Charcoal anatomy of Brazilian species . I . Anacardiaceae

Anthracological studies are firmly advancing in the tropics during the last decades. The theoretical and methodological bases of the discipline are well established. Yet, there is a strong demand for comparative reference material, seeking for an improvement in the precision of taxonomic determination, both in palaeoecological and palaeoethnobotanical studies and to help preventing illegal charcoal production. This work presents descriptions of charcoal anatomy of eleven Anacardiaceae species from six genera native to Brazil (Anacardium occidentale, Anacardium parvifolium, Astronium graveolens, Astronium lecointei, Lithrea molleoides, Schinus terebenthifolius, Spondias mombin, Spondias purpurea, Spondias tuberosa, Tapirira guianensis, and Tapirira obtusa). They are characterized by diffuse-porous wood, vessels solitary and in multiples, tyloses and spiral thickenings sometimes present; simple perforation plates, alternate intervessel pits, rounded vessel-ray pits with much reduced borders to apparently simple; parenchyma paratracheal scanty to vasicentric; heterocellular rays, some with radial canals and crystals; septate fibres with simple pits. These results are quite similar to previous wood anatomical descriptions of the same species or genera. Yet, charcoal identification is more effective when unknown samples are compared to charred extant equivalents, instead of to wood slides.


INTRODUCTION
Charcoal, frequent in archaeological or geological contexts, is an important proxy to the reconstruction of past vegetation and climate, as well as to palaeoethnobotanical interpretations.
Anthracology, the identification of charcoal based on wood anatomical characters, is a discipline primarily developed for palaeovegetation studies related to archaeology, palaeoecology, geology, and others (Chabal 1997, Scheel-Ybert 2004a, Scott and Damblon 2010).It also applies for archaeobotany and for the identification of contemporary unknown charcoal, in which case it can be used for charcoal control.THAÍS A.P. GONÇALVES and RITA SCHEEL-YBERT At present, Brazil is the world's largest producer of charcoal, but a great part of this production still comes from illegal logging of native species (FAO 2014).Almost 90% of charcoal's production goes to the iron and steel industry (Brasil 2012) -around 30-35% of the total output of this production comes from native species (IBGE 2010, ABRAF 2013).Frequently, this illegal production is associated with criminal acts against human beings, often including children and workers under slave-like conditions (Carneiro 2008, IOS 2011a, b).Besides, the production of illegal charcoal is responsible for the emission of c. 5 tons of carbon dioxide caused by improper removal of trees and released gases from carbonization (ABRAF 2009).Controlling charcoal production is therefore very important.
Up to recently, it was virtually impossible for government agents to verify if charcoal loads corresponded indeed to legal extraction of reforested woods or to illegal exploitation of native vegetation.However, this can be done through anthracological analysis (Gonçalves and Scheel-Ybert 2012).Usually, species from exotic genera of distinctive wood and charcoal anatomy, such as Corymbia and Eucalyptus, are cultivated for charcoal production (Gonçalves et al. 2014).Although some agroforestry managed native species can also be legally used (Friederichs et al. 2015), most native species are still illegally exploited.An official document (DOF) containing information about the charcoal origin and the species used to produce it must accompany each charcoal load.Verifying the exactitude of this information depends on the direct verification of the load by inspection agents (Gonçalves and Scheel-Ybert 2012).Hence the importance of recognizing the charcoal anatomy of native species.
In Mediterranean and temperate regions, areas of lower plant diversity, wood anatomy is better known and charcoal identification is usually based on the consultation of atlases (e.g.Greguss 1959, Schweingruber 1990, Vernet et al. 2001).However, despite numerous studies (e.g.Détienne and Jacquet 1983, Mainieri and Chimelo 1989, Barros and Callado 1997, Barros et al. 2001, 2008, Sonsin et al. 2014), wood anatomy of Brazilian species is still incompletely known, especially for extra-Amazonian and non-commercial timber species, and there are heretofore only unpublished studies on charcoal anatomy of extant species (e.g.Gonçalves 2006, 2010, Albuquerque 2012, Pinto 2013).
There is, therefore, a strong demand for anthracological studies of tropical species aiming at charcoal identification, both in the academic fields of archaeobotany, palaeoecology and others, and concerning Brazilian governmental institutions such as Serviço Florestal Brasileiro, IBAMA (Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis), Polícia Militar Ambiental, and INEA (Instituto Estatual do Ambiente), due to the economic importance of charcoal commerce and especially environmental conservation.With the purpose of improving the anatomical knowledge on tropical species and assisting microscopic charcoal identification, we investigate the wood anatomy of carbonized samples from species of the rich Brazilian flora.In this paper, we present results for native species from the Anacardiaceae family.This family is widely distributed in the tropical zone, including about 700 species from 70 genera all around the world (Souza and Lorenzi 2008), with few representatives in temperate regions (Gupta and Agarwal 2008).It comprises trees, shrubs, and rarely woody climbers and herbs (Souza and Lorenzi 2008).Many species are of a considerable economic value, producing edible fruits, chemical compounds used in industry (gums, resins, tannins, and dyes) or medicine, and quality timbers of commercial importance (Record andHess 1943, Paula andAlves 1997).In spite of the impossibility to cover all Brazilian species comprehensively, we expect that this initiative will provide useful information to researchers of different expertise, including forest sciences, environmental conservation, wood technology, wood anatomy, palaeoecology, and palaeoethnobotany.
Charcoal samples were manually split according to the fundamental wood sections (transversal, longitudinal tangential, and longitudinal radial sections) and examined under a reflected light brightfield/ darkfield microscope.Descriptions followed the recommended procedures and terminology by the International Association of Wood Anatomist (IAWA Committee 1989), except for intervessel pits size, for which the internal horizontal diameter of pit apertures was measured (Scheel-Ybert and Gonçalves in press).Anthracologists conventioned to measure the pit aperture (but measuring also the pit chamber diameter when possible) because the carbonization process induces wood cell walls homogenization, frequently making pit chambers indistinguishable.The pit aperture, however, is always distinct.
For quantitative measurements, arithmetic means and amplitude (minimum and maximum values) are given.Micrographs were taken using a JEOL 6300F SEM at University Montpellier II (UM-II, France), a JEOL 5310 at the Institut of Biophysics of the Federal University of Rio de Janeiro (UFRJ, Brazil), and a Leica S440 SEM at the Center of Mineral Technology (CETEM, Brazil).Some authors argued that charcoal is sufficiently conductive to be suitable for SEM studies without any special coating or preparation (Blankenhorn et al. 1972, Cutter et al. 1980).However, we have obtained better results by previously sputter-coating specimens with gold or platinum.

RESULTS
Wood anatomical descriptions for each genus follow.The most important anatomical features of the studied species are presented in Table I.

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THAÍS A.P. GONÇALVES and RITA SCHEEL-YBERT A. occidentale; perforation plates simple with oblique or horizontal end walls; intervessel pits alternate, 7(4-9)µm; vessel-ray pits larger than intervessel pits, with much reduced borders to apparently simple, rounded.Axial parenchyma: scanty paratracheal or vasicentric, sometimes confluent, in 2-4-celled strands.Rays: 1-2-seriate in A. occidentale, exclusively 1-seriate in A. parvifolium; 10(9-13) rays/mm; body ray cells procumbent with one row of upright and square marginal cells in A. occidentale and body cells procumbent with variable thickness and 1-3 rows of upright and square marginal cells in A. parvifolium.Fibres: septate with bordered pits and very thin walls.Secretory elements: absent.Mineral inclusions: absent.Growth rings: boundaries indistinct or absent.Vessels: wood diffuse-porous; solitary (55%), in multiples of 2 (35%), and 3 (10%); tangential diameter 135(60-250)µm; 15(5-30) vessels/mm²; tyloses absent in S. tuberosa, present in the other species; perforation plates simple with oblique and horizontal end walls; intervessel pits alternate, 7(5-10)µm; vessel-ray pits larger than intervessel pits, with much reduced borders Growth rings: boundaries marked by thick-walled fibres in late wood in T. obtusa, indistinct or absent in T. guianensis.Vessels: wood diffuse-porous; solitary (50%) and in multiples of 2 (27%), 3 (18%), and 4 (5%); tangential diameter 90(60-140)µm; 20(10-30) vessels/mm²; tyloses usually present; perforation    Even if vessels tangential diameter and frequency may sometimes differ, it is not possible to distinguish the studied species through charcoal anatomy.Genera, however, or groups of species, can be distinguished by considering a combination of features such as radial canals, helical thickenings, rays width, vessels diameter, and vessels frequency, as proposed in the dichotomous key below.Features such as diameter of intervessel pit apertures and rays frequency do not vary significantly, while the presence of tyloses may depend on the part of the stem analyzed, and that of crystals on environmental aspects.Those are not reliable diagnostic characters, but can be useful to ascertain the identification.is due to methodological requirements.Charcoal analysis in reflected-light microscopy is technically easier and much less time-consuming than traditional techniques of wood analysis involving slides preparation.
Besides, we highlight the unequivocal advantage of comparing similar materials.Charcoal identification is much more effective when unknown samples are compared to charred extant equivalents, instead of to wood slides.This happens not only because of easiness of observation, but also in reason of minor anatomical changes due to carbonization.As a rule, wood structure is very well preserved after carbonization, but some modifications may occur, such as: homogenization of the cell walls; tangential and radial shrinkage (which may induce small changes in vessels diameter and in the size of axial and radial parenchyma cells); and disappearance or modification in cell contents (Prior and Gasson 1993, Kim and Hanna 2006, Gonçalves et al. 2012).
In other cases, however, a few features differed from these previous descriptions, especially regarding vessels tangential diameter and frequency -e.g., in Astronium graveolens vessels are 140% smaller and 80% more frequent, and in Astronium lecontei 75% smaller and 60% more frequent than it was reported by Dechamps (1979); in Schinus terebinthifolius vessels are 60% more frequent than reported by Tavares (1970); in Spondias mombin vessels are 10% smaller than reported by León (2003); in Spondias purpurea vessels are 100% smaller and more frequent than reported by Barajas-Morales and Gómez (1989); in Tapirira guianensis vessels are 60% more frequent than reported by Dechamps (1979).
Intervessel pits are generally smaller than reported in the literature, but the data reported for noncarbonized wood refer to the diameter of chamber, while in this work we have measured pit apertures.
Quantitative variation, particularly lower vessels tangential diameter, and higher vessels frequency, can be explained by the carbonization process, but intraspecific variability and/or ecological factors can also be implied.

CONCLUSIONS
Anatomical description of charcoal aims to improve taxonomical identification and to provide better means to anthracological studies in the tropics.THAÍS A.P. GONÇALVES and RITA SCHEEL-YBERT Descriptions of tropical charcoal anatomy are of utmost importance in different fields of knowledge, such as archaeobotany, palaeoecology, wood technology, and environmental conservation.They are most important as a comparative material for the identification of unknown charcoal pieces from archaeological or sedimentary contexts, which may lead to palaeoenvironmental and palaeoethnobotanical interpretations.
The use of anthracology to control illegal charcoal commerce also shows a strong potential in Brazil.There are as yet few studies with charcoal intended to reduce the indiscriminate use of native species, but they are generally focused in Eucalyptus, which is the most frequently species legally used for charcoal production (e.g.Botrel et al. 2007, Gonçalves et al. 2014).This is the first attempt to directly describe the anatomy of charcoal samples aiming to contribute to the construction of a database that might eventually contribute to prevent the continuity of deforestation and help the sustainability of the charcoal supply chain.The use of anthracology to control illegal charcoal commerce is, therefore, a particularly important application of the present work.
Additionally, this work may contribute to a better knowledge of wood anatomy of native tropical species, which is still incompletely known.Indeed, while this is the first time that charcoal anatomy descriptions are presented for all the studied species, two species are here described for the first time both in charcoal as in conventional wood anatomy: Anacardium parvifolium Ducke and Spondias tuberosa Arruda.