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PREAMBLE

This guide was written as part of teaching (lectures and practical work) on charcoal analysis given as part of the Anatomical and taxonomic diversity in archaeobotany module (QPB22) in the Quaternary, Prehistory and Bioarchaeology course of the MNHN's BEE master's degree.

It aims to present the main criteria to be observed under the microscope in order to determine charcoal from reference collections and archaeological samples. The guide is aimed at students who are not biologists but who nevertheless have a basic knowledge of the development of woody tissues (for the MNHN, follow the QPB09, biology refresher course).

INTRODUCTION

Wood combines a longitudinal and transversal system. To observe wood anatomy, anthracologists make manual cuts in wood/charcoal fragment in three cutting sections (transverse, longitudinal tangential, longitudinal radial). Each plane provides information on a given anatomical element and allows the identification of the taxon1 . The identification is performed under a reflection light microscope with magnifications ranging from X50 to X1000. The anthracologist/xylologist uses identification atlases2 corresponding to the region in which he works and a reference collection of modern charcoal.

This document is an introduction to wood identification. The taxonomic identification range most often reaches the genus (e.g., Salix sp., Acacia sp., Acer sp., Quercus sp.), sometimes the grouping of species (e.g., Prunus type avium/padus; Quercus deciduous or evergreen), or, more rarely, the species. Sometimes, only the subfamily can be proposed (e.g., Maloideae/Maleae).

The guide is organised by major taxonomic group (Gymnosperms/Angiosperms), by type of element (main and secondary) and then by type of tissue/cell.

GYMNOSPERMS

Gymnosperm wood has a homogeneous appearance, because the wood is formed essentially of tracheids which form the main mass of the wood and ensure both the support and the conduction of raw sap. In addition to tracheids, all gymnosperms are composed of horizontal parenchyma (rays). There are no vessels in gymnosperms (figure 1).

TRACHEIDS

Tracheids constitute 90-95% of gymnosperm wood cells. The thickness of the tracheid walls is variable. The transition between initial and final wood of the same year can be abrupt (clearly visible to the naked eye) or not very visible. This is due to the marked contrast between tracheids formed early in the growing season (spring, thin wall, wide cavity/lumen) and those formed late in the season (summer, thick to very thick wall, narrow cavity/lumen).

The transition between initial and final wood (abrupt or continuous) is observed in transversal section (figure 2).

Radial longitudinal section Tangentiel longitudinal section

Transversal section e.g.,

Tracheids

Resin canal Pits Horizontal parenchyma (rays)

Cross-field

Tracheids communicate laterally with each other by simple areolated pits that allow the circulation of sap from one tracheid cell to another (figure 3). Pits in crossing fields [START_REF][END_REF] (ray/tracheid intersections) allow the sap circulation from tracheids to rays (parenchyma cells). [START_REF][END_REF] In gymnosperms, this is the intersection of tracheids with the ray (parenchyma) cells; in angiosperms, the appropriate term is "ray/vessel intersection".

Ring limit

Initial wood

High number of marginal areolated pits

Description

Faible nombre de ponctuations aréolées aux extrémités.

Figure 2 On the left, Pinus sylvestris transversal section (TS), transition is continuous, on the right, Abies alba TS, transition is abrupt.

Final wood Cross-fields pits are important characters to determine gymnosperm wood genus but difficult to look at, especially in archaeological charcoal/wood.

Ring limit

Their type (dimension, number per crossing field, shape) are visible in high magnification (>x500) in radial longitudinal section.

HORIZONTAL PARENCHYMA

The horizontal parenchyma, or ray, is involved in the storage of nutrient reserves in gymnosperms.

Diagnostic elements of the rays are mainly observable on the tangential longitudinal section. Ray width is generally of one cell (uniseriate rays), sometimes of two cells (biseriate rays) in tangential section (figure 5). Their height can vary from 1 to 40 cells, sometimes more. Homogeneous rays are only composed of parenchyma cells. Heterogeneous rays have additional elements as transverse tracheids or resin ducts (Schweingruber 1990).

SECONDARY ELEMENTS

Other elements are not systematically present in gymnosperms wood (figure 6). Their absence/presence can be of great help to identify the genus.

-Presence/absence of axial resin ducts, visible in transverse, longitudinal tangential and radial sections.

-Presence/absence of spiral thickenings that are helical type reinforcements of the internal wall of the tracheids. They are visible in both longitudinal sections.

ANGIOSPERMS

The woody of angiosperms is heterogeneous, complex and very diversified depending on the species. It is composed of four main types of cells: vessels, fibers, horizontal parenchyma (ray) and longitudinal parenchyma.

Vessels

These hollow cells serve to conduct raw sap from the roots up to the leaves. The vessels occupy 5 to 30% of the wood and sometimes contain various deposits such as starch, tannin, resins, crystals and thylls.

The three elements to observe are:

(a) Size, arrangement, and number of vessels 5 , in transversal section allows a first classification into two groups: porous woods and diffuse to semi-porous (or semi-ring) woods. This is the first entry in identification keys: porous wood and semi-ring to diffuse wood (figure 7). 5 We also talk about pores. (b) Perforations at each vessel elements extremity ensure conduction (figure 8). These perforations, visible in both longitudinal sections, can be simple or scalariform (the number of bars is a diagnostic criterion).

Longitudinale tangential section

Radial longitudinal section

(c) Deux types of punctuations can be observed:

Inter-vessel punctuations promote exchanges between joined vessel elements. Some of them may be diagnostic. For example, the punctations of birch (Betula) are very small and numerous. Moreover, their arrangement -alternating or opposite -can also guide the identification (Figure 9).

Porous wood: vessels in the initial wood are much larger in diameter than those in the final wood (ex: Quercus, Fagus, Fraxinus).

Diffuse wood: distribution of vessels in the annual rings is uniform and vessels have the same diameter (ex: Betula, Populus, Acer).

Semi-ring porous wood: vessels have the same diameter throughout the annual ring but are more abundant in the initial wood (ex: Prunus avium). Other types of punctations can be identified in the crossing fields (ray/vessel intersection).

They favor exchanges between vessels and horizontal parenchyma cells. Some of them can be diagnostic of a species. For example, in willow (Salix) and poplar (Populus), tcrossing field punctations are large (figure 10). (a) Cells can be uni-seriate (one cell wide), bi-tri-seriate (2 or 3 cells wide) and multi-seriate (more than 3 cells wide). The number of cells composing the ray, in its height, has to be observed in longitudinal sections (figure 11). (b) Furthermore, when the parenchyma cells are of the same size/shape over the entire height of the ray, the ray is said to be "homogeneous". On the other hand, when the cells are of different sizes, it is called a "heterogeneous" ray. To describe accurately, we count the number of cells lying (i.e. procumbent) and/or upright and/or square in radial longitudinal section (figure 12, figure 13).

Figure 12 Les rayons de Prunus sp. sont multisériés (3-4

cellules de large). En coupe tangentielle (à gauche) et radiale (à droite), les cellules présentent des formes très variables (rayon hétérogène).

FIBERS AND LONGITUDINAL PARENCHYMA

Fibers provide support. They are bundles of resistant cells, arranged in the longitudinal direction, which ensure the rigidity and mechanical resistance of the wood. The libriform fibers are dead elements of fusiform shape, arranged longitudinally, in the direction of the tree axis. They only participate in the support of the tree. They do not have any punctuations. The tracheid fibers have a double function of support and conduction (punctuations present), but are not present in all species (ex : present in Castanea). Longitudinal parenchyma are living wood cells. These cells can be:

-Independent of the vessels (apotracheal parenchyma).

-Associated with vessels (paratracheal parenchyma). The distribution of longitudinal parenchyma cells can be observed in cross section (figure 14, figure 15). When observed under the microscope, it may be difficult to distinguish the fibers from the parenchyma cells. It is then, most often, diffuse apotracheal parenchyma.

SECONDARY ELEMENTS

Helical-type reinforcements of the inner vessel wall, i.e. spiral thickenings, may be present. They are visible in tangential and radial longitudinal sections.

TO SUM UP

The main features to observe to identify Gymnosperms:

• Transition between initial and final wood (transversal/cross section).

• Nature of the rays (homogeneous or heterogeneous) (longitudinal sections).

• Presence, or not, of resin canals (transverse and longitudinal sections).

• Punctuations inside the crossing-fields (radial longitudinal section).

The main features to observe to identify Gymnosperms:

• Arrangement of vessels: diffuse/porous or semi-porous wood (cross section).

• Arrangement of longitudinal parenchyma cells: paratracheal or apotracheal (transverse section). • Nature of the rays (width/height/homogeneous or heterogeneous) (longitudinal sections). • Perforations in vessels (simple, scalariform) (longitudinal sections).

• Presence or not of spiral thickenings in vessels (longitudinal sections).

See also the observation guide below (6.2).

USEFUL REFERENCES

BIBLIOGRAPHIC OR INTERNET RESOURCES

• IDENTIFICATION ATLASES 1 -Benkova, V., Schweingruber, F.H. ( 2004 For gymnosperms, the first entry in the key is the size and shape of the dots in the crossing fields visible at high magnification in radial longitudinal section (p.5). For angiosperms, it is the porosity of the wood in cross-section (p.8-9) followed by the width of the rays in tangential longitudinal section (p.10).

During the practical exercise, you will have to (1) describe the three sections of a modern gymnosperm and angiosperm charcoal using the observation guide (6.3), (2) by yourself, identify a mystery taxon (angiosperm) using the simplified key (6.1) and the atlases provided on the Plateau d'archéobotanique. Rayons clairement multisériés avec nombre de rangées de cellules carrées et dressées marginales.

LES GYMNOSPERMES

Rayons avec souvent 1-2-( 4) rangées de cellules carrées et dressées marginales.

Pores peu fréquents, en ligne radiale de 2 à 10 pores.

Voir 3-5 cellules (largeur rayon), légèrement hétérogène.

Répartition dentritique des pores.

Pores espacés, solitaires ou en file radiale de 2 ou 3 pores.

Voir Pores solitaires ou en file radiale de deux à quelques cellules, ou en groupe. Chez certaines espèces, rayons avec plus de 5 cellules de large.

BOIS DIFFUS A SEMI-POREUX (suite)

Bois poreux avec beaucoup de cernes très étroits qui peuvent paraître similaires à du bois diffus, voir bois poreux.

Petits pores, en files radiales longues, souvent rayons de plus de 4mm de haut.

Rayons très larges, perforations simples et scalariformes.

Comme Fagus , rayons très larges plus nombreux, ponctuations intervasculaires opposées, en lignes horizontales.

Pores groupés, le plus souvent à orientation tangentielle.

Voir bois poreux.

Rayons hétérogènes, perforations scalariformes à nombreux barreaux.

Pores très larges (voir bois poreux).

Voir bois poreux.

OBSERVATION GUIDE

The purpose is to guide you in the observation of your first charcoal samples (of temperate or arid species). For each major type of wood (homoxylate/heteroxylate) are presented:

-the main questions to ask for each observation plan.

-the main cells/tissues to observe.

-the magnification7 to be used for the observations. Please note that the list is not exhaustive. You can add to it or skip observation points depending on your experience. Use this guide, as well as IAWA features (Wheeler, Baas, Gasson 1989) to help you!

Gymnosperm

In cross/transversal section:

1-Is the transition between the initial and final wood continuous or abrupt (X50 à x100)? 2-Are there any resin canals (X50)? 3-Observe differences in thickness cells of initial and final wood at high magnification (X200 à X500). 4-Observe horizontal parenchyma cells at high magnification (X200 à X500).

Longitudinal tangential section: 5-Observe pits in the tracheids at low and high magnification (X50 à X500). 6-What is the number of cells in the height of rays (take into account 5 rays, X200 à X500)? 7-What is the number of cells in the width of rays (take into account 5 rays, X200 à X500)? 8-Do you observe any particular cells inside rays (X500)? Are rays homogeneous or heterogeneous?

Longitudinal radial section: 9-Locate an area where rays and tracheids are crossing (X50), search for pits in the crossing fields (X500 à X1000). 10-What is the shape and the size of the crossing fields pits (X1000)? How many pits/parenchyma cells; are pits big or small?

Angiosperm

In cross/transversal section: 1-Observe the arrangement of vessels in a growth ring. Does the wood appear porous or semiporous to diffuse (X 50)? 2-Do the vessels have a particular organization (groups, solitary, in flame, etc…)? 3-Do the rays appear wide, thin, or uniseriate (X50 to X100)? It has to be confirmed in longitudinal section. 4-Do you notice the presence of longitudinal parenchyma cells? If so, do they have a particular organization in relation to the vessels, or independently of the vessels (X100 to X500)? 5-Observe or try to locate parenchyma cells at high magnification (X200 to X500).

Figure 1

 1 Figure 1 Main cells observed on the three observation planes of a homoxylate wood (gymnosperm).

Figure 3

 3 Figure 3 Transverse and longitudinal views of tracheids in initial and final wood (unknown source).

Figure 4 -

 4 Figure 4-Main type of cross-field pits(Schweingruber 1990).

Figure 5

 5 Figure 5 Homogeneous rays and uniseriate of Abies alba (fir).

Figure 6

 6 Figure 6 Secondary elements in gymnosperms. On the left, in tangential section of Pinus sylvestris (pine), resin ducts are visible in final wood; on the right, spiral thickenings are visible in longitudinal sections in Taxus baccata (yew).

Figure 7

 7 Figure 7 Different wood porosities (drawing: AS).

Figure 8

 8 Figure 8 Schema of a vessel in longitudinal with main elements to look at (drawing: AS).

FigureFigure 9

 9 Figure 10 Cross-field punctuations, Populus (poplar).

  Woody rays are composed of parenchyma cells. Rays are mainly observed in longitudinal tangential and radial sections. The most diagnostic identification features are (a) width/size of the rays and (b) cells arrangement.

Figure 11

 11 Figure 11 Les différents types de rayons chez les angiospermes.

Figure 13

 13 Figure 13 Les rayons d'Acer (érable) sont trisériés dans leur majorité (coupe longitudinale tangentielle, à gauche) et présentent uniquement des rangées de cellules couchées (rayon homogène) en coupe longitudinale radiale (à droite).

Figure 14

 14 Figure 14 Disposition et catégories du parenchyme longitudinal (points noirs) en coupe transversale.

Figure 15

 15 Figure 15 left, cross-section of Quercus (oak), porous wood, presence of banded lines of apotracheal parenchyma in the final wood; right, longitudinal section of ash (Fraxinus), circumvascular paratracheal parenchyma cells are stuck to the vessels.

  ) Russian Wood Anatomy. Paul Haupt, Bern. 2 -Fahn, A., Werker, E., Baas, P. (1986) Wood Anatomy and Identification of Trees and Shrubs of Israel and Adjacent Regions. Israel Academy of Sciences ans Humanities, Jérusalem. 3 -Hather, J. (2000) The identification of northern European woods. Left coast Press, London. 4 -Jacquiot C. (1955) Atlas d'anatomie des bois de conifères. 2 vol. Centre Technique du bois. 5 -Jacquiot C. (1976) Atlas d'anatomie des bois des angiospermes. 2 vol. Centre Technique du bois.

Any unit (genus, family, species, subspecies, etc.) of hierarchical classifications of living things.

Book that gathers the main woody species that can potentially be identified in a given region. The description and photographs of the three observation sections (transverse, longitudinal tangential and radial) are presented. In general, a identification key based on various discriminating anatomical criteria allows a preliminary identification of the species. Atlases are not available for all regions of the world, particularly tropical areas.

-Neumann, K., Schoch, W., Détienne, P.,Schweingruber, F.H., Richter, H. (2000) Woods of the Sahara and the Sahel. Paul Haupt, Berne.

-Parsa Pajouh, D.,Schweingruber, F.H. (1993): Atlas des bois du nord de l'Iran. Publication de l'Université de Téhéran, Tehran. 2 nde édition.

[START_REF] Schweingruber | Anatomie europaïscher Hölzer -Anatomy of european woods[END_REF] Anatomie europaïscher Hölzer -Anatomy of european woods. Eidgenössische Forschungsanstalt für Wald, Schnee und Landschaft, Birmensdorf. Haupt, Bern und Stuttgart.

The following simplified key is dedicated to the support of practical work. It is not translated in English. Nevertheless, the majority of the comparison atlases (see section 5.1) are in English.

Since oculars have a magnification of X10, a magnification of X50 corresponds to an objective of X5. From magnification X20 (or X50), the microscope must be switched to white background.