methine protons directly beneath quinoxaline moieties. A chemical shift around 5.5 ppm indicates a vase conformer, whereas a chemical shift around 3.7 ppm reveals a kite form. [START_REF] Azov | [END_REF] For our four new compounds (1, iso-1, 2 and 3), 1 H NMR investigations carried out in CDCl 3 and [D 8 ]toluene, clearly indicate a vase conformation of the cavitands, the methine protons appear at 5.73 (1), 5.76 and 5.71 (iso-1), 5.70 (2), 5.79-5.71 (3) ppm in CDCl 3 and at 6.14 (1), 6.15 and 6.08 (iso-1), 6.15 (2) and 6.20 and 6.11 (3) ppm in [D 8 ]toluene. Then, we interested in the difference in π-clouded environment between cis-and trans-walled cavitands. For that, we compared the chemical shifts of the CH 3 proton of the POCH 3 moieties in cavitands iso-1 and iso-4. As shown in Figure 2 (b) and (d), the  (differences in the chemical shifts between inward-and outwardoriented POCH 3 ) are 0.66, and 0.88 for iso-1 and iso-4, respectively. These results are important evidences suggesting that the trans-walls create a definite compartment that is more heavily influenced by the π-clouds than the compartment of the cis-walled cavitand.

Phosphites 1 and 3 readily formed complexes with AuCl•S(CH 3 ) 2 . Using a Au/P ratio of 1:1.2 in toluene, after 0.5 h cis-1•(AuCl) 2 and cis-3•AuCl complexes were isolated in 100% and 63% yield, respectively (Scheme 2). The latter gold complexes were tested in cross-and homo-dimerization of alkynes as well as in hydration of di-substituted alkynes. Their performances in catalysis will be ranking with the previous reported trans-4•(AuCl) 2 and trans-6•AuCl complexes. 7b,15 Scheme 2. Synthesis of (a) 1•(AuCl)2 and (b) 3•AuCl complexes.

For the gold-catalyzed terminal alkynes dimerization, the runs were carried out using 1 mol % of cis-1•(AuCl) 2 or trans-4•(AuCl) 2 complex and 2 mol % of AgOTf in toluene at room temperature for 20 h (Scheme 3). Using precatalyst cis-1•(AuCl) 2 , whether for cross-dimerization of ethynylbenzene and 1-octyne or for homo-dimerization of 1-octyne, only 2-octanone (7c), coming from hydration of octyne, was observed in 29% and 35% yield, respectively. These results contrast with those observed when the tests were repeated using the trans-diquinoxaline spanned trans-4•(AuCl) 2 complex. The letter led only to the formation of dimeric products, with a ratio hetero-/homo-dimer of 7a/7b = 75:25 when a mixture of ethynylbenzene and 1-octyne was employed or quantitatively to the dimer 7b in the case of dimerization of 1octyne. These results clearly show the importance of the positioning of the two gold center on the resorcin[4]arene skeleton. Using bis-gold complexes with cis-and trans-walled cavitands led to different type of active species and catalytic outcome.

Differences in activity and regioselectivity could also be highlighted in the gold-catalyzed hydration of internal alkynes using mixed P(III)/P(V) extended cavitands, in which the two phosphorus atoms are cis-or trans-positioning, cis-3•AuCl or trans-6•AuCl complexes, respectively. For hydration of ethynylbenzene, the cis-3•AuCl complex led to low activity and selectivity, the products, 1-phenyl-1-butanone (7d) and 1-phenyl-2-butanone (7e) were obtained in 5% and 10% yield, respectively. While repeating the run with precatalyst trans-6•AuCl, the compounds 7d and 7e were isolated in 2% and 88% yield, respectively. A better regioselectivity was also observed when trans-6•AuCl complex was employed in the hydration of 3-octyne, the ratio 4-octanone (7f)/3-octanone (7g) = 9:91. The ratio 7f/7g decrease to 46:54 when complex cis-3•AuCl was used (Scheme 4). The three cavitands bearing phosphoramidite moieties (cis-2, trans-5 and 8 16 ) were assessed in styrene hydroformylation (Scheme 5). The catalytic systems were generated in situ by mixing ligand with Rh(acac)(CO) 2 precursor and the reactions were carried out in toluene under 20 bar of CO/H 2 (1:1) with a styrene/Rh ratio of 5000.

In a first series of tests, conduced at 80°C during 24h, the cavitand/Rh ratio (P/Rh) was studied (Table 1). Variation of the number of phosphorous atoms per rhodium atom ratio to 2 to 10, as general trends, we observed a decrease of the conversion when the amount of ligand increased. For example, using monophosphoramidite 8, conversions of 64% and 54% were measured when P/Rh ratios of 2 and 10 were employed, respectively (Table 1, entries 7 and9). Di-phosphoramidites cis-2 and trans-8 were more efficient than mono-phosphoramidite 8, conversions of 96%, 99% and 64%, respectively, were observed with a P/Rh ratio of 4 (Table 1, entries 2, 5 and8). Interestingly, the regioselectivity of the reaction was not affected by the presence of excess of ligand for cis-2 (l/b = 35/65) contrary to the other two (Table 1, entries 1-3). It is noteworthy, in the case of ligand trans-5, that the P/Rh ratio drastically affects the regioselectivity of the reaction. The proportion of branched aldehyde increase with the number of extra-added ligand, 2-phenylpropanal was formed in 48% and 78% selectivity with P/Rh ratio of 2 and 10, respectively (Table 1, entries 4 and 10). Note that the regioselectivities were similar when either trans-5 or 8 when were employed in large excess, branched aldehydes were formed in 78% and 76%, respectively (Table 1, entries 6 and9).

Table 1. Evaluation of phosphoramidites cis-2, trans-5, and 8 in styrene hydroformylation: influence of the P/Rh ratio. [a] Entry P/Rh [b] Ligand [a] Reactions were carried out in conditions of Scheme 5 at 80 ºC for 24 h. The conversions and products distributions were determined by GC (ndecane as internal standard) and by 1 H NMR. [b] Ratio of phosphorous atoms per rhodium atom.

In a second series of runs, we investigated the influence of the temperature from 60°C to 140°C after 5 h reaction time with a P/Rh ratio of 2 (Table 2). As general trends, the conversion increase with the temperature, around 120°C, and the regioselectivity of the catalysis change from mainly branched aldehyde at low temperature to mainly linear aldehyde at high temperature. For example, using trans-5 ligand, conversion of 52% with a l/b ratio of 16/84 were observed at 60°C against a full conversion with a l/b ratio of 64/36 at 120°C (Table 1, entries 8 and 13). For the tests carried out at 140°C, lower conversions came from decomposition of the catalyst at high temperature (Table1, entries 7, 14 and 21). We can observe that diphosphoramidite (cis-2 and trans-5) led to higher activities than mono-phosphoramidite 8, especially at low temperature. In fact, at 60°C, conversions of 59%, 52% and 11% were measured when ligand cis-2, trans-5 and 8, were employed, respectively (Table 2, entries 1, 8 and 15). Similarly, a higher regioselectivity toward linear aldehyde was measured when cis-2 and trans-5 were used rather than 8, proportion of 35 %, 51% and 31% of 3phenylpropanal were obtained, respectively (Table 2, entries 2, 9 and 16). Note that during runs, once formed, the in situ generated catalysts were stables as observed by similar catalytic outcome analyzed during the first 5 h of hydroformylation reactions carried out at 120°C (Table 1, entries 4-6, 11-13 and 18-20). We can also observed that, at 60°C, cis-2 led to a faster catalytic system with a higher proportion of linear aldehyde than its homologue diphosphoramidite trans-5, conversion of 59% (linear aldehyde 33%) against 52% (16% of linear aldehyde), respectively (Table 2, entries 1 and 8). The main differences observed between the two di-phosphoramidites, is that cis-2 led to less thermically stable active species than those formed with trans-5. In fact, at temperature of 120°C or higher, lower activities were measured when cis-2 was employed rather than trans-5 (Table 2, entries 5-7 and 12-14) and at the end of the runs carried out with cis-2, carful observation of the reaction mixture shown the formation of traces of black insoluble materials, probably rhodium colloids. [a] Reactions were carried out in conditions of Scheme 5 with P/Rh = 2 (ratio of phosphorous atoms per rhodium atom). The conversions and products distributions were determined by GC (n-decane as internal standard) and by 1 H NMR.

From these experiments, several information about the nature of the catalytic systems generated from the three ligands could be deduced:

-In the case of mono-phosphoramidite 8, due to the steric hindrance generated by the three quinoxaline moieties, formation of RhL 2 complex should be exclude. Generation of singly ligated active species favor the formation of Rh( 3styrenyl)(8)(CO) 2 intermediate over the less stable Rh(ethylphenyl)(8)(CO) 2 species and in fine, favor the formation of the branched aldehyde, 17 at less at low temperature. At high temperature, thermal agitation generates strong steric interactions between the coordinated substrate and the cavity wall; such metal confinement favors the formation of the linear aldehyde. 18 -For the di-phosphoramidite trans-5, due to the distance between the two phosphorus atoms (9.7 Å measured on the RX structure of trans-5•(AuCl) 2 complex 8 ), formation of rhodium-chelate complexes should be exclude. Nevertheless, the proximity of two coordinated rhodium atoms, on the same cavitand, may allow to consider a cooperation between these two metal centers. 19 In fact, the higher activity and the selectivity toward the linear aldehyde regarding ligand 8 are arguments in that sense. Furthermore, when a large excess of trans-5 was employed, in that case, we can assume that only one atom of rhodium is coordinated to the ligand, the second phosphorus atom remained free, regioselectivity of the reactions was similar to that obtained when monophosphoramidite 8 was employed (vide supra).

-With di-phosphoramidite cis-2, according to molecular modeling, the distance between the two phosphorus atoms (around 6.5 Å) was to important to allow the formation of rhodium-chelate complexes.

The shorter distance, compare to the trans-8 isomer, should reinforce the cooperation between the two metallic centers, which led to a more efficient catalytic system as observed at low temperature. However, the shorter distance may be responsible of the low thermal stability of the corresponding binuclear complexes and the formation of rhodium colloids. The cis-positioning of the phosphorus atoms led also to a more open cavitand compare to trans-8, the steric pressure excess by the two quinoxaline walls was reduced and consequently, the proportion of branched aldehyde formed was more important than when isomer trans-8 was employed.

Conclusions

In summary, three cavitands having a cis-arrangement of two quinoxaline walls (cis-1, cis-2, and cis-3) were successfully synthesized. These cavitands, in which two catalytic centers are inwardly oriented, provide new architecture for transition metal catalysis. Gold-catalyzed dimerization and hydration of alkynes and rhodium-catalyzed styrene hydroformylation were carried out with these cis-type cavitands. Comparative studies with analogues trisquinoxaline and trans-diquinoxaline spanned cavitands allow us to evaluate the structure-activity relationship between cis-and trans-walls flanked-cavitands. The catalytic results strongly suggest three salient features. Firstly, cis-positioning phosphorus atoms led to a more open cavitands, which leads to a highly reactive species toward solvent impurities, for example in the dimerization of alkynes, traces of water present in toluene.

Secondly, the less impeded environment around the metal center reduced or modified the regioselectivities of the reactions compare to those observed with trans-spanned ligand. Thirdly, the distance between the two phosphorus atoms is shorter in the case of cisthan trans-cavitands. Although a shorter distance promotes cooperation between the two metal centers, as observed in the styrene hydroformylation, it favors the degradation of the active species and the formation of rhodium colloids. 36.7, 35.9, 34.5, 34.4, 32.8, 32.5, 32.3 (many peaks are overlapped), 31.8, 31.1, 30.2, 30.12, 30.09 (many peaks are overlapped), 30.05, 30.01, 29.78, 29.75, 28.4, 28.3, 23.1, 23.0 8, 34.4, 32.6, 32.3, 31.6, 30.1, 29.8, 28.4, 28.3, 23.1, 14.5, (many

Synthesis of 3: (see Scheme 1(c))

To 1 (148 mg, 0.1 mmol) in toluene (4 mL) at 0 ºC was slowly added a cooled-toluene solution of mCPBA (75%, 23 mg, 0.1 mmol) over 3 min. After stirred at 0 ºC for 1.5 h, the reaction was quenched with saturated aqueous NaHCO3 (2 mL), and stirred at ambient temperature for 30 min. The mixture was transferred into a 50 mL separatory funnel, washed with water (10 mL) and brine (10 mL), dried over Na2SO4, and concentrated in vacuum to give a crude of 143 mg as a white solid material. Purification by short-plugged column chromatography (SiO2, toluene/EtOAc = 9/1) led to 44 mg of 3 in 30% yield as white solid powder. 9, 34.5, 34.4, 33.1, 32.3, 32.1, 31.5, 31.4, 30.1 (many For 3•AuCl Under N2 atmosphere, a solution of 3 (111 mg, 0.074 mmol) in toluene (1.2 mL) underwent addition of AuCl•S(CH3)2 (26 mg, 0.089 mmol), and the reaction mixture was stirred for 30 min, consumption of 3 was monitoring by TLC. After all the volatiles had been evaporated, the crude products were purified by short-plugged silica-gel column chromatography (eluent: hexane/EtOAc, 1/1) to afford 56 mg of 3•AuCl as white powder materials in 63% yield. 1 H NMR (400 MHz, CDCl3) 8.40 (s, 1H), 8.11 (d, J = 8.4 Hz, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.88 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.72 (dd, J = 8.4, 8.4 Hz, 1H), 7.63 (dd, J = 8.4, 8.4 Hz, 1H), 7.56 (s, 1H), 7.53-7.46 (m, 2H) Representative procedure for Au-catalyzed dimerization of terminal alkynes: (see Scheme 3) Under N2 atmosphere, the bis-Au catalyst (0.01 mmol) in a 25 mL two-necked flask was dissolved in toluene (5 mL; containing traces of water), and the starting alkynes of ethynylbenzene (102 mg, 1 mmol) and the other partner 1-octyne (165 mg, 1.5 mmol) were added. After addition of AgOTf (5.0 mg, 0.02 mmol) at room temperature, the reaction was conducted for 20 h. The solvent was evaporated off, and filtered through a short-plugged column chromatography to give a crude product. The crude materials consisted of just starting alkynes and/or product 7a, 7b, and 7c because of clean reaction progress: thus, chemical yields and molar ratios of products were determined in the crude state. All dimeric adducts were identical to the authentic samples that we previously reported. 7b,9 Representative procedure for Au-catalyzed hydration of internal alkynes: (see Scheme 4) Complex 1•(AuCl)2 (17 mg, 0.01 mmol) was added under Ar to a solution of 3-octyne (0.07 mL, 0.5 mmol) in toluene-d8 (1 mL) and H2O (0.05 mL, 2.5 mmol). The mixture was stirred at room temperature for 5 min, AgOTf (3 mg, 0.012 mmol) was then added, and the whole system was immersed in a 50 ºC preheated oil bath. After stirred for 1 h, the reaction mixture was allowed to cool to ambient temperature. Purification by short-plug silica-gel column chromatography with use of a Pasteur pipette and [D8]toluene as eluent gave a colorless C7D8 solution. 1 H NMR spectroscopy determined the chemical yields and product distribution (proportions of 7f and 7g). 9 1 H NMR spectroscopic data for 7f and 7g were identical to those for commercially available authentic sample.

Representative procedure for Rh-catalyzed hydroformylation of styrene: (see Scheme 5) The hydroformylation reactions were carried out in a glass-lined, 50 mL stainless steel autoclave containing a magnetic stirring bar, then the autoclave closed and flushed twice with vacuum/N2. To the vessel were added toluene (8 mL), styrene (10 mmol), n-decane (0.5 mL), 1 mL toluene solution of Rh(acac)(CO)2 (2 μmol), and 1 mL toluene solution of appropriate ligand. After pressurized at 20 bar (CO/H2 1:1) and heated at appropriate temperature for the adequate time, the autoclave was allowed to cool to ambient temperature before being depressurized. The reaction mixture was analyzed by GC using a WCOT fused-silica gel column chromatography (25 m x 0.25 mm) and by 1 H NMR.

Entry for the Table of Contents

  (a) and (b)). The latter up fieldshifted doublet ( = 3.20 ppm) results from anisotropic effects of the aromatic π clouds and the OCH 3 group inwardly oriented. Consequently, in cavitand 1, the two POCH 3 moieties are outwardly oriented as confirmed by the chemical shifts of the outwardly oriented POCH 3 groups in the well know cavitands 4 ( = 3.97 ppm, 3 J PH = 8.7 Hz) and iso-4 (Figure 3 (a);  = 3.98 ppm, 3 J PH = 8.3 Hz and  = 3.10 ppm, 3 J PH = 12.4 Hz) (Figure 2 (c) and (d)). Interestingly, oxidation of only one phosphorus atom of iso-1 could be carried out in the presence of mCPBA (metachloroperbenzoic acid), cavitand 3 was isolated in 30% yield (Scheme 1(c)).[START_REF]The oxidation with mCPBA led to 24% of di-phosphate derivative and 46% of unreacted iso-1 was recovered[END_REF]

Figure 2 .

 2 Figure 2. Portions of 1 H NMR spectra (400 MHz, CDCl3) of (a) 1, (b) iso-1, (c) 4, (d) iso-4, (e) 2, and (f) 5. The peaks labeled with black coloredcircle and black colored-triangle correspond to inward-and outwardoriented POCH3 or PN(CH3)2 groups, respectively.

Figure 3 .

 3 Figure 3. Structures of (a) iso-4 that has one inward P-OMe and one outward P-OMe, and (b) mono-phosphoramidite 8 that was flanked by three quinoxaline moieties.

Scheme 3 .

 3 Scheme 3. Catalytic evaluation of cis-1•(AuCl) 2 and trans-4•(AuCl) 2 in (a) cross-dimerization and (b) homo-dimerization (see Experimental Section).

  Introverted ligand / Cisspanned cavitands / Diquinoxalineextended resorcin[4]arene / Gold and Rhodium catalysis Three new cis-diquinoxaline spanned cavitands were synthetized. These cisdiphosphinated derivatives were assigned in homogeneous catalysis. Results were ranked with those obtained with their trans-diphosphinated analogues. The Structure-activity relationship realized with these cis-or trans-flanked cavitands shows that the cis-or trans-positioning of the catalytic center has a direct effect either on activity or on regioselectivity of a catalytic reaction.

Table 2 .

 2 Evaluation of phosphoramidites cis-2, trans-5, and 8 in styrene hydroformylation: influence of the temperature.[a]

	Entry	Cavitand T/ºC	t/h	Conv./%	Product Distribution Linear/% Branched/%
	1		60	5	59	33	66
	2		80	5	88	35	65
	3		100	5	92	56	43
	4	cis-2	120	0.5	41	66	34
	5		120	2	48	63	37
	6		120	5	69	65	35
	7		140	5	60	62	38
	8		60	5	52	16	84
	9		80	5	75	51	49
	10		100	5	89	58	42
	11	trans-5	120	0.5	48	56	44
	12		120	2	89	63	37
	13		120	5	100	64	36
	14		140	5	100	64	36
	15		60	5	11	18	82
	16		80	5	40	31	68
	17		100	5	82	58	42
	18	8	120	0.5	17	56	44
	19		120	2	69	66	34
	20		120	5	91	64	36
	21		140	5	54	68	32

  All reactions sensitive to air or moisture were carried out in anhydrous conditions under argon or nitrogen atmosphere. Dry solvents were purchased and used without further purifications. All reagents were purchased and used without further purifications. Analytical thin layer chromatography was carried out on Merck silica 60F254. Column chromatography was carried out with silica gel 60 N (Kanto Chemical Co.). Hz C 30 ), 148.5 (C 26 ), 147.3 (d, JC,P = 4.5 Hz, C 17 ), 146.6 (d, JC,P = 2.4 Hz, C 37 ), 140.34 (C 13 ), 140.31 (C 6 ), 140.28 (C 14 ), 140.2 (C 5 ), 137.8 (d, JC,P = 2.6 Hz, C 36 ), 136.54 (C 21 ), 136.45 (C 34 ), 135.9 (C 23 ), 135.7 (C 27 ), 135.4 (C 28 ), 134.8 (C 35 ), 134.4 (C 29 ), 129.8 (C 2 ), 129.7 (C 11 ), 129.5 (C 3 ), 129.4 (C 10 ), 128.5 (C 22 ), 128.3 (C 33 ), 127.8 (C 20 ), 124.1 (C 12 ), 123.1 (C 1 ), 122.6 (C 9 ), 121.7 (C 4 ), 119.1 (C 25 ), 118.0 (C 39 ), 117.7 (C 31 ), 116.8 (C 18 ), 51.9 (d, JC,P = 22.2 Hz, C 41 ), 50.2 (d, JC,P = 2.6 Hz, C 42 ),

		General Methods: LRMS and HRMS were reported on the basis of TOF (time of flight)-MS
		(MADI-TOF or LCMS-IT-TOF), and DART (Direct Analysis in Real
		Time)-MS. 1 H, 13 C{ 1 H} and 31 P{ 1 H} NMR spectra were recorded with a 5
		mm QNP probe at 400 MHz and 100 MHz, respectively. Chemical shifts
		are reported in  (ppm) with reference to residual solvent signals [ 1 H
		NMR: CHCl3 (7.26), toluene (2.08); 13 C NMR: CDCl3 (77.0); 31 P shifts are
		given with respect to external H3PO4]. Signal patterns are indicated as s,
		singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. Assignment
		of 13 C NMR was carefully performed with labeling the corresponding
		numbers those were listed in 13 C NMR spectra.
		Synthesis of 1 and iso-1: (see Scheme 1 (a) and (b)) To the Schlenk tube
		charged with a solution of the tetra-hydroxy cis-cavitand (407 mg, 0.3
		mmol) in dry toluene (3 mL) under N2 at 135ºC, EtN(iPr)2 (0.52 mL, 3.0
		mmol) and P(OCH3)3 (0.29 mL, 2.4 mmol) were added. After stirred for 6 h,
		the reaction mixture was cooled to room temperature and concentrated to
		give 457 mg of crude products as pale yellow solid materials. Purification
		by silica-gel column chromatography (eluent: hexane/EtOAc, 19/1)
		afforded 195 mg of 1 ("out-out") as white solid materials in 44% yield and
		98 mg of iso-1 ("in-out") as white solid materials in 22% yield.
		Data for 1: 58 (s,
		1H), 7.49 (d, J = 8.2 Hz, 2H), 7.18 (dd, J = 8.1, 7.4 Hz, 2H), 7.06-7.01 (m,
		2H), 6.32 (s, 1H), 6.14 (t, J = 8.1 Hz, 2H), 4.87 (t, J = 7.8 Hz, 2H), 3.65 (d,
		3 JPH = 8.6 Hz, 6H, POCH3), 2.46-2.35 (m, 8H), 1.50-1.29 (m, 72H), 0.97-
		0.92 (m, 12H) ppm; 13 C{ 1 H} NMR (100 MHz, CDCl3) 153.2 (C 15 ), 153.03
		(C 10 ), 152.98 (C 7 ), 152.9 (C 8 ), 147.3 (C 17 ), 147.2 (C 21 ), 147.10 (C 5 ), 147.06
		(C 6 ), 140.03 (C 13 ), 137.6 (d, JC,P = 2.6 Hz, C 19 ), 136.5 (C 12 ), 135.3 (C 20 ),
		129.6 (C 1 ), 129.4 (C 4 ), 128.4 (C 2 ), 128.2 (C 3 ), 123.8 (C 18 ), 122.9 (C 14 ),
		122.3 (C 13 ), 119.2 (C 22 ), 118.1 (C 16 ), 117.4 (C 9 ), 50.4 (d, JC,P = 2.6 Hz, C 23 ),
		35.9, 34.4, 32.9, 32.3 (many peaks are overlapped), 30.1 (many peaks are
		overlapped), 29.8 (many peaks are overlapped), 28.4, 28.3, 23.1 (many
		peaks are overlapped), 14.5 (many peaks are overlapped) ppm; 31 P{ 1 H}
		NMR (162 MHz, CDCl3) 127.3 ppm; MS (ESI) m/z: 1512 [M + Cl] -; IR
		(neat): 2922, 2851, 1606, 1578, 1482, 1412, 1332, 1158, 1029, 759 cm -1 ;
		HRMS (ESI) calcd for C90H118N4O10P2Cl: 1511.8017 [M + Cl] -, Found:
		1511.8000.
		Data for iso-1: 1 H NMR (400 MHz, CDCl3) 8.36 (s, 1H), 7.99 (d, J = 8.0
		Hz, 1H), 7.97 (d, J = 7.8 Hz, 1H), 7.84 (d, J = 8.1 Hz, 1H), 7.64 (d, J = 8.2
		Hz, 1H), 7.64-7.51 (m, 4H), 7.39 (s, 1H), 7.28 (s, 1H), 7.22 (s, 2H), 7.15 (s,
		1H), 7.11 (s, 1H), 6.31 (s, 1H), 5.76 (t, J = 7.8 Hz, 1H), 5.71 (t, J = 7.8 Hz,
		1H), 4.53-4.46 (m, 2H), 3.87 (d, 3 JPH = 8.6 Hz, 3H, POCH3), 3.21 (d, 3 JPH =
		12.6 Hz, 3H, POCH3), 2.29-2.17 (m, 8H), 1.41-1.27 (m, 72H), 0.90-0.87 (m,
	To the best of our	12H) ppm; 1 H NMR (400 MHz, [D8]toluene) 8.82 (s, 1H), 8.03 (d, J = 8.3
	knowledge, this work represents the first examples in	Hz, 1H), 7.81 (d, J = 8.3 Hz, 1H), 7.69 (s, 1H), 7.66 (s, 1H), 7.63 (s, 1H),
	homogeneous catalysis of comparative studies involving ligands	7.56 (s, 2H), 7.50 (s, 1H), 7.42 (d, J = 8.2 Hz, 1H), 7.39 (d, J = 8.2 Hz, 1H),
	Experimental Section

built on extended cavitands having a cis-or a trans-positioning of two active centers. Further developments of hybrid catalysts grafted on extended cavitands are ongoing and will be reported in due course. 1 H NMR (400 MHz, CDCl3) 8.41 (s, 1H), 7.98 (d, J = 8.2 Hz, 2H), 7.82 (d, J = 8.2 Hz, 2H), 7.62-7.52 (m, 4H), 7.38 (s, 2H), 7.21 (s, 3H), 7.17 (s, 1H), 6.46 (s, 1H), 5.73 (t, J = 7.8 Hz, 2H), 4.54 (t, J = 7.8 Hz, 2H), 3.88 (d, 3 JPH = 8.6 Hz, 6H, POCH3), 2.30-2.17 (m, 8H), 1.43-1.27 (m, 72H), 0.90-0.87 (m, 12H) ppm; 1 H NMR (400 MHz, [D8]toluene) 8.82 (s, d1H), 7.85 (d, J = 8.2 Hz, 2H), 7.78 (s, 2H), 7.69 (s, 2H), 7.66 (s, 2H), 7.7.26 (dd, J = 8.2, 7.3 Hz, 1H), 7.18 (dd, J = 8.2, 7.3 Hz, 1H), 7.05-7.01 (m, 2H), 6.38 (s, 1H), 6.15 (t, J = 8.1 Hz, 1H), 6.08 (t, J = 8.1 Hz, 1H), 4.91 (t, J = 7.9 Hz, 1H), 4.75 (t, J = 7.9 Hz, 1H), 3.72 (d, 3 JPH = 8.6 Hz, 3H, POCH3), 2.43-2.32 (m, 8H), 2.04 (d, 3 JPH = 12.4 Hz, 3H, POCH3), 1.48-1.29 (m, 72H), 0.95-0.93 (m, 12H) ppm; 13 C{ 1 H} NMR (100 MHz, CDCl3) 153.3 (C 40 ), 153.23 (C 8 ), 153.19 (C 32 ), 153.1 (C 19 ), 153.0 (C 16 ), 152.9 (C 7 ), 152.7 (C 15 ), 152.4 (d, JC,P = 1.2 Hz, C 38 ), 148.8 (C 24 ), 148.6 (d, JC,P = 6.2

  JCP = 18.8 Hz, two peaks are overlapped, C 23 C 24 ), 35.

	(many
	peaks are overlapped), 14.5 (many peaks are overlapped) ppm; 31 P{ 1 H}
	NMR (162 MHz, CDCl3) 125.6, 110.6 ppm; MS (ESI) m/z: 1512 [M + Cl] -;
	IR (neat): 2922, 2851, 1578, 1482, 1412, 1352, 1146, 1029, 760 cm -1 ;
	HRMS (ESI) calcd for C90H118N4O10P2Cl: 1511.8021 [M + Cl] -, Found:
	1511.8021.
	Synthesis of 2: (see Scheme 1 (a)) To the tetra-hydroxy cis-cavitand
	platform (272 mg, 0.2 mmol) in a 20 mL Schlenk tube under Ar were
	added toluene (2 mL) and Et3N (0.13 mL, 0.96 mmol). After the mixture
	was stirred for 10 min, P[N(CH3)2]3 was added and the reaction mixture
	was heated at 75°C for 1 h. The mixture was allowed to cool to room
	temperature, filtered, washed with toluene, and then the filtrate was
	concentrated in vacuum to give 309 mg of crude products. Purification by
	short-plugged column chromatography (eluent, CHCl3 only) afforded 138
	mg of white solid materials, which were recrystallized from EtOH/EtOAc
	(8 mL/4.5 mL) to yield 2 in 40% (120 mg) as colorless crystals. 1 H NMR
	(400 MHz, CDCl3) 8.39 (s, 1H), 7.99 (dd, J = 8.5, 8.4 Hz, 2H), 7.80 (dd, J
	= 8.2, 1.4 Hz, 2H), 7.61-7.51 (m, 4H), 7.32 (s, 2H), 7.19 (s, 1H), 7.18 (s,
	2H), 7.12 (s, 1H), 6.33 (s, 1H), 5.70 (t, J = 8.2 Hz, 2H), 4.55 (t, J = 7.6 Hz,
	2H), (d, 3 JPH = 10.6 Hz, 12H, N(CH3)2), 2.28-2.17 (m, 8H), 1.41-1.27
	(m, 72H), 0.90-0.87 (m, 12H) ppm; 1 H NMR (400 MHz, [D8]toluene) 8.83
	(s, 1H), 7.88 (d, J = 8.2 Hz, 2H), 7.75 (s, 2H), 7.72 (s, 1H), 7.68 (s, 2H),
	7.60 (s, 1H), 7.48 (d, J = 8.2 Hz, 2H), 7.19 (ddd, J = 8.2, 8.2, 1.0 Hz, 2H),
	7.05-7.01 (m, 2H), 6.15 (t, J = 8.1 Hz, 2H), 4.97 (t, J = 7.4 Hz, 2H), 2.52
	(d, 3 JPH = 10.2 Hz, 12H, N(CH3)2), 2.49-2.09 (m, 8H), 1.51-1.30 (m, 72H),
	0.96-0.93 (m, 12H) ppm; 13 C{ 1 H} NMR (100 MHz, CDCl3) 153.3 (C 15 ),
	153.1 (C 10 ), 153.0 (C 7 ), 152.6 (C 8 ), 149.8 (two peaks are overlapped, C 17
	C 21 ), 149.3 (two peaks are overlapped, C 5 C 6 ), 140.3 (d, 4 Jcp = 9.3 Hz, C 13 ),
	137.7 (C 19 ), 136.5 (C 12 ), 135.7 (C 20 ), 134.5 (C 1 ), 129.2 (C 3 ), 128.3 (C 4 ),
	123.9 (C 18 ), 122.6 (C 14 ), 121.8 (C 11 ), 119.1 (C 22 ), 117.8 (C 16 ), 117.0 (C 9 ),
	35.4 (d, 2

  JP,H = 13.9 Hz, 3H), 3.99(d, 3 JP,H = 11.4 Hz, 3H), 8H), 72H), 12H) ppm; 13 C{ 1 H} NMR (100 MHz, CDCl3) 153.6 (two peaks are overlapped, C 19 C 38 ), 153.1 (C 6 ), 152.8 (two peaks are overlapped, C 32 C 40 ), 152.7 (C 5 ), 152.5 (C 13 ), 152.0 (C 14 ), 146.8 (d, JC,P = 6.4 Hz, C 17 ), 146.3 (d, JC,P = 6.4 Hz, C 30 ), 144.3 (d, JC,P = 5.5 Hz, C 24 ), 143.8 (d, JC,P = 4.8 Hz, C 26 ), 140.44 (C 20 ), 140.36 (C 37 ), 140.3 (C 35 ), 140.2 (C 33 ), 138.0 (d, JC,P = 1.7 Hz, C 16 ), 137.1 (d, JC,P = 4.8 Hz, C 7 ), 136.1 (C 23 ), 135.9 (C 22 ), 135.8 (C 29 ), 135.5 (C 15 ), 135.4 (C 18 ), 133.6 (d, JC,P = 3.6 Hz, C 31 ), 130.0 (C 36 ), 129.9 (C 21 ), 129.8 (C 34 ), 129.7 (C 28 ), 129.3 (C 27 ), 128.8 (C 3 ), 128.1 (C 10 ), 127.8 (C 11 ), 123.7 (C 2 ), 123.0 (C 9 ), 122.8 (C 4 ), 122.5 (C 12), 119.2 (C 1 ), 118.4 (d, JC,P = 3.8 Hz, C 18 ), 117.0 (d, JC,P = 4.3 Hz, C 39 ), 116.5 (C 25 ), 56.0 (d, 2 JC,P = 6.0 Hz, C 42 ), 54.7 (d, 2 JC,P = 2.9 Hz, C 41 ),36.0, 34.4, 33.2, 32.3 (many peaks are overlapped), 31.8, 30.4, 30.1 (many peaks are overlapped), 30.0 (many peaks are overlapped), 29.8, 29.7, 23.0 (many peaks are overlapped), 14.5 (many peaks are overlapped) ppm;

	, 7.39 (s, 1H), 7.21 (s, 1H), 7.20 (s, 1H), 7.19 (s, 1H), 7.15 (s, 1H), 6.73 (s, 1H), 5.81 (t, J = 8.0 Hz, 1H), 5.77 (t, J = 8.3 Hz, 1H), 4.51 (t, J = 8.1 Hz, 1H), 4.50 (t, J = 8.2 Hz, 1H), 4.07 (d, 3 31 P{ 1 H} NMR (162 MHz, CDCl3) 112.8, -7.60 ppm; MS (ESI) m/z: 1760 [M + Cl] -; IR (neat): 2921, 2849, 1483, 1408, 1328, 1145, 1041, 914, 759 cm -1 ; HRMS (ESI) calcd for C90H118AuCl2N4O11P2: 1759.7320 [M + Cl] -, Found: 1759.7350.

Acknowledgments

JSPS Grant-in-Aid for Scientific Research (C), Grant Number 19K05426, which supported TI in this work, is gratefully thanked for generous funding. The authors thank Dr. Toshiyuki Iwai and Dr. Takatoshi Ito at ORIST for gentle assistance with HRMS. Prof. Dr. Schramm, M. P. at CSULB are gratefully thanked for helpful discussions. SH gratefully acknowledges the University of Carthage and the Tunisian Ministère de l'Enseignement Supérieur et de la Recherche Scientifique for the financial support.

____________

Supporting Information (see footnote on the first page of this article):

The 1 H and 13 C{ 1 H} NMR spectra of all new compounds.

Supporting Information

1 H NMR and 13 C NMR spectra for all new compounds of cis-1, cis-2, cis-3, 1•(AuCl) 2 and 3•AuCl.


Diego López-Onaindia
email: diego.lopez.onaindia@mail.com

Aurore Schmitt
email: aurore.schmitt@cnrs.fr

Juan F Gibaja
email: juan.gibaja@eehar.csic.esorcid.org

M Eulàlia Subirà
email: eulalia.subira@uab.catorcid.org

Non-metric dental analysis of human interactions around the Pyrenees during the Neolithic and their biological impact

Keywords: Exchange, dental morphology, biological affinities, Western Mediterranean, V-III millennium cal BCE

Background: Interactions across the Pyreneans during the Middle-Neolithic (V-IV millennium BCE) have been described for a long time. Nevertheless, except for a few examples and attempts to describe it, the biological impact of these interactions on the human groups' make-up is not yet understood. The present work analyses the biological affinities of different groups from both sides of these mountains that represent the Populations of the Middle-and Late-Neolithic by means of the dental morphology.

Methods:

We present novel dental morphological data of 221 individuals from 11 archaeological sites. These data have been analysed and compared to those from previously published 20 Iberian sites and one French site. Data were recovered following the ASUDAS protocol, and MMD biological measure was calculated between groups.

Results: Our results suggest that there were some differences between the analysed populations. These differences are observed at each side of the Pyrenees, but also across them. Concretely, the coastal groups across the mountains show more affinities between each other than the inland groups. In addition, the differences between groups decrease by the end of the Neolithic.

Conclusions: Therefore, our results indicate that the intense trade activities registered in the coastal area between both sides of the Pyrenees would have had the greatest biological impacts in the homogenisation of the groups. Although less intense, the across mountain network and coastal to mountain area trade networks to the south of Pyreneans, also influenced the biological make-up of the groups.

Introduction

Anatomical variations have been used to understand the biological impact of population interactions and migrations, to determine or to discriminate the geographic or biological origin of human populations, both ancient and modern, for a long time now [START_REF] Howells | Cranial variation in man; a study by multivariate analysis of patterns of difference among recent human populations[END_REF][START_REF] Berry | The use of non-metric variations of the cranium in the study of scandinavian population movements[END_REF][START_REF] Milne | Discrete trait variation in Western Australian Aboriginal skulls[END_REF][START_REF] Rubini | Biological Homogeneity and Familial Segregation in the Iron Age Population of Alfedena (Abruzzo, Italy), Based on Cranial Discrete Traits Analysis[END_REF][START_REF] Crubézy | Microévolution d'une population historique: Etude des caractères discrets d'une population Missiminia (Soudan, III-IVe siècle)[END_REF]Hanihara and Ishida, 2001a, 2001b, 2001c;[START_REF] Puişoru | Analysis of mandibular variability in humans of different geographic areas[END_REF][START_REF] Ricaut | Cranial Discrete Traits in a Byzantine Population and Eastern Mediterration Population Movements[END_REF][START_REF] Burnett | Six new examples of the bipartite trapezoid bone: Morphology, significant population variation, and an examination of pre-existing criteria to identify bipartition of individual carpal bones[END_REF]. Concretely, in archaeological collections teeth are amongst the best-preserved osteological materials. This occurs mainly due to the hardness of the enamel, which is 96% mineral in composition (Simmer & Fincham, 1995;Nanci, 2008).

The study of dental morphology is based on the analysis of the phenotypic variation of teeth, by considering discrete traits such as cusps, tubercles, grooves and ridges from the crown and roots (Scott & Turner, 1997). These anatomical variations of teeth have increasingly been studied since the first quarter of the 20th century and applied to understand migration movements and origins of different human populations (Hrdlička, 1921;[START_REF] Gregory | The Origin and Evolution of the Human Dentition[END_REF][START_REF] Campbell | The Dentition and Palate of Australian Aboriginal[END_REF][START_REF] Shaw | The Teeth, the Bony Palate, and the Mandible in the Bantu Races of South Africa[END_REF][START_REF] Dahlberg | The changing dentition of man[END_REF][START_REF] Dahlberg | The dentition of the American Indian[END_REF][START_REF] Pedersen | The East Greenland Eskimo dentition[END_REF]. Since the 1990's a selection of traits identified by Turner and collaborators in their ASUDAS protocol (1991) has mainly been used to score variability in ancient populations of different periods and regions [START_REF] Irish | Who Were the Ancient Egyptians? Dental Affinities Among Neolithic Through Postdynastic Peoples[END_REF][START_REF] Coppa | Origins and Spread of Agriculture in Italy: A Nonmetric Dental Analysis[END_REF][START_REF] Desideri | When Beakers Met Bell Beakers: an analysis of dental remains[END_REF][START_REF] Irish | Other" possibilities? Assessing regional and extra-regional dental affinities of populations in the Portuguese Estremadura to explore the roots of Iberia's Late Neolithic-Copper Age[END_REF][START_REF] López-Onaindia | Prehistoric funerary complexity in northern Iberia studied by using dental morphology[END_REF][START_REF] López-Onaindia | Biological Differences Related to Cultural Variability during the Neolithic in a Micro-Geographical area of the Iberian Peninsula[END_REF], 2019). This protocol includes 36 standardised traits, chosen because of the lack of sexual dimorphism and because they are overall considered to be neutral to selection (Scott and Turner, 1997).

Although it has been determined that severe environmental stress during development might affect the expression of certain crown traits (Riga et al., 2014), the significant genetic inheritance in the expression of these discrete traits is consistent (Scott and Turner, 1997;[START_REF] Hlusko | Elucidating the evolution of hominid dentition in the age of pheonics, modularity, and quantitative genetics[END_REF]. This is further backed by the evidences that the heritability estimates for several traits in Australian twin studies are higher than 0.60 [START_REF] Higgins | Strong genetic influence on hypocone expression of permanent maxillary molars in South Australian twins[END_REF][START_REF] Hughes | Twin studies of dental crown morphology: genetic and environmental determinants of the cusp of Carabelli[END_REF], 2013;[START_REF] Hughes | An overview of dental genetics[END_REF], and in any case between 0.40-0.80 [START_REF] Paul | Patterns of heritability across the human diphyodont dental complex: Crown morphology of Australian twins and families[END_REF]. Even, some syndromes characterised by strong genetic components show particular dental morphology manifestations that are inherited in the families (Skrinjaric et al., 2016). In addition, in recent years several studies have analysed the capability and reliability of dental non-metric traits for determining population dynamics and biological relationships between groups compared to genetic data. In this sense, the works by Hubbard and collaborators (2015) and Rathmann and Reyes-Centeno (2020) are worth mentioning. Comparing nine dental traits to 41 neutral nuclear STRs in several Kenyan populations, they were able to reconstruct a similar population history with both methods, thus validating the applicability of the dental traits as proxies for inferring biological distances between human populations [START_REF] Hubbard | Do Nuclear DNA and Dental Nonmetric Data Produce Similar Reconstructions of Regional Population History? An Example From Modern Coastal Kenya[END_REF]. In addition, the work by Rathmann and Reyes-Centeno (2020) has confirmed this utility, but also remarked that there are certain sets of traits that are more useful and significant than other combinations. Nonetheless, although that as an analysis of population history this tool is reliable, it is not always as useful to predict ancestry at individual's level [START_REF] Delgado | Variation in dental morphology and inference of continental ancestry in admixed Latin Americans[END_REF].

The first Neolithic evidences in the Northwestern Mediterranean date from the middle of the 6 th millennium cal BCE. The origin of these populations and the process known as Neolithisation has been debated throughout the last decades. These debates have been based on two different points of view for the interpretation of Neolithisation, the autoctonists and diffusionists, in the way to evaluate in combination the last Hunter-Gatherer communities from the Mesolithic and the first Farmers from the Neolithic [START_REF] Vicent | The Island Filter Model revisited[END_REF]Zilhao, 1997;[START_REF] Bernabeu | Una visión actual sobre el origen y difusión del Neolítico en la península Ibérica[END_REF][START_REF] Binder | Le Mésolithique à trapèzes et la néolithisation de l'Europe sudoccidentale[END_REF]. Nevertheless, in the latest years, mixed proposals have also been suggested and encouraged by diverse data [START_REF] Isern | The Neolithic Transition in the Iberian Peninsula: Data Analysis and Modeling[END_REF][START_REF] Perrin | Beyon the sea… The Neolithic transition in the southwest of France[END_REF]). In the case of the Northeast of the Iberian Peninsula there is little information regarding the moment of transition, due to the relative scarceness of sites from the end of the 7 th and beginnings of the 6 th millennium cal BCE. This rare presence of remains might be related to a climatic cooling event around 8,200 BP and the abandonment of several territoires [START_REF] Berger | The 8200 cal BP abrupt environmental change and the Neolithic transition: A Mediterranean perspective[END_REF][START_REF] Morales | Las últimas evidencias mesolíticas del NE peninsular y el vacío preneolítico[END_REF], but further radiocarbon dating on Mesolithic materials must confirm this correlation.

Taking into account the archaeological data recovered to date, the Neolithisation in the Northwestern area of the Mediterranean can be linked to two events [START_REF] De Lagrán | Recent Data and Approaches on the Neolithization of the Iberian Peninsula[END_REF]. The first of the events, after 5,800 cal BCE, is characterised by pottery with Impressa-Ligur style decoration (Bernabeu et al. 2011;Oms, 2014). These first Neolithic evidences are scarce, and related to an economy based on agriculture and livestock, dominated by herds of goats (Capra hircus) and emmer wheat (Triticum disccocum) crops. These archaeological sites are localised in zones near the Mediterranean sea; both in Southeast France, represented by the cases of Peito Signado and Pont de Roque-Haute [START_REF] Briois | L'habitat néolithique ancien de Peiro Signado à Portiragnes (Hérault)[END_REF][START_REF] Guilaine | Pont de Roque-Haute[END_REF], and in the East of the Iberian Peninsula with the sites of Guixeres de Vilobí and El Barranquet [START_REF] Bernabéu | Las primeras producciones cerámicas: el VI milenio cal AC en la Península Ibérica[END_REF][START_REF] Oms | La neolitización del nordeste de la Península Ibérica: datos radiocarbónicos y culturales de los asentamientos al aire libre del Penedès[END_REF].

The second event was related to the presence of Cardial decoration in the pottery, and is documented after 5,600-5,500 cal BCE [START_REF] Guilaine | Du concept de céramique imprimée méditerranéenne à la genèse du Cardial[END_REF][START_REF] Oms | The Neolithic in Northeast Iberia: Chronological phases and 14 C[END_REF]. The sites that represent this moment are also found near the coastal and pre-coastal areas of the Northwest Mediterranean, and in isolated cases also in inland areas. At this time the economy was dominated by the culture of naked wheat (Triticum aestivun/durum) and barley (Hordeum vulgare), and by herds of ovicaprids (Ovis and Capra) (Saña, 2013), while the bovine and swine domestication was still of little importance.

The quantity of burials discovered in the Iberian Peninsula between the middle of the 6th and the middle of the 5th Millennium cal. BCE is fairly anecdotal [START_REF] Bosch | La cova sepulcral del Neolític antic de l´Avellaner, Cogolls, Les Planes d'Hostoles[END_REF][START_REF] Blasco | Cardial, epicardial y postcardial en Can Sadurní (Begues, Baix Llobregat)[END_REF]Gonzalez et al. 2011;[START_REF] Oms | First evidence of a collective human inhumation from the Cardial groups (Cova Bonica, Barcelona, NE Iberian Peninsula)[END_REF]). In the northeast of the peninsula, only around 10 sites with funerary structures of this period are known to date [START_REF] García | Los enterramientos neolíticos del yacimiento de Paternabidea (Ibero)[END_REF]García and Sesma, 2007;[START_REF] Pou | L'enterrament del Neolític antic de la Plaça de la Vila de Madrid (Barcelona), una estructura funeraria del VIè milleni aC[END_REF][START_REF] Quintana | Nuevos datos sobre la secuencia de uso sepulcral de la cueva de Santimamiñe (Kortezubi, Bizkaia)[END_REF][START_REF] Oms | The Neolithic in Northeast Iberia: Chronological phases and 14 C[END_REF]2017;Garcia-Sagastibeltza et al., 2020a;2020b). Nevertheless, the situation changes if the focus moves towards the second half of the 5th Millennium cal BCE. It was then when a proliferation of funerary evidence becomes available, either in the form of necropolises or as isolated burials in different areas of Northwestern Mediterranean, and in the Northeast of the Peninsula [START_REF] Martín | Les sociétés du Néolithique moyen en Catalogne et leur gestion funéraire[END_REF][START_REF] Gibaja | El neolítico en Cataluña[END_REF], Gibaja et al., 2016 a).

As regards to the funerary structures, between the end of the 5 th and a long period during the 4 th millennium cal BCE, two main burial groups can be identified in the Northeast of the Iberian Peninsula: burial pits dug in the soil, and sepulchres made of stone cists. The pit structures are usually found in areas near the coast, while the cysts are found in the inland and Pyreneans. These two groups equally exhibit certain variations concerning their morphology, size, and the employed constructive elements [START_REF] Martín | La question de l'homogénéité des pratiques funéraires dans la « Culture des Sepulcres de Fossa » du Néolithique moyen. Révision des typoplogies a la lumiere des dernieres découvertes[END_REF]. At the same time, some of the burials were not made ex profeso, but by re-using different kinds of structures; we refer to the re-use of silos in which the wastes include one or several individuals or the re-use of mines as burial places, which is the case of the Prehistoric Mines of Can Tintorer/Gavà [START_REF] Villalba | Las sepulturas neolíticas del complejo de Can Tintorer y el modelo social de la población minera[END_REF][START_REF] Bosch | Intervencions arqueològiques a les mines de Gavà (sector serra de les Ferreres). Anys 1998-2009. De la variscita al ferro: neolític i antiguitat[END_REF]. These kinds of funerary structures are also found in southern France from the beginning of the Middle Neolithic [START_REF] Schmitt | Les inhumations du site du Clos de Roque (Saint-Maximin-la-Sainte-Baume[END_REF], which mainly comprises the Midi-Pyrénées, Languedoc and Provence-Alpes-Côte d'Azur areas.

Although there is some diversity on the funerary gestures, there are some common characteristics in both sides of the Pyreneans for the main defining structures of the mentioned cultures. In these "classic" structures, burials usually contain one or two individuals: either men, women, or child.

Bodies were usually deposited in supine de cubitus with folded legs and arms in variable positions.

Occasionally bodies are found fully stretched or in lateral de cubitus. Nevertheless, in both sides of the Pyreneans there were variations of these patterns. Certain graves exhibit bodies with no anatomical connection, or displaying only a partial connection, in unsual positions, as if they had been thrown in the pit. Some graves contain multiple burials, where the bones from the primary burials were displaced towards the sides of the tomb or withdrawn from the tomb to make room for those buried later [START_REF] Perrin | Le Chasséen, des Chasséens[END_REF][START_REF] Allièse | Quelques réflexions sur les practiques funéraires du nord-est de la péninsule Ibérique à la fin du Ve et au début du Ive millénaire[END_REF][START_REF] Martín | La question de l'homogénéité des pratiques funéraires dans la « Culture des Sepulcres de Fossa » du Néolithique moyen. Révision des typoplogies a la lumiere des dernieres découvertes[END_REF][START_REF] Schmitt | Le traitement des défunts dans le midi de la France entre 4400 et 3500 avant notre ère[END_REF][START_REF] Gibaja | Mirando a la muerte: Las prácticas funerarias durante el Neolítico en el noreste peninsular[END_REF]2018;2019).

Recent studies on funeral taphonomy have provided important information about certain aspects that help better understand these funerary behaviours. For example, today we know that these individuals were not simply deposited in the bottom of the funerary structure, but occasionally placed in the interior of the tomb within rigid boxes, probably wooden, or in bags [START_REF] Allièse | Les pratiques funéraires dans la nécropole néolithique de Can Gambús-1 (Sabadell, Espagne) : de la préparation du corps à la fermeture de la tombe[END_REF].

During this period, there are evidences of exchange networks at different levels that connect both sides of the Pyrenees [START_REF] Gibaja | L'utilisation de l'outillage lithique taillé destiné à l'acquisition et à la transformation des aliments au Néolithique moyen en Méditerranée nordoccidentale[END_REF][START_REF] Terradas | Des réseaux d'échanges entre les vallées du Rhône et de l'Èbre: un point de vue du nord-est de la péninsule Ibérique[END_REF]. According to the evidence, these exchanges were stronger on an East-to-West direction than in the opposite sense [START_REF] Gibaja | L'utilisation de l'outillage lithique taillé destiné à l'acquisition et à la transformation des aliments au Néolithique moyen en Méditerranée nordoccidentale[END_REF][START_REF] Terradas | Des réseaux d'échanges entre les vallées du Rhône et de l'Èbre: un point de vue du nord-est de la péninsule Ibérique[END_REF]. In this context, researchers have found imported materials in the sepulchres from the Northeast of the Iberian Peninsula. Thes materials include Barrémo-Bédoulian Flint from Vaucluse (Provence) [START_REF] Binder | Silex blond et complexité des assemblages lithiques dans le Néolithique liguroprovençal[END_REF][START_REF] Léa | Les industries lithiques du Chasséen en Languedoc occidental. Caractérisation per l'analyse technologique[END_REF][START_REF] Briois | Les industries de pierre taillée néolithiques en Languedoc occidental[END_REF][START_REF] Gassin | Lithic Management in the Chassey Culture Neolithic[END_REF][START_REF] Gibaja | Between sardinia and Catalonia: contacts and relationships during the Neolithic[END_REF], polished axes originated in the Alps [START_REF] Léa | Raw, pre-heated or ready to use: discovering specialists supply system for flint industries in mid-Neolithic (Chassey culture) communities in Southern France[END_REF][START_REF] Vaquer | Le rôle du silex bédoulien du Vaucluse dans la compréhension de la chronologie et des relations culturelles du Néolithique moyen solsonien. In: Darreres investigacions al Pre-pirineu lleidatà 2009-2011[END_REF][START_REF] Pétrequin | Les haches en jades alpins pendant les V e et IV e millénaires. L'exemple de l'Espagne et du Portugal dans une perspective européenne[END_REF], and Obsidian blades and nuclei from Sardinia [START_REF] Gibaja | Between sardinia and Catalonia: contacts and relationships during the Neolithic[END_REF][START_REF] Terradas | Neolithic diffusion of obsidian in the western Mediterranean: new data from Iberia[END_REF]. On the other hand, pieces made from Variscite exploited at Can Tintorer mines from Gava (Barcelona) appeared in Chasséen sites to the west of the Rhone [START_REF] Terradas | Des réseaux d'échanges entre les vallées du Rhône et de l'Èbre: un point de vue du nord-est de la péninsule Ibérique[END_REF].

The relationship between these cultural changes with demographic changes and the biological composition of the involved populations has therefore been a subject of interest in different fields of physical anthropology [START_REF] Schmitt | La population Chasséenne du gisement du Crès (Hérault): caractéristiques métriques et anatomiques[END_REF][START_REF] Allièse | Les sépultures de la Bòbila Madurell-Can Gambús (Vallès occidental). Ėclairages sur les practiques funéraires du nord-est de la péninsule Ibérique à la fin du Ve et au début du IVe millénaire[END_REF][START_REF] Luyer | Évolution dentaire dans les populations humaines de la fin du Pléistocène et du début de l'Holocène (19000 -5500 cal. BP) : une approche intégrée des structures externe et interne des couronnes pour le Bassin aquitain et ses marges[END_REF]Lopez-Onaindia and Subirà, 2017). In these latest years, the study of this subject from a paleogenetic perspective has added incredible amounts of information. From the first studies, focused on mitochondrial DNA [START_REF] Hervella | Ancient DNA from Hunter-Gatherer and Farmer Groups from Northern Spain Supports a Random Dispersion Model for the Neolithic Expansion into Europe[END_REF][START_REF] De La Rua | Ancient DNA in the Cantabrian fringe populations: A mtDNA study from Prehistory to Late Antiquity[END_REF][START_REF] Szécsény-Nagi | The maternal genetic[END_REF] to nuclear genome studies [START_REF] Olalde | The genomic history of the Iberian Peninsula over the past 8000 years[END_REF][START_REF] Villalba-Mouco | Survival of Late Pleistocene Hunter-Gatherer Ancestry in the Iberian Peninsula[END_REF][START_REF] Rivollat | Ancient genome-wide DNA from France highlights the complexity of interactions between Mesolithic hunter-gatherers and Neolithic farmers[END_REF], all of them indicate that the process was complicated and heterogeneous both geographically and diachronically. In this sense, it has been reported that the hunter-gatherer signal is preserved in the Neolithic group, but that the intensity of this signal varies amongst regions [START_REF] Olalde | A Common Genetic Origin for Early Farmers from Mediterranean Cardial and Central European LBK Cultures[END_REF][START_REF] Olalde | The genomic history of the Iberian Peninsula over the past 8000 years[END_REF][START_REF] Szécsény-Nagi | The maternal genetic[END_REF][START_REF] Villalba-Mouco | Survival of Late Pleistocene Hunter-Gatherer Ancestry in the Iberian Peninsula[END_REF][START_REF] Rivollat | Ancient genome-wide DNA from France highlights the complexity of interactions between Mesolithic hunter-gatherers and Neolithic farmers[END_REF].

In addition, our previous results based on dental morphology suggest that there is a difference in the biological composition of the Neolithic groups from different regions of Northern Iberia [START_REF] Subirà | Cultural Changes in Funeral Rites During the Neolithic in the Northeast of the Iberian Peninsula? The Cave of Pantà de Foix (Barcelona)[END_REF][START_REF] López-Onaindia | Prehistoric funerary complexity in northern Iberia studied by using dental morphology[END_REF][START_REF] López-Onaindia | Biological Differences Related to Cultural Variability during the Neolithic in a Micro-Geographical area of the Iberian Peninsula[END_REF]. We have observed that these differences have both a microreographical [START_REF] López-Onaindia | Biological Differences Related to Cultural Variability during the Neolithic in a Micro-Geographical area of the Iberian Peninsula[END_REF] and cultural [START_REF] López-Onaindia | Prehistoric funerary complexity in northern Iberia studied by using dental morphology[END_REF][START_REF] López-Onaindia | Biological Differences Related to Cultural Variability during the Neolithic in a Micro-Geographical area of the Iberian Peninsula[END_REF] component. We specially were able to distinguish how the populations from the North East Ebro basin and the pre-pyrenean region differ from the coastal Mediterranean population in their biological composition [START_REF] López-Onaindia | Prehistoric funerary complexity in northern Iberia studied by using dental morphology[END_REF][START_REF] López-Onaindia | Biological Differences Related to Cultural Variability during the Neolithic in a Micro-Geographical area of the Iberian Peninsula[END_REF].

The aim of this work is to analyse the biological relationship amongst both Middle and Late Neolithic groups from both sides of the Pyreneans. This topic has scarcely been explored to date. In this work we aim to understand the effects on the populations of these geographic region of the demic migrations that affected the biological composition of the Iberian populations, the implications of the cultural similarities observed in both sides of the Pyreneans during the Neolithic and the effect of trade activities in the biological make up of these groups.

Materials and methods

This work presents novel data from 221 individuals and 3,977 human teeth from 11 southern French Neolithic and Chalcolithic sites (Table 1). In addition, these data have been used in combination to already published 20 Iberian sites and the French Les Crès site (255 individuals and their 3,661 teeth) [START_REF] Schmitt | La population Chasséenne du gisement du Crès (Hérault): caractéristiques métriques et anatomiques[END_REF][START_REF] López-Onaindia | Prehistoric funerary complexity in northern Iberia studied by using dental morphology[END_REF][START_REF] López-Onaindia | Biological Differences Related to Cultural Variability during the Neolithic in a Micro-Geographical area of the Iberian Peninsula[END_REF][START_REF] Pascual | La població neolítica barcelonina des de la morfologia dental. Dades de la població de la serralada prelitoral[END_REF][START_REF] Subirà | Cultural Changes in Funeral Rites During the Neolithic in the Northeast of the Iberian Peninsula? The Cave of Pantà de Foix (Barcelona)[END_REF] (Supplementary Table S1). Thus, the whole corpus of this work includes dental morphological data from 31 Neolithic and Chalcolithic sites from the Northwest Mediterranean region (Figure 1). S1).

The Middle-Neolithic from Languedoc-Midi Pyrénées is formed by the samples from, La Terrasse (La Vimona, Zac Agora), Narbons, Saint Gely-du-Fesc, et Le Crès. La Terrasse site is formed by hundreds of pits in the area of Tolouse, of which 20 were burials (Gandelin & Vaquer, 2008;[START_REF] Marlière | Les sépultures de Cugnaux, La Vimona et la ZAC Agora[END_REF]. Narbons is located to the southeast of Montesquieu-de-Lauragais (Haute-Garonne) and provided six pit structures at the time of the archaeological intervention (Tchérémissinof et al., 2005). Four of these structures resulted to be sepulchres. Three are attributed to the Chasséen culture, and number 1020 is situated at the end of Middle Neolithic. The interventions in Saint Gely du Fesc were held in 1996 (Hérault), provided four burial pits in a concentrated area of less than 25m 2 , each of them containing a single individual (Schmitt et al., in press). Radiocarbon dates on each subject indicate that three belonged to the end of Middle Neolithic and one to the beginning of Middle Neolithic ("Néolithique moyen I" according to the French chronological system for the south of France). Le Crès site (Hérault) provided 190 negative structures, of which 31 were burials that were excavated during 2000 and 2001 [START_REF] Loison | Le Crès. Habitats préhistoriques en bordure de l'Orb. Structures domestiques et sépulture du Chasséen ancien[END_REF][START_REF] Loison | Diversité des pratiques funéraires et espaces sépulcraux sectorisés au Chasséen ancien sur le site de Crès à Béziers (Hérault): croisement des données archéologiques et anthropologiques[END_REF]. 30 of these pits correspond to Chasséen burials and contained 49 individuals from this period. Moreover, compared to other sites this represented a wide variety of tomb typologies, from simple pits to protomegalithic structures.

The Middle-Neolithic from Provence group includes the samples from Clos de Roque, Les Clavelles, Les Martins and Le Collet Redon. This last site is the sole collective burial of this group, located in La Couronne [START_REF] Schmitt | Un exemple inédit en Provence de sépulture collective du Néolithique récent/final: le site de Collet-Redon (Martigues)[END_REF]; the radiocarbon dates give a chronological interval situated between Middle and Late Neolithic. The burial provided a minimum number of 11 individuals in primary and secondary deposits. The tomb is installed in a quadrangular feature measuring 2.4 x 2.2 m. The walls of the pit are built with piled up stones. Les Martins is a site excavated in 1986 that produced two Middle-Neolithic burials (St. 6 and St. 8), consisting of individual burials in pits with a child and an adult male [START_REF] D'anna | L'habitat de plein air en Provence : recherches récentes[END_REF][START_REF] Van Willigen | Chronologie relative et chronologie absolue du Néolithique moyen dans le sud-est de la France: l'apport de l'analyse des données et de la modélisation chronologique bayésienne[END_REF]. In the case of Clos de Roque, 6 pits containing individual burials were discovered in 2011 [START_REF] Remicourt | Les occupations pré-et protohistoriques du Clos de Roque à Saint-Maximin-la-Sainte-Baume dans le Var[END_REF][START_REF] Schmitt | Les inhumations du site du Clos de Roque (Saint-Maximin-la-Sainte-Baume[END_REF][START_REF] Olalde | The Beaker phenomenon and the genomic transformation of northwest Europe[END_REF]. From these six individuals, four belong to the beginning of the Middle-Neolithic (called "Néolithique moyen I" (St. 2209(St. , 2215;;2205, 2224)).

Les Clavelles is an unpublished site. It provided a circular pit composed of two layers. 5 individuals were buried in this feature. An unpublished radiocarbon date on human bone indicates its belonging to the end of the 5th millennium cal BCE. The hypogeum of "les Crottes" was excavated in 1966. It is composed of 5 layers. Two of them are funerary collective deposits, level 2 and 5 [START_REF] Courtin | Le Néolithique de la Provence. Éitions Klincksieck[END_REF][START_REF] Sauzade | Les Sépultures du Vaucluse, du Néolithique à l'Age du Bronze[END_REF]. The latest dates from the last quarter of the 4th millennium cal BCE.

The remains from the caves of Bélesta and Montou form the Montbolò group, in the Roussillon area. La Caune de Bélesta was excavated in 1983, when remains of a collective burial were identified together with many pieces of pottery [START_REF] Claustre | La Cauna de Belesta : une tombe collective il y a 6000 ans[END_REF] that are attributed to the Montbolò culture.

Montou is located in Corbères-les-Cabanes (Eastern Pyrenees), and it represents a collective burial like Bélesta [START_REF] Claustre | Complément à l'étude de la céramique néolithique de la galerie close de Montou[END_REF][START_REF] Valentin | La gestion de l'espace sépulcral Néolithique moyen de la grotte de Montou (Pyrénnées-orientales): un essai d'interprétation[END_REF].

The last site analysed for the work is Grôtte Mykolas cave which was excavated in 2007 [START_REF] Chancerel | La grotte du Piale (dite Mykolas) commune du Bugue (Dordogne)[END_REF]. This site is located to the North of Bugue (Aquitania) and its chronological affiliation corresponds to the Late-Neolithic age, the only French site from this period. According to the initial report an MNI of 9 individuals was calculated, but dental remains indicate that there were at least 12 inhumated individuals.

The analysis of the teeth was carried out following the Arizona State University's Dental Anthropology System (ASUDAS) [START_REF] Turner | Scoring Procedures for Key Morphological Traits of the Permanent Dentition: The Arizona State University Dental Anthropology System[END_REF] for scoring 35 non-metric dental traits. In individuals with full dentition, these 35 traits provide 121 variables for individual. Most traits are not just present or absent, but ranked in a scale of expression, and the level at which they are represented in each of the antimers of an individual may vary. For this reason, the antimere showing the highest level of expression, which is believed to be the one representing the trait's maximum potential for the individual and be genetically more reliable, is selected in each individual [START_REF] Turner | Expression count: a method for calculating morphological dental trait frequencies by using adjustable weighting coefficients[END_REF][START_REF] Turner | Scoring Procedures for Key Morphological Traits of the Permanent Dentition: The Arizona State University Dental Anthropology System[END_REF].

In order to identify correlations between traits, Kendall's Tau-b analysis was done on the ranked data [START_REF] Irish | The Mean Measure of Divergence: Its Utility in Model-Free and Model Bound Analyses Relative to the Mahalanobis D2 Distance for Nonmetric Traits[END_REF] with IBM SPSS 20.00. A correlation was considered strong when the Tau-b value is superior to 0.5, and for it to be statistically significant we considered that the N must be >10 and the p value <0.05. This step is necessary to edit the database before calculating the biological distances, as one of the traits of the correlated pair was eliminated in order to avoid differential weighting of the underlying dimensions that might yield erroneous results [START_REF] Sjøvold | Non-metrical divergence between skeletal populations: the theoretical foundation and biological importance of C.A.B. Smith's mean measure of divergence[END_REF][START_REF] Irish | The Mean Measure of Divergence: Its Utility in Model-Free and Model Bound Analyses Relative to the Mahalanobis D2 Distance for Nonmetric Traits[END_REF].

The ranked ASUDAS values were later dichotomised into presence or absence values, based on their reported morphological thresholds (Nichols, 1989) and according to standard procedure [START_REF] Turner | Expression count: a method for calculating morphological dental trait frequencies by using adjustable weighting coefficients[END_REF][START_REF] Turner | Late Pleistocene and Holocene population history of East Asia based on dental variation[END_REF]. From these dichotomised data, frequencies for the expression of traits were calculated for the different samples and groups.

Together with one of each pair of correlated traits, those traits that presented no frequency differences in the various samples were excluded from further analysis, because they were noninformative. The Mean Measure of Divergence (MMD) [START_REF] Smith | Coefficients of biological distance[END_REF] was calculated between samples as an among-samples phenetic dissimilarity measure. The formula used to calculate the MMD contains Freeman and Tukey's angular transformation to correct for low or high trait frequencies and small sample sizes [START_REF] Harris | Calculation of Smith's Mean Measure of Divergence for Intergroup Comparisons Using Nonmetric Data[END_REF]. Nevertheless, each trait used must have been scored at least 10 times (including present and absent) in each sample. In order to determine if two samples are significantly different, the MMD has to be compared to its standard deviation (SD). Therefore, if MMD>2xSD, the null hypothesis of P1=P2 is rejected at the 0.025 level. In some cases, when the internal variability of a sample resulted greater to that between the two samples compared negative MMD values might result. These values are statistical artefacts, and therefore, they were set to 0 [START_REF] Vargiu | Italian populations during the Copper Age: assessment of biological affinities through morphological dental traits[END_REF][START_REF] Irish | The Mean Measure of Divergence: Its Utility in Model-Free and Model Bound Analyses Relative to the Mahalanobis D2 Distance for Nonmetric Traits[END_REF].

The matrices obtained with the MMD calculation were graphically represented in Multi-Dimensional Scaling (MDS) [START_REF] Kruskal | Multidimensional scaling[END_REF] using PAST software [START_REF] Hammer | PAST: Paleontological Statistics Software Package for Education and Data Analysis[END_REF]). This representation is useful to obtain further information beyond if two samples are significantly different or not. The representations offer a stress value that indicates the accuracy with which the matrix has been represented. Usually, stress values <0.10 are considered excellent, while ≥0.15 are unacceptable [START_REF] Borgatti | Multidimensional Scaling[END_REF]. Moreover, Principal Component Analysis (PCA) were carried out based on the same traits used to calculate the MMD. These analyses usually provide complementary information on which traits are more involved in the distribution observed on the MDS representations.

Due to the different conservation state and sample size of the collections included in this work, it was necessary to carry out a series of different analyses including different variables and samples at each of them to maximise the information. Two different sets of MMD calculations were carried out: one that included all the French and Iberian samples except for the Montbolò group, and was based on 10 traits. The other excluded some more French and Iberian samples, but included Italian samples [START_REF] Coppa | Origins and Spread of Agriculture in Italy: A Nonmetric Dental Analysis[END_REF] in order to get a broader context on the relationships observed with the previous analysis.

The Montbolò samples were especially small, and after trait editing to avoid the non-informative and correlated variables, very few variables could be included in MMD calculation. For this reason, this group was compared with Fisher's exact test to the Iberian and French samples, for all those traits scoring more than 5 observations, to see which groups present less differences with it.

Results

The number of times a trait was scored in each group, the frequency for the presence of the traits and dichotomic breaking-point for each of them are shown in Table 2. Some traits provided significant correlation values when analysed with Kendal's Tau-b. These traits were: UM2 cusp5 was correlated to UP4 m&d cusps (Tau-b=0.716, N=25) and UM3 cusp5 (Tau-b=0.642, N=19); on the other hand UM1 Metacone and UM3 Carabelli's tubercle were also correlated (Tau-b= 0.725, N=12).

The MMD analysis based on 10 variables that included both French and Iberian samples (Table 3), showed a complex scenario with significant differences among samples from each side of the Pyrenees, while there are no differences between some of the samples across the mountains. Those samples presenting significant differences at the 0.025 level are the Languedoc-Midi Pyrenees group and the Late Neolithic Mediterranean groups (Roaix, Cova del Pantà de Foix and Can Sadurní). On the other hand, those samples that presented no differences with any other sample were the Middle-Neolithic sample from Provence and Grôtte Mykolas.

When this matrix was represented (Figure 2) we obtained a MDS plot that provided a grouping of samples to the right end of the horizontal axis where two sites from the Iberian Peninsula with pit burials were registered (Los Cascajos, Early-and Middle-Neolithic from Navarre, and Bòbila Madurell) and a Chalcolithic cave from the Mediterranean coast of the same area (Cova del Pantà de Foix). On the other extreme we observe the Middle-Neolithic sample from Languedoc and Midi-Pyrénées area, and the rest of the samples appear in the centre between them. Therefore, the biggest differences are observed between Middle-Neolithic samples from both sides of the Pyreneans.

The PCA analysis for the same traits provides a similar representation to the MDS with the two first components (83.5% of the total variance explained): the Languedoc/Midi-Pyrénées sample was the most divergent from the rest, which was determined by higher frequencies of LM1 cusp6 and LM2 cusp 7 (Table 4). On the opposite side the Middle-Neolithic samples from Provence, Bòbila Madurell and Cascajos together with Cova del Pantà de Foix were characterised for having high frequencies for LP4 cusp number and relatively high presences of Hypocone in both UM1 and UM2.

When we excluded the smallest samples from the analysis (Grôtte Mykolas, Middle-Neolithic from Provence, and Cova del Pantà de Foix) and compared the other samples to Italian samples using 8 traits we obtained the matrix shown in Table 5. With the distance data, we observed that Italian samples significantly differed from all Iberian and French samples. In the same sense, the French and Iberian samples differed between each other too. Nevertheless, these distance values were smaller than those any of both groups presented compared to the Italians.

The MDS representation of this matrix (Figure 3 When correlations between the main components in the PCA analysis and the variables were analysed (Table 6), we observed that the Italian groups present UM2 cusp5 and UM1 Carabelli's tubercle in higher frequencies than Iberian and French groups, while the latter had overall more presence of UI2 shovelling, this differentiation occurs in the first component. The second component split the French and Iberian samples, although not in the same manner as the MDS. We observe the Languedoc and Solsonian Middle-Neolithic groups on one side and the other groups (Middle-and Late-Neolithic samples) on the other corner. In this sense, the most significant trait was the presence of multiple lingual cusps in LP4, which was most common in Late-Neolithic samples and the Middle-Neolithic samples of Bòbila Madurell and Los Cascajos.

The Montbolò sample did not show differences for most of the 18 analysed traits with the rest of samples when this was analysed by Fisher's exact test (Table 7). Moreover, when Bonferroni's correction was applied to the significance level (p<0.0002777), this sample only differed significantly in one trait from Los Cascajos sample: LM1 Cusp5.

Discussion

Although there are many cultural similarities between the two main Middle-Neolithic cultures from Catalonia and Mediterranean France (Sepulcres de Fossa Culture and Chasséen), our results indicate that in the biological background of the defined human groups there is some variance. This variance, thought, is not a simple divergence between French and Iberian samples. As it has previously been mentioned, many studies support a dual dispersion model for the Neolithic in the Northeastern area from the Mediterranean [START_REF] Guilaine | De la vague à la tombe[END_REF]Cruz-Berrocal, 2013).

In their recent work, Molist and collaborators (2016) analyse different material evidence between the Chasséen and the Sepulcres de Fossa cultures. This study suggests that although the affinities and the common elements between both cultures, each of them has singular elements related to the previous background in the territories. Nevertheless, they do also observe that the similarities between the two groups are greater than that each of them has with any other surrounding area. Moreover, both share common characteristics that can be interpreted as heritage from the Montbolò and Molinot cultures that spread through the eastern Pyrenees. Finally, as it has previously been mentioned, according to many evidence pieces, the exchange networks between these groups at both sides of the Pyrenees were strongly rooted [START_REF] Gibaja | Between sardinia and Catalonia: contacts and relationships during the Neolithic[END_REF][START_REF] Terradas | Neolithic diffusion of obsidian in the western Mediterranean: new data from Iberia[END_REF]2016;[START_REF] Molist | El "Chassense" y los "Sepulcros de Fosa de Cataluña": relaciones complejas entre culturas arquológicas vecinas[END_REF]).

Ancient DNA (aDNA) analyses of the region show that although there is an important new genetic composant in the Neolithic individuals from Southwestern Europe, there is a persistence of the Hunter-Gatherer component too [START_REF] Villalba-Mouco | Survival of Late Pleistocene Hunter-Gatherer Ancestry in the Iberian Peninsula[END_REF][START_REF] Brunel | Ancient genomes from present-day France unveil 7,000 years of its demographic history[END_REF][START_REF] Rivollat | Ancient genome-wide DNA from France highlights the complexity of interactions between Mesolithic hunter-gatherers and Neolithic farmers[END_REF]Seguin-Orlando et al., 2021). Focusing in the Neolithic individuals from the Iberian Peninsula different studies show complex scenarios: while in some cases the Neolithic samples have signals similar to those from Eastern Mediterranean and LBK culture [START_REF] Olalde | A Common Genetic Origin for Early Farmers from Mediterranean Cardial and Central European LBK Cultures[END_REF], other studies show mixed signals in the different areas of the Iberian Peninsula (de la Rúa et al., 2015;Szécsény-Nagy et al., 2017). Therefore, a mixed model would be the most accurate one explaining the current evidences. In this sense, some of our previous results evidence slight biological differences amongst some Iberian samples [START_REF] López-Onaindia | Prehistoric funerary complexity in northern Iberia studied by using dental morphology[END_REF][START_REF] López-Onaindia | Biological Differences Related to Cultural Variability during the Neolithic in a Micro-Geographical area of the Iberian Peninsula[END_REF]. These works, in the same way as aDNA works remark, show that in the Northeast of the Iberian Peninsula there was biological variability during the Neolithic. These differences are observed sometimes related to funerary practices [START_REF] López-Onaindia | Prehistoric funerary complexity in northern Iberia studied by using dental morphology[END_REF], where the individuals buried in Megaliths significantly diverged from the rest of groups. Moreover, and still related to a different funerary structure represented by stone cists burials, we observed differences between mountain and coastal groups in the east of the Peninsula [START_REF] López-Onaindia | Biological Differences Related to Cultural Variability during the Neolithic in a Micro-Geographical area of the Iberian Peninsula[END_REF]. In addition, a first exploration of the relationship between Iberian Hunter Gatherers and first farmers analysing dental morphology suggested that there were some affinities between them, suggesting a certain degree of continuity [START_REF] López-Onaindia | Heirs of the glacial maximum: Dental Morphology suggests Mesolithic human groups along the Iberian Peninsula shared the same biological origins[END_REF].

The present results show that, overall, those groups from coastal areas or valleys near the coast present differences with fewer groups than non-coastal samples. These differences of the inland groups are present at each side of the Pyreneans and between sides. Exchanges of materials and therefore potential population movements and mixtures are well recorded for the Neolithic in western Europe. The presence of raw materials like the Barrémo-Bédoulian flint (honey flint) from Vaucluse (Provence, France) in cist-like and pit burials from Northeastern Iberia and Southern France [START_REF] Binder | Silex blond et complexité des assemblages lithiques dans le Néolithique liguroprovençal[END_REF][START_REF] Léa | Les industries lithiques du Chasséen en Languedoc occidental. Caractérisation per l'analyse technologique[END_REF][START_REF] Briois | Les industries de pierre taillée néolithiques en Languedoc occidental[END_REF][START_REF] Gibaja | Prácticas funerarias durante el neolítico. Los enterramientos infantiles en el noreste de la Península Ibérica (Neolithic Funeral rituals: child burials in the Northeastern Iberian Peninsula)[END_REF][START_REF] Gassin | Lithic Management in the Chassey Culture Neolithic[END_REF]; the presence of polished axes originated in the Alps in Iberian sites [START_REF] Léa | Raw, pre-heated or ready to use: discovering specialists supply system for flint industries in mid-Neolithic (Chassey culture) communities in Southern France[END_REF][START_REF] Vaquer | Le rôle du silex bédoulien du Vaucluse dans la compréhension de la chronologie et des relations culturelles du Néolithique moyen solsonien. In: Darreres investigacions al Pre-pirineu lleidatà 2009-2011[END_REF][START_REF] Pétrequin | Les haches en jades alpins pendant les V e et IV e millénaires. L'exemple de l'Espagne et du Portugal dans une perspective européenne[END_REF]; and the evidence of the trade of variscite from Gavà to the Rhone's valley [START_REF] Terradas | Des réseaux d'échanges entre les vallées du Rhône et de l'Èbre: un point de vue du nord-est de la péninsule Ibérique[END_REF] are good examples for this trans-pyrenean trade. This trade seems especially intense throughout the coast compared to more mountainous regions [START_REF] Terradas | Áreas de aprovisionamiento, territorios de subsistencia y producciones técnicas en el Neolítico Antiguo de La Draga[END_REF], which is further supported by the discovery of obsidian from Sardinia in some Catalan sites [START_REF] Gibaja | Between sardinia and Catalonia: contacts and relationships during the Neolithic[END_REF][START_REF] Terradas | Neolithic diffusion of obsidian in the western Mediterranean: new data from Iberia[END_REF].

If we assume a more intense coastal trading, the populations from these regions would be exposed to a more diverse biological background than those from inner land. In contrast, the sample from Los Cascajos, although it is inland, shows affinities similar to those from the Catalonian coastal samples, separating from the Pyrenean samples. In this case, aDNA analyses have shown that Los Cascajos is a sample with high genetic variability and would not be an isolated group [START_REF] Hervella | Early Neolithic funerary diversity and mitochondrial variability of two Iberian sites[END_REF]. Los Cascajos is situated in the Ebro basin, where there was an intense exchange network that intensely ran Northwest to Southeast and vice versa following the river, from inland to the Mediterranean coast [START_REF] Cava | La circulación de materias primas líticas en la transición Mesolítico/Neolítico antiguo en el País Vasco. Los abrigos de Mendandia[END_REF][START_REF] Baldellou | Variscita de Can Tintorer en el Neolítico Antiguo del Valle Medio del Ebro[END_REF][START_REF] Montes | Enredados en la Malla Neolítica de la Cuenca del Río Ebro. Redes, Continuidades y Cambios[END_REF][START_REF] Tarriño | Materias primas líticas[END_REF]. This network was more intense than the trades documented between coastal valley and mountainous areas in the Catalan region [START_REF] Villalba | Les mines neolítiques de Can Tintorer (Gavà). Excavacions 1978-1980[END_REF]Bosch and Borrel, 2009).

Although the following interpretations should be taken cautiously due to the lack of interobserver error test between our samples and the Italian data [START_REF] Coppa | Origins and Spread of Agriculture in Italy: A Nonmetric Dental Analysis[END_REF], all French and Iberian samples significantly diverge from the Italian groups. However, these differences are smaller with the Neolithic than with the Hunter-Gatherer sample. Concretely, the Languedoc-Midi Pyrenees and Solsonian samples (inland) are the most divergent from the Italian Neolithic one, while Bòbila Madurell (Vallès area) and Los Cascajos present slighter differences with it. Hence, it is plausible that some of the migration from the East that arrived into French and Iberian territory with the spread of the agriculture had the same substrate as the Italian. Nevertheless, this seems to have been less intense than that observed in the Italian Peninsula [START_REF] Coppa | Origins and Spread of Agriculture in Italy: A Nonmetric Dental Analysis[END_REF]. Furthermore, the Italian Palaeolithic-Mesolithic sample diverges significantly from the Iberian one when dental morphology is analysed [START_REF] López-Onaindia | Heirs of the glacial maximum: Dental Morphology suggests Mesolithic human groups along the Iberian Peninsula shared the same biological origins[END_REF], which would result in different previous biologic backgrounds in each area.

Finally, it must also be mentioned that in those two Middle-Neolithic groups that diverge most from the rest, Solsonian area and Languedoc-Midi Pyrenees, there are representations of burials done in stone slab cists, while in the other studied areas these are not present. In the case of the Late-Neolithic, these samples are also diverse, but show similar affinities with most Middle-Neolithic samples. This trend has previously been observed in our works, as groups get uniform between them towards the Bronze Age [START_REF] López-Onaindia | Prehistoric funerary complexity in northern Iberia studied by using dental morphology[END_REF][START_REF] López-Onaindia | Biological Differences Related to Cultural Variability during the Neolithic in a Micro-Geographical area of the Iberian Peninsula[END_REF]. This trend towards the uniformity of the biological make-up of the population by the end of the Neolithic is also observed in the genetic studies (Szécsény-Nagy et al., 2017;[START_REF] Olalde | The genomic history of the Iberian Peninsula over the past 8000 years[END_REF][START_REF] Villalba-Mouco | Survival of Late Pleistocene Hunter-Gatherer Ancestry in the Iberian Peninsula[END_REF]. These studies also show that the Hunter-Gatherer component becomes more important at this period.

In summary, our results are compatible with a dual pattern model for the dispersion of the Neolithic in the Northwest of the Mediterranean, although the impact of incoming eastern farmers would not be as important as in other areas of the Mediterranean. In addition, we observe that this effect is similar in the Iberian Peninsula and South of France, and that the affinities that are observed in material culture between the Chasséen and Sepulcres de Fossa cultures have a parallel in biological means. Lastly, the similarities between inhabitants from coastal areas are greater, as probably these were more open to external influences related to exchange networks; moreover, the inner groups also present some variation in funerary culture with the presence of burials in cists.

The

  French samples belong both to the Middle-and Late-Neolithic, and almost the full extension (West-to-East) of the Southern part of the country. The Middle-Neolithic samples are grouped in three ensembles regarding their geographical and cultural characteristics: Middle-Neolithic from Languedoc-Midi Pyrénées; Middle-Neolithic from Provence-Alpes-Côte d'Azur; and Montbolò Neolithic (Supplementary Table

  ) shows the Italian Upper-Palaeolithic and Mesolithic sample appeared separated from all the other samples and the Italian Neolithic sample stands in the centre of the plot, while in the other corner French and Iberian samples appeared separated between them as well. From the Iberian and French samples those showing more affinities to the Italian Neolithic were Cascajos (Early-and Middle-Neolithic from Navarre), Bòbila Madurell (Middle-Neolithic from Catalonia) and Roaix (Late-Neolithic from Provence), while the Langedoc and Solsonian samples appeared farther.

Acknowledgments

We would like to thank F. Valentin (UMR 7041, Arscan, Ethnologie préhistorique), M. Gandelin,Y. Tchérémissinoff, R. Donat (INRAP, France), and P. Courtaud (UMR 5199 PACEA, Bordeaux) for providing the samples analysed for this work and their help on concrete questions about the contexts. We are especially thankful the museums and archaeological deposits: Maison du Patrimoine Françoise-Claustre and Château-Musée de Bélesta. Lastly great thanks to the curators from these centres Gilles Peyre and Tarek Kuteni for their help during the study of the materials. Moreover, this work has been funded by the project HAR2015-67323-C2-1-P of Ministerio de Ciencia y Economia, and been posible thanks to the 2017 SGR 1630 Grup de recerca en Antropologia Biològica (GREAB) and project PID2020-112513RB-I00 of Ministerio de Ciencia e Innovación. DLO was supported by the Basque Governments postdoctoral Fellowship (POS_2019_1_0024) and MSC Actions project N° 895713.