Pierre Rambeau

Emilie Faure

Alexis Théron

Jean-François Avierinos

Chris Jopling

Stéphane Zaffran

Adèle Faucherre

Reduced aggrecan expression affects cardiac outflow tract development in zebrafish and is associated with bicuspid aortic valve disease in humans

Keywords:

Hemodynamic forces have been known for a long time to regulate cardiogenic processes such as cardiac valve development. During embryonic development in vertebrates, the outflow tract (OFT) adjacent to the ventricle comes under increasing hemodynamic load as cardiogenesis proceeds. Consequently, extracellular matrix components are produced in this region as the cardiac cushions form which will eventually give rise to the aortic valves. The proteoglycan AGGRECAN is a key component of the aortic valves and is frequently found to be deregulated in a variety of aortic valve diseases. Here we demonstrate that aggrecan expression in the OFT of developing zebrafish embryos is hemodynamically dependent, a process presumably mediated by mechanosensitive channels. Furthermore, knockdown or knockout of aggrecan leads to failure of the OFT to develop resulting in stenosis. Based on these findings we analysed the expression of AGGRECAN in human bicuspid aortic valves (BAV). We found that in type 0 BAV there was a significant reduction in the expression of AGGRECAN. Our data indicate that aggrecan is required for OFT development and when its expression is reduced this is associated with BAV in humans.

Introduction

Arterial valve leaflets are composed of three distinct layers of Extra-Cellular Matrix (ECM) called fibrosa, spongiosa and ventricularis. The spongiosa layer is particularly rich in proteoglycans which will provide compressive properties to the tissue and allow the leaflet to change shape during the cardiac cycle. Indeed, valves are submitted to extreme hemodynamic forces such as shear stress and cyclic strain that will subsequently regulate both their development and function. Studies in chick and zebrafish have shown that disruption of the hemodynamic forces results in cardiac valves defects including defects of the outflow tract (OFT) cushion formation and severe heart defects associated with a total absence of valves [START_REF] Menon | Altered hemodynamics in the embryonic heart affects outflow valve development[END_REF][START_REF] Hove | Intracardiac fluid forces are an essential epigenetic factor for embryonic cardiogenesis[END_REF]. Among valve diseases, bicuspid aortic valve (BAV) is one of the most common pathologies found in patients, occurring in 1-2% of living births and are frequently associated with aortic stenosis, regurgitation, endocarditis and calcified valves [START_REF] Padang | Genetic basis of familial valvular heart disease[END_REF].

AGGRECAN is one of the major members of the large proteoglycans found in cartilage and provides the ability to resist compressive loads [START_REF] Lincoln | Hearts and bones: shared regulatory mechanisms in heart valve, cartilage, tendon, and bone development[END_REF]. A recent transcriptomic study of human BAV has shown that AGGRECAN expression is decreased in BAV patients with mild calcification compared with calcified tricuspid aortic valve (TAV) [START_REF] Padang | Comparative transcriptome profiling in human bicuspid aortic valve disease using RNA sequencing[END_REF]. Zebrafish possess 2 aggrecan paralogues, aggrecanA (acana) and aggrecanB (acanb). Here we show that acana expression in the zebrafish OFT is dependent on hemodynamic forces and that knockdown or knockout of acana during development induces cardiovascular defects. Moreover, we observe that in humans, AGGRECAN (ACAN) expression is reduced in type 0 BAV compared to normal TAV.

Materials and methods

Zebrafish strains and husbandry

Zebrafish were maintained under standardized conditions [START_REF] Westerfield | The zebrafish book, A Guide for the Laboratory Use of Zebrafish (Danio rerio)[END_REF] and experiments were conducted in accordance with the European Communities council directive 2010/63. The Tg(fli1a:GFP)y1 line was provided by the CMR[B]. ISH were performed as described previously [START_REF] Brend | Zebrafish whole mount high-resolution double fluorescent in situ hybridization[END_REF][START_REF] Thisse | High-resolution in situ hybridization to whole-mount zebrafish embryos[END_REF] (see Supplementary information). For RR treated fish, the proteinase K treatment was reduced to 15 min.

Ruthenium Red treatment

Morpholinos and injections

Morpholino oligonucleotides were obtained from Gene Tools (Philomath, OR, USA) and injected into one-cell stage embryos (see Supplementary information).

CRISPR/Cas9

Acana target sequences were identified using ZiFiT online software [START_REF] Sander | Zinc Finger Targeter (ZiFiT): an engineered zinc finger/target site design tool[END_REF]. 150 pg of acana gRNA was co-injected with nls-Cas9 protein (N.E.B) (see Supplementary information).

Cardiovascular parameters analysis

Cardiovascular parameters were determined using the MicroZebraLab™ software from ViewPoint [START_REF] Parker | A multi-endpoint in vivo larval zebrafish (Danio rerio) model for the assessment of integrated cardiovascular function[END_REF] (see Supplementary information).

Real-time quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)

Human aortic valve tissues were collected after surgery by the Department of cardiac surgery at "La Timone" Hospital, Marseille, France. The protocol was evaluated and authorised by the "CPP Sud Méditerranée" n°: 13.061. and by the "Agence de la biomedicine" n°PFS14-011. (see Supplementary information).

Results

AggrecanA is expressed in the zebrafish OFT and is dependent on hemodynamic forces

To determine the expression pattern of aggrecan, we performed in situ hybridization (ISH) on 4 days post-fertilization (dpf) embryos using antisense acana and acanb probes. Acana showed a high expression in craniofacial cartilage as previously described [START_REF] Kang | Molecular cloning and developmental expression of a hyaluronan and proteoglycan link protein gene, crtl1/hapln1, in zebrafish[END_REF], however we were also able to observe a clear expression of acana in the OFT (Fig. 1.A). In contrast, we could not detect any acanb expression in this cardiac structure (Fig. 1.B). In order to confirm that acana expression was localised to the OFT, we performed double fluorescent ISH on 4dpf larvae using antisense probes targeting acana and elastin b (elnb), an abundant component of the zebrafish OFT [START_REF] Miao | Differential expression of two tropoelastin genes in zebrafish[END_REF]. In this manner we were able to observe that acana expression co-localised with elnb expression in the OFT (Fig. 1.C-H). Because ECM composition can be modulated by hemodynamic forces, we sought to determine whether this was the case for acana expression in the OFT. To achieve this, we used a previously described morpholino targeting tnnt2 which effectively stops the heart from beating [START_REF] Sehnert | Cardiac troponin T is essential in sarcomere assembly and cardiac contractility[END_REF]. In this manner we found that tnnt2 morphants display an apparent lack of acana expression in the OFT (Fig. 1.I). Previous research has indicated that mechanosensitive ion channels (MSC) can detect hemodynamic forces and trigger valve formation [START_REF] Heckel | Oscillatory flow modulates mechanosensitive klf2a expression through trpv4 and trpp2 during heart valve development[END_REF]. To determine whether MSC could mediate acana expression in response to hemodynamic forces, we employed the nonselective MSC blocker Ruthenium Red (RR). In this manner we could observe that larvae incubated with RR showed a dose responsive decrease of acana expression in the OFT (Fig. 1.J-L).

AggrecanA is involved in cardiac development

Due to its expression in the OFT, we speculated that acana may play a role in cardiac development. To answer this question we adopted a morpholino (MO) mediated approach targeting an internal splice site. Injection of this MO produced a phenotype characterized by defective cardiogenesis including a larger atrium and associated edema at 3dpf (Fig. 1.M-N, P-Q). To confirm the specificity of the phenotype, we performed several control experiments (Suppl. Fig. 1 and Suppl. information). We also implemented a previously described transient CRISPR/ Cas9 knockout strategy [START_REF] Willems | The Wnt Co-receptor Lrp5 is required for cranial neural crest cell migration in zebrafish[END_REF][START_REF] Moriyama | Evolution of the fish heart by sub/neofunctionalization of an elastin gene[END_REF]. In this manner we were able to observe zebrafish embryos displaying a similar cardiac phenotype to that observed when acana was knocked-down with a MO (Fig. 1.O,R). Although this approach will result in a mosaic knockout of acana, all embryos which displayed a cardiac phenotype tested positive for KO of acana by T7 assay (n = 6/44) (Suppl. Fig. 2). Together these results indicate that loss of acana either by knockdown or knockout leads to perturbed cardiogenesis.

To better understand how the OFT has been affected, we analysed this structure in Tg(fli1a:GFP)y1 larvae which express GFP in all endothelial cells. At 3dpf, the wild-type OFT displays a typical "pear-shape" structure (Fig. 2.A). By contrast, in the acana morphants this structure has failed to develop (Fig. 2.B). In parallel, we also analysed a number of cardiovascular parameters such as heart rate and stroke volume. No differences were observed in the heart rate between the wild-type and acana morphants (Suppl. Fig. 3). However, the cardiac output and stroke volume were significantly decreased in acana morphants when compared to controls (Fig. 2.C and Suppl. Fig. 3). We also recorded high-speed movies of the beating embryonic heart and observed that in acana morphants there was a significant amount of blood regurgitation between the ventricle and the atrium, most likely caused by failed OFT development, forcing blood back through the AV canal (Suppl. movies M1 and M2).

ACAN expression is reduced in human BAV patients

Based on our findings in zebrafish, we hypothesised that reduced AGGRECAN (ACAN) expression may also be associated with aortic valve diseases such as BAV. Firstly, we determined the relative abundance of ACAN in the aortic valve by qPCR and were able to detect ACAN expression during foetal valve development in human (Suppl. Fig. 4). By 13 weeks of gestation, the ACAN expression greatly increased and this expression was even more abundant in adult valves. To assess the possibility that ACAN expression could be reduced in BAV patients, we performed RT-qPCR analysis using RNA extracted from aortic valves surgically removed from patients diagnosed type 0 "pure" BAV. As a control we used RNA extracted from normal TAV. We observed a significant (16 fold) decrease in the expression of ACAN in the type 0 BAV samples when compared to the control TAV samples (Fig. 2.D).

Discussion

Here we have shown that hemodynamically dependent acana expression is required for OFT development in zebrafish. Moreover, we showed that a MSC could be the sensor of these hemodynamic forces. It has recently been shown that the Trpv4 MSC, a target of RR, is involved in atrioventricular valve development in response to hemodynamic forces [START_REF] Heckel | Oscillatory flow modulates mechanosensitive klf2a expression through trpv4 and trpp2 during heart valve development[END_REF]. However, because RR is non-specific, we cannot determine the true identity of the MSC at this juncture. Because the analogous region in humans will give rise to the aortic valves, we also analysed ACAN expression during human aortic valve development and in patients who suffer from type 0 BAV we found a significant reduction when compared to TAV.

Because of the rarity of this condition, we were only able to analyse relatively few patients, however the difference was in excess of 16 fold. It will therefore be necessary to expand this cohort to determine fully the reduction in the expression of AGGRECAN associated with BAV type 0. At present little is known about the genetic causes of BAV, with only a handful of genes thus far identified and, as one can imagine, there is even less known about what causes the different types of BAV. Although we cannot categorically state that reduced AGGRECAN expression is the root cause of type 0 BAV, its association with this condition does appear to be significant. Decreased AGGRECAN expression with BAV type 0 may not be so surprising considering it is required to strengthen and provide rigidity to the developing valves, and when this is lost the developmental process will malfunction. Why AGGRECAN expression is reduced in BAV type 0 remains unclear at this juncture, however it is possible that defective hemodynamics or defective mechanosensation of these forces during OFT development could be involved with this condition.

Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijcard.2017.09.174.

Fig. 1 .

 1 Fig. 1. Acana is hemodynamically expressed in the OFT and is involved in heart development. (A-B) ISH of 4dpf larvae using antisense acana (A) and acanb (B) probes. Acana is expressed in the OFT (A, yellow arrowhead). Acanb expression was not detected in the OFT (B, yellow arrowhead). (C-E) Confocal maximal projection images of double fluorescent ISH on 4dpf WT larvae using antisense acana (C, red) and elnb (D, green) probes (Blue: DAPI, yellow arrowheads indicate the OFT). (F-H) Same larvae at higher magnification (dashed rectangle in E). The merged images (E,H) indicate that acana is expressed in the OFT labelled by elnb. Scale bars E: 100 μm; H: 20 μm. (I) ISH of 4dpf tnnt2-MO injected larvae using an antisense acana probe. Acana expression was not detected in the OFT (yellow arrowhead). (J-L) ISH of 4dpf larvae using an antisense acana probe on nontreated (J, control, n = 19), or treated specimen with 10 μM (K, n = 15) and 20 μM (L, n = 6) of Ruthenium Red (RR). Black arrowheads indicate the OFT. (M-O) Bright field images of non-injected (M), acana-MO (N) and CRISPR/Cas9 injected (O) 3dpf larvae. Both morphant and transient knockout display heart edema (black arrowheads). (P-R) myl7 ISH showing the morphology of a wild-type heart (P) or of hearts from acana-MO (Q) and CRISPR/Cas9 injected (R) 3dpf larvae. Both morphant and transient knockout display a larger atrium (v: ventricle; a: atrium).

Fig. 2 .

 2 Fig. 2. Knockdown of acana disrupts OFT development and AGGRECAN expression is modified in human BAV patients. (A,B) Confocal maximal projection image of 3dpf Tg(fli1a:GFP)y1 OFT from WT (A) and acana morphant (B) larvae. White dashed lines indicate the OFT. (C) Graph showing the difference in stroke volume between noninjected (NI, n = 13) and acana morphants (n = 12). ***p b 0.001 (Student's t-test) (D) Graph showing relative mRNA expression of ACAN between TAV controls (n = 4) and BAV Type 0 (n = 2). **p b 0.05 (nonparametric Mann and Withney test). Error bars represent mean ± SEM in all histograms.

Acknowledgements A.F is currently supported by a Labex ISCT postdoctoral fellowship with previous support provided by a Fondation Lefoulon-Delalande postdoctoral fellowship. P.R is supported by the Labex ISCT PhD program. C.J is supported by an INSERM ATIP-AVENIR grant and a Marie Curie CIG (PCIG12-GA-2012-332772). A.F, P.R and C.J are members of the Laboratory of Excellence «Ion Channel Science and Therapeutics» supported by a grant from the ANR. Work in C.J lab is supported by a grant from the Fondation Leducq and by the Fédération pour la Recherche sur le Cerveau (FRC). Work in S.Z lab is supported by INSERM, the Fédération Française de Cardiologie, and the Association Française contre les Myopathies (AFM-Telethon).

Conflict of interest

The authors report no relationships that could be construed as a conflict of interest.


Wilson R Lourenço
email: wilson.lourenco@mnhn.fr

Mohamed Lamine Kourim

Salah Eddine Sadine

Vachon

Una nuova specie africana del genere Leiurus Ehrenberg, 1828 (Scorpiones: Buthidae)

Keywords: Scorpion, new species, Leiurus hoggarensis sp. n., Buthidae, Algeria, Hoggar. Riassunto Scorpione, nuova specie, Leiurus hoggarensis sp. n., Buthidae, Algeria, Hoggar

A new species of buthid scorpion belonging to the genus Leiurus Ehrenberg 1828 is described on the basis of four males and six females collected in the region of Amesmessa Tamanrasset in the south of Algeria. The new species, Leiurus hoggarensis sp. n., most certainly corresponds to the Leiurus population previously cited by Vachon from both the Hoggar and the Tassili N'Ajjer as Leiurus quinquestriatus. Several characteristics, however, attest that this population is unquestionable distinct from these found in Egypt, and both species can be distinguished by a distinct coloration pattern, different morphometric values and different number of teeth on the pectines. The type locality of the new species represents the most westerly record of the genus Leiurus in Africa, and the new species also inhabit a more mesic zone when compared to the central compartment of the Saharan desert. Leiurus hoggarensis sp. n., apparently does not present characteristics of a psamophilic species and may be considered as a lithophilic species. This is the 12 th species to be described for this buthid genus.

Introduction

As already outlined in several previous publications [START_REF] Lourenço | Description of a new species of Leiurus Ehrenberg, 1828 (Scorpiones, Buthidae) from the South of Jordan[END_REF][START_REF] Lourenço | The African species of the genus Leiurus Ehrenberg, 1828 (Scorpiones: Buthidae) with the description of a new species[END_REF][START_REF] Lourenço | One more African species of the genus Leiurus Ehrenberg, 1828 (Scorpiones: Buthidae) from Somalia[END_REF], the genus Leiurus Ehrenberg, 1828 was represented over many decades by a single species, Leiurus quinquestriatus, containing two subspecies, L. quinquestriatus quinquestriatus (Ehrenberg, 1828) and L. quinquestriatus hebraeus (Birula, 1908). Leiurus quinquestriatus seems to be a common species of desert faunas in certain regions of Egypt, Sinai and Sudan, although the precise identity of some regional populations from these areas requires yet further investigations [START_REF] Lowe | A review of the genus Leiurus Ehrenberg, 1828 (Scorpiones: Buthidae) with description of four new species from the Arabian Peninsula[END_REF]. Contrarily, L. hebraeus Birula, 1908 (now recognized as a valid species) is largely distributed in Israel and nearby countries [START_REF] Levy | Leiurus quinquestriatus hebraeus (Birula, 1908) (Scorpiones; Buthidae) and its systematic position[END_REF]. Leiurus species are globally infamous since they secrete one of the most noxious venoms among buthid scorpions in general, and are responsible for very serious human incidents (for details refer to [START_REF] Lourenço | One more African species of the genus Leiurus Ehrenberg, 1828 (Scorpiones: Buthidae) from Somalia[END_REF]. Because of its infamous reputation as a very dangerous scorpion, the toxins of both L. quinquestriatus and L. hebraeus have been the subject of numerous biochemical studies (for references see [START_REF] Simard | Venoms and Toxins[END_REF][START_REF] Hammock | Structure and neurotoxicity of venoms[END_REF]. Neverthless, many aspects of the taxonomy of the genus Leiurus remained confused for many decades (for more details see [START_REF] Lourenço | The African species of the genus Leiurus Ehrenberg, 1828 (Scorpiones: Buthidae) with the description of a new species[END_REF][START_REF] Lourenço | One more African species of the genus Leiurus Ehrenberg, 1828 (Scorpiones: Buthidae) from Somalia[END_REF].

Only in recent years, totally new species were finally described for the genus Leiurus. The description which really changed most conservative views about this group of scorpions was that of Leiurus jordanensis Lourenço, Modry et Amr, 2002 described from Jordan [START_REF] Lourenço | Description of a new species of Leiurus Ehrenberg, 1828 (Scorpiones, Buthidae) from the South of Jordan[END_REF]. Just a few years later, Leiurus savanicola Lourenço, Qi et Cloudsley Thompson, 2006[START_REF] Lourenço | The African species of the genus Leiurus Ehrenberg, 1828 (Scorpiones: Buthidae) with the description of a new species[END_REF] was described from Cameroon, representing the second confirmed species from Africa. In a more recent contribution, [START_REF] Lowe | A review of the genus Leiurus Ehrenberg, 1828 (Scorpiones: Buthidae) with description of four new species from the Arabian Peninsula[END_REF] proposed, in a very extensive article, a full revision of the genus Leiurus, but dealing mainly with the populations from the Arabian Peninsula. The status of some old species was revalidated, one recently described species was placed in synonymy, one subspecies was raised to species and four new species were described. This raised the total number of species in the genus Leiurus to ten. The characters used by these authors to define the species, as well as the proposed dichotomic key are rather difficult to be used. Nevertheless, we globally agree with these authors and, in particular with their opinion about the African species, stated as follows: "Our findings show that, like many other scorpion genera, Leiurus is comprised of an assemblage of allopatric or parapatric species spread across different regions separated by physiographic barriers, each adapted to local environments and substrates. Additional species diversity may emerge when other local populations are analysed in more detail, for example those in southern Sinai and in more central parts of North Africa". Moreover, after the study of the original syntypes used in the description of Leiurus quinquestriatus, these same authors [START_REF] Lowe | A review of the genus Leiurus Ehrenberg, 1828 (Scorpiones: Buthidae) with description of four new species from the Arabian Peninsula[END_REF] suggest as follows: "The syntypes include assorted material from the Sinai, the Nile Valley in Egypt and Sudan, and the desert region of Egypt east of the Nile. These could represent more than one species if the populations in the Sinai are distinct from those of the Nile Valley". Once again we globally agree with this suggestion.

It is most obvious that the African populations of Leiurus have been largely neglected and still require intensive further studies. If [START_REF] Vachon | Etude sur les Scorpions[END_REF] associated the few specimens he studied from Hoggar to L. quinquestriatus this can be attributed to both the typical incertitude of this author, but also to the very limited material (mainly fragments) he disposed. Again [START_REF] Vachon | Scorpions, Mission scientifique au Tassili des Ajjer[END_REF] in a synopsis of the scorpions from the Tassili N'Ajjer, another mountain range in southern Algeria, cited Leiurus quinquestriatus from this locality, but based on the study of two very young juveniles. None of these specimens was located in the collections of the Museum in Paris and probably were deposited in other collections such as that of the Institute Pasteur of Algeria. In his monograph on the scorpions of North Africa, [START_REF] Vachon | Etude sur les Scorpions[END_REF] also referred to several specimens collected in the Fezzan (Libya) as L. quinquestriatus. It is quite possible however, that this population corresponds to Buthus quinquestriatus libycus Birula, 1908 (= Leiurus quinquestriatus libycus). Nevertheless, only the study of more fresh material from Libya will allow a confirmation to this suggestion. In the present contribution we describe a new species based on material collected in the region of the Hoggar Massif in the south of Algeria. This raises the number of Leiurus species to twelve.

Methods

Illustrations and measurements were obtained using a Wild M5 stereomicroscope with a drawing tube and ocular micrometer. Measurements follow [START_REF] Stahnke | Scorpion nomenclature and mensuration[END_REF] and are given in mm. Trichobothrial notations follow [START_REF] Vachon | Etude des caractères utilisés pour classer les familles et les genres de Scorpions (Arachnides)[END_REF] and morphological terminology mostly follows [START_REF] Hjelle | Anatomy and morphology[END_REF]. Etymology: specific name makes reference to the Hoggar, the region where the new species was found. Diagnosis. Scorpion of large size when compared with the other species of the genus, having a maximum total length of 77.7 mm for male and 94.6 mm for female. Ground colour yellow to orangeyellow with the body and pedipalps almost totally orangeyellow. Male carapace with a brownish which covers the ocular tubercle; metasomal segment V only slightly infuscate, including in juvenile specimens; other metasomal segments orangeyellow. Ocular tubercle strongly prominent. Pectines with 32 to 34 and 26 to 29 teeth for males and females respectively. Median carinae on sternites IIIIV moderately to strongly marked; sternite VII with mediate intercarinal surface presenting a thin granulation. Pedipalp fingers with 1112 or 1212 rows of granules for both males and females. Description based on male holotype and paratypes. (Morphometric measurements in Table I). Coloration. Ground colour yellow to orangeyellow; body and pedipalps almost totally orangeyellow; legs yellow. Male carapace orangeyellow with a brownish spot which covers the ocular tubercle. Mesosoma tergites with some infuscations in male, absent from female. Metasoma orangeyellow on segments I to IV; segment V slightly infuscate, including on juveniles. Vesicle yellow with reddish tonalities on lateral sides; aculeus yellow at the base and dark red at its extremity. Venter yellow to slightly orange yellow without spots. Chelicerae yellow without any dark reticulated spots; teeth dark red. Pedipalps yellow to orangeyellow overall except for the rows of granules on chela fingers which are dark red. Legs yellow with some zones slightly orangeyellow.

Morphology. Prosoma: Anterior margin of carapace with a weak concavity. Carapace carinae moderately to strongly developed; central median and posterior median carinae moderate to strong; anterior median carinae strong; central lateral carinae moderate to strong; posterior median carinae moderate to strong, terminating distally in a small spinoid process that extends very slightly beyond the posterior margin of the carapace. All carinae better marked on males. Intercarinal spaces with very few irregular granules, and the reminder of the surface almost smooth, in particular laterally and distally. Median ocular tubercle in a central position and strongly prominent; median eyes large in size and separated by about two ocular diameters. Four/five pairs of lateral eyes; the fourth and fifth are vestigial. Mesosomal tergites III pentacarinate; IIIVI tricarinate. All carinae strong, granular, better marked on male; each carina terminating distally in a spinoid process that extends slightly beyond the posterior margin of the tergite. Median carinae on I moderate, on IIVI strong, crenulated. Tergite VII pentacarinate, with lateral pairs of carinae strong and fused; median carinae present on the proximal half in female and on the 2/3 on male, moderate to strong. Intercarinal spaces weakly to moderately granular. Lateral carinae absent from sternite III; moderate to strong on sternites IVVI; strong, crenulate on VII; median carinae on sternites IIIIV moderate to strong. Pectines long; pectinal tooth count 3434 on male holotype and 2928 for female paratype (see diagnosis for variation). Metasomal segments IIII with ten carinae, moderately crenulate; lateral inframedian carinae on I moderate; on II present on the posterior third; on III limited to a few posterior granules; IV with eight carinae. Dorsal and dorsolateral carinae moderate, without any enlarged denticles distally. All the other carinae moderate to weak on segments IIV. Segment V with five carinae; ventromedian carinae with several slightly spinoid granules distally; anal arch with three slightly spinoid lobes, better marked in female. Dorsal furrows of all segments weakly developed and smooth; intercarinal spaces almost smooth, with only a few granules on the ventral surface of segment V. Telson smooth; subaculear tubercle absent; aculeus as long as vesicle. Chelicerae with two reduced denticles at the base of the movable finger [START_REF] Vachon | De l'utilité, en systématique, d'une nomenclature des dents des chélicères chez les Scorpions[END_REF]. Pedipalps: Trichobothrial pattern orthobothriotaxic, type A [START_REF] Vachon | Etude des caractères utilisés pour classer les familles et les genres de Scorpions (Arachnides)[END_REF]; dorsal trichobothria of femur in configuration [START_REF] Vachon | Sur l'utilisation de la trichobothriotaxie du bras des pédipalpes des Scorpions (Arachnides) dans le classement des genres de la famille des Buthidae Simon[END_REF]. Femur pentacarinate; all carinae moderately β crenulate. Patella with seven carinae; all carinae moderately to weakly crenulate; dorsointernal carinae with 23 spinoid granules distally. Chelae slender, with elongated fingers; all carinae weakly marked, almost vestigial. Dentate margins of fixed and movable fingers composed of 1112 or 1212 almost linear rows of granules in both sexes. Legs: Ventral aspect of tarsi with short spiniform setae more or less arranged in two rows. Tibial spurs present on legs III and IV, moderately marked. Pedal spurs present on all legs, strongly marked.

Relationships.

Based on the key supplied by [START_REF] Lowe | A review of the genus Leiurus Ehrenberg, 1828 (Scorpiones: Buthidae) with description of four new species from the Arabian Peninsula[END_REF], the new species seems to present affinities with L. quinquestriatus 'typicus' normally only distributed in Egypt, mainly Sinai, and perhaps also in Sudan. Nevertheless the two species differs by a number of characters: I) distinct patterns of pigmentation, with the population from Hoggar showing a more orangeyellow colour; II) quite distinct morphometric values for specimens of a similar global size; III) lower numbers of pectinal teeth counts. Moreover, the geographic distributions of the African populations are not continuous. The future examination of material from Libya should confirm the existence of an intermediate population between Egypt and Algeria.

Ecology

As already outlined in a recent publication [START_REF] Lourenço | Scorpions from the region of Tamanrasset, Algeria. Part I. A new species of Buthacus Birula, 1908 (Scorpiones: Buthidae)[END_REF], the Region of El Ahaggar (Tamanrasset) which corresponds to the El Ahaggar National Park is very important in surface. It is located in the Central Massif of the southeastern Algerian region (Fig. 15) and covers a total area of ca. 450,000 km 2 . The main locality in this area is Tamanrasset [START_REF] Wacher | lnventaire de la faune sahelosaharienn[END_REF]. The very diverse geomorphological features are constituted by the Regs, Ergs, Stone Plateaux (Figs. 1617) but also very high summits such as the Tahat with more than 3000 meters, constituting the highest mountain in Algeria [START_REF] Sahki | Guide des principaux arbres et arbustes du Sahara central (Ahaggar et Tassili)[END_REF]. The region of El Ahaggar, as all the others regions around Tamanrasset is characterized by a typical arid climate with mild winters but also important thermal amplitudes between the day and the night [START_REF] Kourim | Biodiversité faunistique dans le Parc National de l'Ahaggar[END_REF]. The hottest months range from June to August. Rain fall is extremely rare in the region of El Ahaggar and the average values can vary extremely accordingly to the year; very important dry periods can be observed over more than three years. Maximum rain is generally observed during the hot period from June to August [START_REF] Hamdine | Conservation du Guépard (Acinonyx jubatus Schreber, 1776) de la région de l'Ahaggar et du Tassili n'Adjjer en Algérie[END_REF]. The new species described here was collected in the region of Amesmessa which is located about 450 Km NW of Tamanrasset. This site is located in a region of mountains with quite many sand deposits which are the consequence of gold mining. The new Leiurus species appears as the most common scorpion in the region of study, representing up to 76.24% in the region of Tamanrasset and up to 84.72% in the region of Amesmessa. Leiurus hoggarensis sp. n., apparently does not present characteristics of a psamophilic species and may be considered as a lithophilic species (Fig. 18).

Figs. 14 .

 14 Figs. 14. Leiurus hoggarensis sp. n. Habitus. 12. Male holotype. 34. Female paratype.

59.

  Leiurus hoggarensis sp. n. Male holotype (58). Female paratype (9). 5. Chelicera, dorsal aspect. 6. Cutting edge of movable finger, showing rows of granules. 7. Idem, detail of the extremity. 89. Metasomal segment V and telson, lateral aspect.Taxonomic treatmentFamily Buthidae C. L. Koch, 1837 Genus Leiurus Ehrenberg, 1828Leiurus hoggarensis sp. n. (Figs. 114)Type material: Algeria, AmesmessaTamanrasset (21°03' N -02°28' E), 326/X/2015 (M. L. Kourim). Male holotype, 2 males and 3 females paratypes deposited in the Muséum national d'Histoire naturelle, Paris, France. 1 male and 3 female paratypes deposited in the University of Ghardaïa, Algeria.

Figs. 1014 .

 1014 Figs. 1014. Leiurus hoggarensis sp. n. Male holotype. Trichobothrial pattern. 1011. Chela, dorsoexternal and ventral aspects. 1213. Patella, dorsal and external aspects. 14. Femur, dorsal aspect.

Fig. 15 .

 15 Fig. 15. Map of Algeria showing in detail the region of El Ahaggar (Tamanrasset), with the type locality of Leiurus hoggarensis sp. n. (black star).

Figs. 1617 .

 1617 Figs. 1617. Aspects of the biotopes found in the region of El Ahaggar with typical sites in Amesmessa where the Leiurus hoggarensis sp. n. was collected.

Fig. 18 .

 18 Fig. 18. A preadult male of Leiurus hoggarensis sp. n. alive in its natural habitat.

Table I .

 I Morphometric values (in mm) of the male holotype and female paratype of Leiurus hoggarensis sp. n.

		Male holotype	Female paratype
	Total length	77.7	94.6
	Carapace:
	length	8.4	10.5
	anterior width	5.8	7.2
	posterior width	9.5	12.5
	Mesosoma length	17.8	20.7
	Metasomal segment I:
	length	6.7	8.2
	width	5.6	6.2
	Metasomal segment II:
	length	8.2	9.8
	width	5.2	5.3
	Metasomal segment III:
	length	8.3	10.3
	width	4.7	4.9
	Metasomal segment IV:
	length	9.1	11.4
	width	4.3	4.6
	Metasomal segment V:
	length	10.5	12.5
	width	4.0	4.6
	depth	3.6	3.9
	Telson length	8.7	11.2
	Vesicle:
	width	3.4	4.2
	depth	3.2	3.8
	Pedipalp:
	Femur length	8.9	11.1
	Femur width	2.2	2.7
	Patella length	9.8	12.3
	Patella width	2.8	3.2
	Chela length	15.8	19.9
	Chela width	2.5	3.2
	Chela depth	2.6	3.3
	Movable finger:
	length	11.2	14.4

Acknowledgements

We are most grateful to Mohamed Kraimat (University of Ghardaïa) for his assistance both during the field work and the preparation of this article, and in special to EliseAnne Leguin (MNHN, Paris) for her assistance in the preparation of the photos and plates.


Alexa Dufraisse
email: alexa.dufraisse@mnhn.fr

Sylvie Coubray

Olivier Girardclos

Noémie Nocus

Michel Lemoine

Jean-Luc Dupouey

Dominique Marguerie

approach to past firewood exploitation and woodland management reconstructions. Dendrological reference dataset modelling with dendro-anthracological tools

Keywords: firewood management, dendro-anthracological tools, anthraco-typology, anthraco-group, deciduous oak

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

Introduction

Anthracology and past woodland reconstruction

The questions raised by relationships between people and the environment in time and space can be explored by archaeological, ethnographic or environmental approaches.

The management of the environment for (plant or animal) food strategies reflects, to some extent, human societies and their organization, their lifestyles, their perception of the environment and the landscape in which they operate.

Forest exploitation in order to produce wood material for multiple needs, is perceptible at different scales: the tree, the woodland and the landscape (Michon, 2005(Michon, , 2015)); Humans R e v i s e d m a n u s c r i p t

"domesticate" the tree by modifying its architecture, its growth cycle, its production and its reproductive functions. This domestication also concerns the forest ecosystem transformed by practices. Wood stands are shaped by and for societies living in them as a result of the installation of fields, herds and villages in forest areas. Variable spatial patterns results from this articulation between forest and agriculture, the landscapes.

Firewood management contributes to this "domestication". It is part of a complex system closely related to social organization, technical and economic systems, and the environment itself (Chabal, 1997;[START_REF] Picornell Gelabert | People, trees and charcoal: somes reflections about the use of ethnoarchaeology in archaeological charcoal analysis[END_REF][START_REF] Dufraisse | Firewood and woodland management in their social, economic and ecological dimensions[END_REF][START_REF] Salavert | Understanding the impact of socio-economic activities on archaeological charcoal assemblages in temperate areas: comparative analysis of firewood management of two Neolithic societies of Western Europe (Belgium, France)[END_REF]. Thus, archaeological charcoal fragments, residues of firewood selected and transported by humans, are valuable ecofacts reflecting use, techniques and woodland management, and are themselves conditioned by environmental resources (available wood resources, i.e. biodiversity and biomass).

In forest science, the criteria characterizing wood stands are the composition (dominant and secondary species), stand density (number of stems per hectare), structure (distribution of age and diameter classes of trees) and modes of regeneration (seeded or vegetative renewal) [START_REF] Rondeux | La mesure des arbres et des peuplements forestiers[END_REF]. The methods usually employed in dendrochronology to extract this information are not suited to anthracological material. In dendrochronology, samples usually come from timber wood and generally from trunks and/or branches or roots, the wood is not charred, the methods are based on statistical tools that require at least 50 consecutive rings and it is possible to individualize the signals (study of distinct elements). In anthracology, fragments derive from trunks and/or branches or roots, the wood is charred, fragmented and incomplete as it is partially reduced to ashes, the fragments present on average less than five rings and result from the exploitation of many indistinguishable individuals [START_REF] Dufraisse | Charcoal anatomy potential, wood diameter and radial growth[END_REF][START_REF] Marguerie | Short Tree ring series: the study materials of the dendro-anthracologist[END_REF]. Consequently, in the absence of adequate tools, charcoal analysis is most often limited to the study of a list of taxa and their relative frequency without exploiting the information contained in the wood anatomy. The identification of the morphological characteristics of harvested firewood (part of the tree, age, R e v i s e d m a n u s c r i p t shape, etc.) still raises methodological problems even though it is a fundamental element for characterizing firewood exploitation techniques and reconstructing the populational and environmental parameters of wood stands.

In order to address this need to learn more about forest exploitation and practices, the ANR DENDRAC project "Development of dendrometrical tools used in anthracology: study of the interactions between Man, resources and environments" aims to convert dendroecological data measured on fresh wood material from modern-day oak wood standscorresponding to different types of historical woodland practices -into parameters adapted to charcoal analysis, using a method similar to that developed by A. Billamboz, termed dendrotypology [START_REF] Billamboz | Applying dendro-typology to large timber series[END_REF][START_REF] Billamboz | Regional patterns of settlement and woodland developments: Dendroarchaeology in the Neolithic pile dwellings on Lake Constance (Germany)[END_REF]. His method consists in establishing a typological classification of tree-ring series according to their growth patterns. The application of a similar method in anthracology involves associating the identification of the taxa with the examination of dendrological and anatomical parameters; a concept that leads to the notion of dendro-anthracology (Marguerie et al., 2010). Deciduous European oak (Quercus petraea/robur) was chosen for its abundance in temperate forests, its anatomy with clearly identifiable growth rings and its representativity in anthracological spectra.

In the present study, we postulate that the characteristics of an assemblage of treerings can be exploited, without taking into account tree-ring series in terms of time series like in dendrochronology. The first step consisted in developing dendro-anthracological tools based on morpho-anatomical features. The second step was to convert dendroecological data to form an anthraco-typological grid, which could then be used as a key approach for the interpretation of archaeological charcoal assemblages. This approach was applied to three modern-day wood stands: a coppice under standard, a high forest and a young stand formed by a mixture of seeded and coppice trees. Analysis was conducted at different levels:

the whole tree, and trunks and branches separately, in order to model different modes of wood exploitation.

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The dendro-anthracological tools

Growth rate is a widely used dendro-anthracological parameter, but the successive tree-rings width series of each charcoal fragment must be localized as precisely as possible on the stem cross-section. In that aim, different dendro-anthracological tools are proposed in order i) to distinguish sapwood from heartwood which provides information about the minimal age of the wood (heartwood formation i.e. duraminisation starts when deciduous oak is around 25 years old) ii) to localize the tree-ring series in respect to the center of the stem, iii)

to model dendroecological data from modern wood stands into dendro-anthracological parameters adapted to charcoal analysis.

The Heartwood-Sapwood discriminating tool

In some species the coloration of heartwood due to the deposition of lignins and polyphenols makes heartwood recognizable, but the charcoalification process that occurs during carbonization obliterates the colour difference, making this feature unusable in anthracology. Fortunately, in some Angiospermae, such as deciduous European oak (Quercus petraea/robur), the formation of tyloses (cellulose walls expansions) in earlywood vessels is an important feature of the changeover of sapwood to heartwood. However, tylosis formation also occurs in sapwood and increases with the formation of heartwood, from 0% of tyloses in the cambial region and close to 100% in the heartwood. Thus, we quantify the number of vessels sealed by tylosis in order to establish discriminating thresholds between sapwood and heartwood (Fig. 1a) [START_REF] Dufraisse | Contribution of tyloses quantification in early wood oak vessels to archaeological charcoal analyses: estimation of a minimum age and influences of physiological and environmental factors[END_REF]. Trunks and branches of ten deciduous oak trees from 15 to 60 years old were sampled in three stations in order to evaluate the number of earlywood vessels with tylosis in sapwood and heartwood. For an application to archaeological charcoal (tyloses are preserved until 800°C), at least one tree ring and 15 vessels must be counted. The best strategy is to count 50 vessels spread over 3-4 tree rings. Thresholds of less than 65% for sapwood and up to 85% for heartwood are R e v i s e d m a n u s c r i p t significant. Besides the discrimination of sapwood and heartwood, the process of heartwood formation starts when deciduous oak is about 25 years old. The absence of heartwood is thus an indication of the exploitation of young wood (trunks or branches).

The pith estimation tool

The pith estimation tool is used to measure the distance between the charcoal fragment and the center of the stem (or the missing pith), named the "charcoal-pith distance".

This measurement is taken with the trigonometric pith estimation tool based on measurements of the angle and the distance between two ligneous rays (Fig. 1b) [START_REF] Dufraisse | Mesurer les diamètres du bois de feu en anthracologie. Outils dendrométriques et interprétation des données[END_REF]Paradis-Grenouillet et al., 2013). This tool was evaluated on fresh and carbonized oak wood discs with different angle values and distances between ligneous rays. This work enables us i) to propose exclusive criteria (angle < 2° and distance < 2 mm) for reducing the margin of error and improving results in archaeological applications, ii) to establish correction factors linked to the trigonometric tool itself (underestimation of distance values between 5 and 10 cm) and the shrinkage which occurs during charcoalification, iii) to highlight that there are no reliable measurements for charcoal-pith distances beyond 12.5 cm, i.e. diameter of 25 cm [START_REF] Dufraisse | Mesurer les diamètres du bois de feu en anthracologie. Outils dendrométriques et interprétation des données[END_REF][START_REF] Martinez | Correction factors on archaeological wood diameter estimation[END_REF].

The values were ordered into diameter classes chosen to be compatible with standards used in dendrometrical plans by foresters [START_REF] Gaudin | Dendrométrie des peuplements[END_REF][START_REF] Deleuze | Estimer le volume total d'un arbre, quelles que soient l'essence, la taille, la sylviculture[END_REF].

For Angiospermae the conventional wood cuts are 4 cm, 7 cm, 20 cm, etc. Two cuts were added at 2 and 10 cm for more accurate interpretation of charcoal diameters, that is to say ]0-2] cm, ]2-4] cm, ]4-7] cm, ]7-10] cm, ]10-20] and >20 cm. For Gymnospermae it is more appropriate to add a cut at 14 cm, namely ]0-2] cm, ]2-4] cm, ]4-7] cm, ]7-10] cm, ]10-14] cm, ]14-20] cm and >20 cm. Therefore, an Analysis Diameter model (ADmodel) was developed, based on the fact that a trunk is biologically considered to be a stack of cones (Fig. 1c) [START_REF] Dufraisse | Les Habitats littoraux néolithiques des lacs de Chalain et Clairvaux (Jura, France): collecte du bois de feu, gestion de[END_REF][START_REF] Dufraisse | Charcoal anatomy potential, wood diameter and radial growth[END_REF][START_REF] Dufraisse | Mesurer les diamètres du bois de feu en anthracologie. Outils dendrométriques et interprétation des données[END_REF]. These cones are hollow and their thickness corresponds to the amplitude of the diameter classes. It is based on a calculation table that provides the respective distribution of these cones in terms of volume. The ADmodel breaks down unburnt wood diameter into an expected distribution of charcoal-pith distances. In return, the ADmodel is a helpful tool to interpret the distribution of charcoal-pith distances from a charcoal assemblage as unburnt wood diameter (UWD). However, this model does not reconstruct the initially quantity of burnt wood [START_REF] Théry-Parisot | Charcoal analysis and wood diameter: inductive and deductive methodological approaches for the study of firewood collecting practices[END_REF]. In the present study, only the ADmodel running into UWD decomposition is used. In the present study, each cone thickness was also characterized by a growth rate (cumulated tree-ring width divided by the number of tree rings) and its sapwood/heartwood affiliation.

Material and Method

The general analytical protocol consists in sampling modern-day oak woodlands corresponding to specific archaeological questions, removing logs from felled trees, cutting wood discs from logs and producing experimental charcoal assemblages (Fig. 2). Various kinds of datasets were produced: i) dendrometrical plans to characterize tree morphology and wood stands (composition, structure stand density, regeneration modes), ii) dendrochronological data from wood discs, iii) anthracological data modelled with the dendro-anthracological tools. With respect to historical woodland practices and to answer to specific archaeological issues such as the distinction branch/ trunk or coppice /high forest three "contrasted" deciduous oak stands managed by the National Forestry Office (ONF) in France were chosen (Fig. 3). The first one is " Les Cagouillères ", located in the Vienne department, on a limestone plateau (altitude: 115m). It is in an old abandoned coppice woodland, about 62 years old, currently undergoing conversion to high forest. The second stand is "Bogny-sur-Meuse" located in the Ardennes department. This is a coppice-under-standard growing on an acidic brown soil on schists, about 68 years old. The third stand is "Le Bois de l'Or", also located in the Ardennes department, near Bogny-sur-Meuse. This is a young stand, about 15 years old, formed by a mixture of even aged seeded and coppice trees (altitude: 350m).

Stand analysis

In order to characterize the wood stands, forest inventory and dendrometric data were compiled. The basal area increment (m²/ha), stand density (number of stems per hectare), dominant height of the trees and the average square diameter were recorded distinguishing trees with diameters of more and less than 7.5 cm (table 1, Fig. 4).

Dendrometry and tree ring analysis

The dendrological information for each tree, such as diameter, age, growth rate and radial growth trend, was defined at breast height on the field and from disc located at 1.30 m above ground, as is usual in dendroecology. However, the nature and representativeness of archaeological samples are different in dendroecology and anthracology. Consequently, for R e v i s e d m a n u s c r i p t the conversion in anthracological data according to anthracological constraints, the dendrological data were measured in the whole tree.

For the study of tree ring-climate relations in sessile oak, six is the number of optimal trees to sample. For our purpose, and taking into account our archaeological questions, one to five trees were felled and registered meter by meter, one dominant tree in the coppiceunder-standard at "Bogny-sur-Meuse", four dominant stems from distinct multi-stem trees at "Les Cagouillères" and five coppice shoots and five seeded trees at "Le Bois de l'Or".

For each tree, the total height, the height of the first large branch insertion on the stem and the height of the crown base were recorded as well as the diameter at breast height and regeneration modes. The set of tree morphology indicators is presented in table 1 and figure 4.

In order to estimate the relative proportion of trunk and branches for each tree and each stand, each tree was cut into logs of 1-metre-long including branches with a diameter of more than 4 cm. A code was attributed to each log according to its position in the tree (height, number of branches, location in the branch). Length and circumference (at three points) of each log were measured to calculate the mean diameter and the volume. Branches with a diameter of less than 4 cm were packed into bundles according to two diameter classes; 0-2 cm and 2-4 cm. Each bundle was weighed. Sub-samples of wood were collected from each bundle to estimate the density of the wood and then to calculate the volume of each bundle.

In order to characterize each tree and then each wood stand at different levels (whole tree, and trunks and branches separately), one disc was removed from the extremity of each log. In the present study, a subsample of the set of discs was taken by selecting discs at different heights in the trunk and in the crown (23 discs for the four trees at "Les R e v i s e d m a n u s c r i p t

Cagouillères", 14 discs for the tree at "Bogny-sur-Meuse" and 77 discs for the 10 trees at "Le Bois de l'Or" (Table 2a,2b).

The tree-ring widths (discriminating earlywood and latewood) of each disc were measured to the nearest 0.01 mm using a LINTAB measurement device and associated TSAP software (Frank Rinn, Heidelberg, Germany) along 5 radii and averaged in order to reduce intra-tree variability.

Each tree-ring was then associated with a diameter class (calculated by the cumulated ring widths) and sapwood/heartwood. Thus, the proportion of sapwood and heartwood was characterized by averaging tree-ring numbers, tree-ring width and wood volume.

The usual dendro-anthracological parameters were first independently considered to obtain a "whole tree" estimation, and then the trunks and branches were separated. i) the distribution of growth-ring width, ii) the proportion of sapwood/heartwood, iii) the distribution of the decomposed unburnt wood diameters (UWD) were recorded.

Results

3.1. Dendrological features of the three wood stands (Fig. 4).

For the four sampled 62 years-old trees from "Les Cagouillères", the average diameter at breast height is 20.75 cm. The average tree height is 17.7 m and 90,4% of the wood volume is from to the trunk. The low proportion of branches, with a diameter of less than 7 cm, reflects an undeveloped crown (probably due to competition, a consequence of the abandonment of forest management). The dominant tree at "Bogny-sur-Meuse" is 68 years old with a diameter of 33 cm at breast height. The height is 20.3 m with a first large branch at 7.7 m and a more developed crown; branches represent 37.4% of the tree volume and can reach a diameter of 20 cm. The trees at "Le Bois de l'Or" are 14 years old, their diameters average 10.21 cm, 8.6 m high, the trunk forms 78.38% of the volume and the diameter of branches less than 7 cm in diameter. Thus, the tree at "Bogny-sur-Meuse" is less R e v i s e d m a n u s c r i p t

slender than trees at "Les Cagouillères" and "Le Bois de l'Or" (see the height/diameter ratio, table 1).

In the three sampled stands, trunk volume is always predominant and branches are poorly represented. The diameter 20-40 cm class is the best represented at "Bogny-sur-

Meuse" whereas the 10-20 cm diameter class characterizes "Les Cagouillères". The main volume at "Le Bois de l'Or" is distributed between 7-10 diameter but a few trees reach 11 cm and thus belong to the 10-20 cm diameter class.

Radial growth rate and growth trends are different in each stand. Tree-ring widths average 1.23 mm/year at "Les Cagouillères" coppice, and the growth trend has been decreasing over the past 20 years due to strong competition between shoots, intra-tree and between stools. At "Bogny-sur-Meuse", growth-ring widths average 1.35 mm/year and the growth trend has been decreasing slightly over the past 20 years. At "Le Bois de l'Or", growth-ring widths average 2.99 mm/year and are marked during the 1 to 10 first years by a steady increase in the coppice trees while seeded trees are characterized by narrowest rings than coppice from around the pith to 6-7 years, followed by an intensive growth period before a relatively sudden decrease (for more details, see [START_REF] Girardclos | Improving identification of coppiced and seeded tress in past woodland management by comparing growth and wood anatomy of living sessile oaks (Quercus petraea)[END_REF].

Simple dendro-anthracologial parameters

Growth rate

The distribution of the growth rates indicates differences at stand and tree levels (Fig. 5a). First, the difference in growth rate observed in § 3.1 and only based on one disc localized at 1.30 m in the trunk, is conserved when the whole tree is taken into account, what is more realistic for anthracology. The growth rate at "Les Bois de l'Or" is the highest, followed by "Bogny-sur-Meuse" and "Les Cagouillères". For a same stand, we also note a significant difference between trunks and branches, the latter being characterized by a lower rate. Moreover, considering the different parts of the trunk (base, top, upper part in the crown), we note that the annual ring-width in the top of the bole is wider than in the lower R e v i s e d m a n u s c r i p t part, and that the growth rate of the trunk localized in the crown is comparable with branches (Fig. 5b). However, this latter observation is less clear at "Le Bois de l'Or".

Sapwood/heartwood

The trees at "Le bois de l'Or", less than 15 years old, are characterized by the absence of heartwood, contrasting with "Bogny-sur-Meuse" and "Les Cagouillères" (Fig. 6).

However, at "Les Cagouillères", heartwood formation is not yet initiated in branches.

Conversely, the trunk and branches of the dominant tree at « Bogny-sur-Meuse » are characterized by heartwood and sapwood. The relative proportion of sapwood in trunk is less important at "Bogny-sur-Meuse" than at "Les Cagouillères". Likewise, the average number of sapwood tree-rings is less important at "Bogny-sur-Meuse". Nevertheless the average sapwood ring width is higher at "Bogny-sur-Meuse" reflecting more vigorous growth.

Diameters

The unburnt wood diameters (UWD) were decomposed with the ADmodel, according to the relative volume of each hollow cone composing the logs (Fig. 1c,7).

The raw dendrological data indicate that there is little overlap between the diameters of branches and trunks. In fact, the low proportion of the trunk represented in the smallest diameter classes corresponds to the upper part of the trunk localized in the crown. Therefore, for each wood stand, the distribution of the decomposed UWD of branches is clearly distinct from the trunk. Besides, as the volume of branches is weak, the wood diameter pattern for whole trees does not show clear differences with that of the trunk. The first combination consisted in assessing growth trends by characterizing each wood stand. In dendroecology, growth trends are obtained by combining tree-ring width and cambial age. Given that i) the analysis of tree-ring patterns in segment of cambial age is considered relevant for studying forest dynamics and development [START_REF] Haneca | Growth trends reveal the forest structure during Roman and Medieval times in Western Europe: a comparison between archaeological and actual oak ring series (Quercus robur and Quercus petraea)[END_REF] ii)

the distance of the charcoal from the pith can be estimated by the charcoal-pith tool, we combined tree-ring width with diameter classes. For the dendrological data, average tree-ring width was calculated for each cambial age. For the modelled anthracological data, average tree-ring width was calculated for each diameter class (Fig. 8).

The radial growth trends of the three wood stands are different and the modelled anthracological data correspond well to their dendrological characteristics. Even though the anthracological data are smoother because of the calculation of average ring width per diameter class, the radial growth trend is consisting of i) a strong increase in the radial growth of trees at "Le Bois de l'Or", reflecting a free juvenile growth, ii) the increase followed by a decrease at "Les Cagouillères" due to the high density of trees over a long period of time, iii) a slight decrease in the life of the tree at "Bogny-sur-Meuse" due to a managed coppice-under-standards. However, the differences observed between seeded and coppice trees at "Le Bois de l'Or" are no longer evident.

The branches at "Les Cagouillères" and "Bogny-sur-Meuse" are characterized by a lower growth rate than in the corresponding trunks (cf. § 3.2.1.) and by a downward growth.

In contrast, the young seeded and coppice trunks at "Le Bois de l'Or", with diameters comparable to the branches, are characterized by a clearly higher growth rate and a more upward growth.

The radial growth rate of whole trees is lower in the first diameter classes than in the trunk considered separately, as it includes the low growth rates of branches. Then, radial growth increases from the boundary of the step between diameters of trunks and branches.

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Diameter classes versus heartwood/sapwood

The second combination aims to improve the interpretation of the distribution of the decomposed UWD by associating them with the presence or absence of heartwood, and the sapwood/heartwood ratio in each diameter class, as decomposed by the ADmodel (Fig. 9).

The distribution of heartwood/sapwood according to the diameter classes shows specific patterns for the different wood stands and possible exploitation modes (whole trees, trunks/branches separately).

At "Les Cagouillères", where branches are characterized by the absence of heartwood, the volume of the trunk is mainly distributed in the penultimate diameter class.

The pattern of the whole trees is similar to that of the trunk, as branches only represent 9.58% of the volume. At "Bogny-sur-Meuse", the same pattern is observable but the main volume is represented in the last two diameter classes. However, regarding the whole tree, sapwood is better represented in the small diameter classes than at "Les Cagouillères", as branches account for 37.4% of the tree volume.

While the mature trees contain a central heartwood core (reflected by heartwood in the small diameter classes) and peripheral sapwood (reflected by sapwood in the largest diameter classes), the absence of heartwood in trunks from "Le Bois de l'Or" and in branches from "Les Cagouillères" is in agreement with young trunks and young branches respectively (less than 25 years old for oak). They are characterized by small diameters with sapwood, and the absence of heartwood and of large diameters. The biggest branches of the tree from "Bogny-sur-Meuse", i.e. 10-20 cm, contain small amounts of heartwood, and traces of heartwood in the smaller classes. The third combination consists in combining tree-ring width with the decomposed UWD and their respective affiliation to sapwood or heartwood (Fig. 10).

Globally, the pattern between whole trees and trunks from a same stand is similar. This is less obvious at "Bogny-sur-Meuse" where no disc from the upper part of the trunk without heartwood has yet been studied. However, we can expect the same pattern, characterized by sapwood and heartwood in all the diameter classes, and a lower average tree-ring width in sapwood corresponding to the external rings, which is coherent with the growth dynamic of trees [START_REF] Fritts | Tree Rings and Climate[END_REF].

The exploitation of branches only is clearly distinct, with a low growth rate and the absence of heartwood in the case of young branches, as at "Les Cagouillères". If branches are a little older as at "Bogny-sur-Meuse", heartwood is absent in the largest classes of diameter. Lastly, the young vigorous seeded and coppice trees are characterized by a high growth rate in sapwood, while heartwood is absent.

Discussion and application to charcoal assemblages

The dendrological characteristics of each wood stand, discriminating branches, trunks and whole trees, were defined with the help of the dendro-anthracological tools. The dendroanthracological parameters (growth rate, heartwood/sapwood, diameters) were recorded independently of each other and then combined, forming anthraco-types (Fig. 11).

First of all, annual ring width was considered individually. Considering the whole tree and the trunk, ring width distribution is significantly different among stands. However, the distribution between seeded and coppice trees at "Le Bois de l'Or" is not significantly different. Likewise, the distribution between branches at "Les Cagouillères" and "Bogny-sur-Meuse" is not different. In each stand, branches are characterized by a lower growth rate than in the trunk. This observation is in agreement with the study of the variation of annual tree-ring width along the stem marked by a slight increase from the base to the top of the At the scale of a charcoal assemblage, these data can be obtained by measuring each tree ring of each charcoal fragment and averaging them (per fragment). However, their interpretation remains problematic at this stage as they may come from different wood stands, trunks and/or branches, and it is not possible to distinguish them. In addition, it is difficult to interpret growth rate without contemporary, diachronic or modern-day reference standards.

The presence or the absence of heartwood and the proportion of sapwood/heartwood are good indicators of the maturity of the wood. In anthracology, sapwood and heartwood can be distinguished using the proportion of vessels sealed by tylosis [START_REF] Dufraisse | Contribution of tyloses quantification in early wood oak vessels to archaeological charcoal analyses: estimation of a minimum age and influences of physiological and environmental factors[END_REF]. Then each fragment can be affiliated to sapwood or heartwood. However, if although the absence of heartwood reflects the exploitation of young trees, it is difficult to interpret sapwood and heartwood proportions as external and internal sapwood are not differentiated.

Unburnt wood diameter (UWD) was decomposed using ADmodel. In a charcoal assemblage, charcoal diameters are obtained by measuring the charcoal-pith distance. The results indicate a diameter limit between branches and trunks for each wood stand, with almost no overlap, which is in agreement with the literature [START_REF] Deleuze | Estimer le volume total d'un arbre, quelles que soient l'essence, la taille, la sylviculture[END_REF]. However, the exploitation of whole trees is difficult to distinguish from the exploitation of trunks on account of the low branch volume. Consequently, if we hypothesize the exploitation of whole trees, the proportion of branches will be inconspicuous and difficult to distinguish from the exploitation of trunks. In addition, as regards the exploitation of different wood stands, it is R e v i s e d m a n u s c r i p t problematic to differentiate branches and young trunks solely on the basis of diameter distribution.

Thus, growth rate, heartwood/sapwood and wood diameters are three parameters that can be applied to charcoal assemblages. However, their use independently of each other is somewhat limited and sometimes difficult to interpret despite their information potential.

A first combination consisted in associating heartwood/sapwood and diameter parameters in order i) to differentiate the two kinds of sapwood: external sapwood in mature woods, and internal sapwood (absence of heartwood) in young woods ii) to improve the interpretation of the distribution of the decomposed UWD. Specific patterns were recorded according to wood stands and the exploitation modes (whole trees, trunks/branches separately). Young woods (trunk or branches) are characterized by absence of heartwood and small diameter classes, whereas mature wood is characterized by heartwood in small diameter classes and sapwood in the largest ones. In the scope of application to charcoal assemblages, this first combination yields four groups of charcoal fragments depending on their position in the wood: i) small diameter associated with sapwood corresponding to young woods, ii) small diameter associated with heartwood corresponding to the internal part of mature woods, iii) large diameter associated with heartwood corresponding to the middle part of mature woods and iv) large diameter associated with sapwood corresponding to the external part of mature woods.

The association of growth rates with the sapwood/heartwood ratio can provide information about the vigour of wood stands and tree morphology. For example, the proportion of sapwood is higher in trunks from "Les Cagouillères" (high forest) than in the trunk of the dominant tree at "Bogny-sur-Meuse" (coppice-under-standard). However, average sapwood ring-width and sapwood width are higher at "Bogny-sur-Meuse" than in "Les Cagouillères" (Fig. 7). This observation shows that i) for a same age (Bogny: 68 years R e v i s e d m a n u s c r i p t old, Cagouillères: 62 years old), the most vigorous trees have a more extensive sapwood surface [START_REF] Lebourgeois | Les chênes sessile et pédonculé (Quercus petraea Liebl. et Quercus robur L.) dans le réseau RENECOFOR : rythme de croissance radiale, anatomie du bois, de l'aubier et de l'écorce[END_REF] ii) sapwood width is higher in coppice-under-standard than in high forest [START_REF] Dhôte | Profil de la tige et géométrie de l'aubier chez le Chêne sessile (Quercus petraea Liebl.)[END_REF]. Thus, the under-representation of sapwood in the trunk of the tree in "Bogny-sur-Meuse" is probably due to a larger tree diameter, 33 cm as opposed to 20.75 cm.

The third combination consists in associating tree-ring width and diameters (distribution of the decomposed unburnt wood diameters). For an application to charcoal assemblage, each tree ring is associated with a charcoal-pith distance, then to a diameter class and finally an average tree-ring width is calculated for each diameter class. Radial growth trends appear to be preserved keeping with dendrological radial growth. An original pattern marked by a low growth rate along the smallest diameter classes followed by a higher rate in the largest diameter classes may be a characteristic of the exploitation of whole trees. However, as it is often the case in dendroecology, one pattern may correspond to several scenarios. Here for example, a partial clearing of the wood stand could lead to a comparable growth trend. Thus interpretations have to be associated with the results established by other disciplines. In addition, an initial distinction between young trunks (coppice) and young branches becomes possible as their growth rate and growth trend differ (high rate and upward trend for coppice, low rate and downward trend for branches).

However, no further distinction is visible between coppice and seeded trees at "Le Bois de l'Or". In fact, only the proportion of earlywood is only significant when radius is up to 1,6 cm [START_REF] Girardclos | Improving identification of coppiced and seeded tress in past woodland management by comparing growth and wood anatomy of living sessile oaks (Quercus petraea)[END_REF].

The last combination is the association of all the dendro-anthracological parameters:

heartwood/sapwood, tree-ring width and diameters. Besides the distinction between young and mature woods based on the association between heartwood/sapwood and diameters, it becomes possible to discriminate branches from trunks among young woods. Indeed, branches are characterized by sapwood, a low growth rate and rather downward growth, R e v i s e d m a n u s c r i p t whereas young trunks (coppice and seeded trees) are characterized by sapwood, a high growth rate and rather upward growth.

Specific patterns appear depending on the stand and the potential types of wood exploitation (trunks and/or branches). Thus, anthracological types could be defined forming an interpretative grid which can act as a useful key for the interpretation of archaeological charcoal assemblages. Moreover, the recorded dendrological information is not the same depending on the position in the tree. For example, the information recorded in tree-ring width depends on the position of the charcoal fragment; tree-ring width and growth trend in young woods may be a good indicator of the origin of the wood in the tree (crown or bole)

whereas stand characteristics (stand density according to strong or low competition between trees) are more perceptible in trunk, i.e. large diameter of mature wood (Marguerie and Hunot, 2007).

These results entail a new approach to anthracological material. Charcoal fragments have to be sorted according to their position in the stem cross-section and in the tree. For that purpose, an anthracological key based on dendro-anthracological parameters and forming anthraco-groups is proposed (Fig. 12).

Each oak fragment is characterized by a charcoal-pith distance, sapwood/heartwood affiliation and annual tree-ring width. The first division at the threshold of a diameter of 7 cm is often used by foresters and corresponds to the diameter limit between branches and trunks in deciduous oak forest. Concerning tree-ring width, charcoal fragments with regular and irregular tree-ring width series are taken into account separately. For example in northern France, according to V. Bernard (1998, p. 96), narrow rings are less than 0.7 mm/year and large rings are between 0.7 and 3 mm/year for deciduous oak. Very large rings, up to 3 mm, can be also considered (i.e. 12 groups).

The use of this anthracological key enables us to sort charcoal fragments according to their position in the tree. Then, measurements of each batch can be processed separately.

R e v i s e d m a n u s c r i p t

To close, it is important to make several remarks concerning the dendroanthracological tools and their applications.

i)

The application of dendro-anthracological tools requires a minimum transversal plane size of about 4 mm x 4 mm and at least one whole growthring. The optimal number of fragments to analyze is around 100 per sampling unit (structure, layer, etc. according to the problematic).

ii)

The choice of the diameter classes, chosen to be compatible with standards used in dendrometrical plans by foresters, seems to be relevant. However, a charcoal fragment can be classified in a class or the other when the value of the charcoal-pith distance is close to a limit but usually the interpretation is not affected.

iii) Given that it exists a boundary between the diameters of trunks and branches within a wood stand and that the part of the trunk located in the crown presents the same dendrological characteristics as a branch, it is more relevant and accurate for charcoal analysis to distinguish bole from crown than trunk from branch when considering oak and probably more generally Angiospermea. However, by Gymnospermea, the trunk can be easily followed until the apex with a clear separation of the branch material. Thus this distinction bole/crown or trunk/branch has to be adapted according to the architecture of the tree. In addition, variations in growth rates are often considered and interpreted in terms of environmental (light, soil or climate)

and human factors (clearings or woodland management). However, we have to keep in mind that they can also result from a change in exploitation techniques (whole trees, trunks, branches). The use of the anthracological key may allow for the classification of the growth-ring width signal and thus bring more accurate information.

R e v i s e d

m a n u s c r i p t iv) Shrinkage during charcoalification leads to lower tree-ring width. This process is not consistent, depending on sapwood/heartwood and charcoal-pith distance. A preliminary study on shrinkage offers promising results in order to propose correction factors (Garcia [START_REF] Martinez | Correction factors on archaeological wood diameter estimation[END_REF].

v)

The relative frequency of the different taxa in charcoal assemblages is representative of the used biomass (wood volume). In the same way, the use of the dendro-anthracological parameters is based on the assumption that charcoal fragments represent the different parts of trees proportionally to their volume, with their dendrological characteristics (growth, ratio sapwood/heartwood, diameter). That is why the ADmodel is based on wood volume (and not on the number of fragments). However, we stress that, this model cannot reconstruct the quantity of initially burnt wood.

vi)

As for the interpretation of tree-ring width (Marguerie, 1992, p. 72;Marguerie and Hunot, 2007), several conditions are required to interpret the dendroanthracological parameters: charcoal assemblages must come from numerous trees, tree-ring series are randomly distributed in the transversal sections of charcoal fragments, ring series must be numerous enough and with a homogeneous width, acquisitions areas are subjected to the same climatic influences and the geological substratum must be homogeneous.

Conclusion

In line with the work of D. Marguerie (Marguerie, 1992, Marguerie and Hunot, 2007[START_REF] Marguerie | Short Tree ring series: the study materials of the dendro-anthracologist[END_REF], Marguerie et al., 2010), combining charcoal identification and dendrological examination, the aim of this study was to improve methods to assess whether it was pertinent to develop quantitative measurements, such as estimating pith-charcoal distance, and whether the combination of dendro-anthracological parameters provides new information on wood exploitation and forest management.

R e v i s e d m a n u s c r i p t

Besides the measurement of tree-ring width, the present study is based on the development of three anthracological tools consisting in i) measuring charcoal-pith distance,

ii) discriminating heartwood/sapwood and iii) modelling dendrological data to make them compatible with charcoal analysis. Three dendro-anthracological parameters i.e. growth ring width, charcoal-pith distances and heartwood/sapwood, modelled with ADmodel, were tested on modern-day oak wood stands chosen with respect to historical woodland practices: a coppice-under-standard, an old coppice undergoing conversion to high forest and a young stand formed by a mixture of seeded and coppice trees. For a more realistic representation of dendrological data according to anthracological constraints, different levels of analysis were considered: the whole tree, and trunks and branches separately, allowing us to further consider various modes of wood exploitation.

The dendro-anthracological parameters taken into account independently of each other provide interesting results but rather limited interpretation, especially for tree-ring width or sapwood/heartwood. Indeed the dendrological information cannot be interpreted in the same way depending on its position in the tree. For example, growth conditions and thus paleo-environmental information are essentially recorded in the trunk. In contrast, the combination of the dendro-anthracological parameters highlights specific patterns between organs, stands and regeneration modes, and enables us to establish an anthraco-typology forming an interpretative grid. A major result here is the identification of the position of the charcoal fragment belonging to young woods or internal/middle/external parts of mature woods and the distinction between branches and young trunks when associated with the tree-ring width. These results lead to the establishment of an anthracological key aiming to sort charcoal fragments into anthraco-groups according to their position in the tree and their growth rate.

Finally, these results offer new opportunities for the interpretation of archaeological charcoal assemblages as well as the development of new dendro-anthracological tools adapted to species other than deciduous oak.

R e v i s e d

m a n u s c r i p t also grateful to two anonymous reviewers for their valuable remarks and suggestions which helped to improve this publication.

Captions

Table 1 Dendrological characteristics of each wood stand and sampled trees.

  undergoes both mass loss and charcoal fragmentation. Consequently, the distribution of the charcoal-pith distances does not indicate unburnt wood diameter.

  3.3. Combination of dendro-anthracological parameters 3.3.1. Decomposed UWD versus tree-ring width R e v i s e d m a n u s c r i p t

  3.3.3. Tree-ring width versus diameter classes versus sapwood/heartwood

  s c r i p t trunk and a strong decreasing in the upper part of the trunk (in the crown). These results are similar to those of[START_REF] Dhôte | Profil de la tige et géométrie de l'aubier chez le Chêne sessile (Quercus petraea Liebl.)[END_REF], based on 82 Quercus petraea distributed in five regions in France. Consequently, growth conditions are mainly recorded in the trunk and branches should be avoided for palaeo-environmental reconstruction. This result fits with the method of D.Marguerie and J.-Y. Hunot (2007) whose the principle is to keep only tree-ring width measurements based on charcoal with large charcoal-pith distance.

Fig. 1 .

 1 Fig. 1. Dendro-anthracological tools.

Fig. 2 .

 2 Fig. 2. General analytical protocol developed in the ANR DENDRAC program. Experimental charcoal assemblages are not considered in this paper.

Fig. 4 .

 4 Fig.4. Main dendrological characteristics of the wood stands: modes of regeneration, average age, average diameter at breast height, relative proportion of trunks and branches (expressed according to volume), distribution of the diameters of trunks and branches (each log and its volume was attributed to an unburnt wood diameter class), average growth rate and growth trends (tree-ring width measurements were taken on each disc at a height of 1.30 m, along 5 radii and averaged).

Fig. 5 .

 5 Fig. 5. Annual ring-width was averaged from 5 radii in each disc. (a) Distribution of annual ring-width (maximum and minimum values, 1st and 3rd quartiles and median) considering whole trees (white), trunks (brown) and branches (green). (b) Distribution of the annual ringwidth along the trunks and in branches.

Fig. 6 .

 6 Fig.6. Relative proportion of heartwood (brown) and sapwood (yellow) for each stand, considering whole trees, trunks and branches. The volume proportion of sapwood and heartwood was estimated for each disc, then each log and tree, and averaged for each wood stand. The average number of sapwood tree-rings, average sapwood width (cm) and average sapwood growth rate (mm/year) are indicated in boxes when heartwood is present.

Fig. 8 .

 8 Fig. 8. Dendrological data (simple line): average tree-ring width calculated by cambial age. Modelled anthracological data (solid line): average tree-ring width calculated for each diameter class.

Fig. 10 .

 10 Fig. 10. Average tree-ring width according to the diameter classes (decomposed UWD) and their respective affiliation to sapwood (yellow) or heartwood (brown).

Fig. 11 .Fig. 12 .

 1112 Fig. 11. Anthraco-typology for deciduous oak: an interpretative grid for archaeological charcoal assemblages.

Table 2a

 2a Analyzed wood discs and dendrological characteristics: Bogny-sur-Meuse; Les Cagouillères Table2bAnalyzed wood discs and dendrological characteristics: Bois de l'Or.

Acknowledgment

The authors thank the Agence Nationale de la Recherche (ANR JCJC 200101 DENDRAC, dir. A. Dufraisse) for financing this study and Louise Byrne for English correction. They are