22 IAWAIAWA Journal Journal 36 (1), 36 (1),2015: 2015 22–35 WOOD ANATOMY OF TETRACLINIS ARTICULATA FROM ITS NATURAL DISTRIBUTION AREA IN SOUTHEAST SPAIN Luis G. Esteban1,*, Paloma de Palacios1, Alberto García-Iruela1, Elena Román- Jordán1, Francisco G. Fernández1, Sandra Díaz Fernández1 and María Conde2 1Universidad Politécnica de Madrid, Escuela Técnica Superior de Ingenieros de Montes, Ciudad Universitaria s/n, 28040 Madrid, Spain 2CIFOR-INIA, Departamento de Productos Forestales, Carretera de la Coruña Km 7.5, 28040 Madrid, Spain *Corresponding author: e-mail: [email protected] ABSTRACT For the first time, the wood anatomy ofTetraclinis articulata (Vahl) Masters has been studied using representative samples from its natural distribution area in Spain, in Sierra de Cartagena (Region of Murcia). Mature wood was collected from five individuals representative of the forest stand and their anatomy was compared with other genera of the Cupressaceae. Axial tracheids without helical thickenings, low homogeneous rays, cupressoid pits and the absence of normal axial resin canals are characteristic features of this monotypic genus, as they are of most other Cupressaceae genera. An obvious warty layer separates this wood from the genera sharing its territory (Cupressus and Juniperus) and its semi-spherical, slightly anastomosed warts distinguish it from other, geographi- cally distant genera (Actinostrobus and Callitris). The presence of traumatic axial resin canals is reported for the first time and supports the occurrence of this feature outside the Pinaceae. The wood anatomical diversity within the clade comprising Tetraclinis, Microbiota and Platycladus, as reconstructed by molecular analysis, is discussed. Keywords: Cupressaceae,warty layer, traumatic resin canals. INTRODUCTION Tetraclinis Mast. is a monotypic genus of the Cupressaceae, T. articulata (Vahl) Masters, comprising trees that can reach a height of 15 metres, although due to human action they do not normally exceed 10–12 metres and are frequently limited to bushy or frutescent shrubs (Ceballos & Ruiz de la Torre 1979) (Fig. 1). The natural distribution of the spe- cies is restricted to north Africa, southeast Spain and the island of Malta. In Africa it covers an area of nearly 800,000 hectares: 600,000 in Morocco, 160,000 in Algeria and 22,000 in Tunisia. Reports of the species in Libya and the Ahaggar Mas- sif, in central Sahara, appear to be confused with cypresses (Charco 1999). In Spain it occurs scattered over four areas (Ibáñez et al. 1989) (Fig. 2): 1) to the northeast of Portman, between Peña del Águila (387 m) and Monte de las Cenizas (337 m), 310 © International Association of Wood Anatomists, 2015 DOI 10.1163/22941932-00000082 Published by Koninklijke Brill NV, Leiden Downloaded from Brill.com10/07/2021 12:26:39AM via free access Esteban et al. – Tetraclinis articulata from southeast Spain 23 1 3 2 Figure 1–3. Tetraclinis articulata. – 1: Small Tetraclinis articulata tree in Monte de las Cenizas, Region of Murcia (Spain). – 2: Geographical distribution in the natural area of the Region of Murcia (Spain). – 3: Sapwood and heartwood markedly different. Downloaded from Brill.com10/07/2021 12:26:39AM via free access 24 IAWA Journal 36 (1), 2015 catalogued individuals occupy an area of around 325 hectares; 2) in Cerro de la Cam- pana, in Valle de Escombreras, there are just 50 spontaneous individuals that have regrown from old stumps and 30 more planted trees growing among Aleppo pines (Pinus halepensis); 3) the largest population, in Algameca Grande, west of Cartagena, has more than 345 individuals catalogued in just 100 hectares, some of them more than 11 metres in height; 4) north of Peña del Águila, 58 individuals have been inven- toried, most of them regrown from old stumps, but no trees are more than five metres heigh (Ibáñez et al. 1989). Intense overgrazing and the traditional use of the timber for firewood and char- coal production have brought the species to the brink of extinction. In the first half of the 20th century only a few examples of regrowth from old stumps were visible and regeneration was completely lacking (Jiménez Munuera 1903; Huguet del Villar 1938; Rigual & Esteve 1953; Templado 1974). The decline of livestock grazing, the end of charcoal production, and the protection given to the species have allowed some recovery (Ibáñez et al. 1989), although progress is very slow due to the difficulty of regeneration from seeds and the semi-arid conditions in the region (Costa Tenorio et al. 1990). The rootwood and burrwood of T. articulata, with their characteristic black spots on a reddish brown background, have commonly been used in cabinetmaking. This continues to be an important industry in the ancient city of Mogador (now Essaouira), in Morocco. The wood anatomy of Tetraclinis has been described by various researchers, although their descriptions were based on a limited number of samples, mostly of unknown provenance. Strangely, Castellarnau (1883), in his study of the Spanish conifers, did not describe Tetraclinis and Peraza (1964) was the first to make a complete description of the species in the Spanish stands, using a single sample from Murcia. Saint-Laurent (1926) described Tetraclinis under the name of Callitris articulata (Vahl) Murbeck using a sample from Algeria. The provenance and number of the samples used by Peirce (1937), Phillips (1948), Huber and Rouschal (1954), Greguss (1955) and Jacquiot (1955) are not known. Tetraclinis has since been described by Schweingruber (1990) and Heinz (2004). Like Peraza (1964), Esteban and Guindeo (1988) and Esteban et al. (1996, 2002) described a sample from Murcia. The scarce information about the earlier samples described makes it impossible to determine whether the wood was mature or juvenile or came from branches or trunks and therefore neither the qualitative nor the quantitative features can be compared. A complete description of this wood with samples collected in its natural regions of provenance using mature trunk wood will complement molecular phylogeny studies of this species. Gadek et al. (2000), Little (2006) and Yang et al. (2012) recovered a clade formed by Microbiota Kom., Platycladus Spach and Tetraclinis. The aims of this study were to: 1) describe the wood anatomy of Tetraclinis using trees collected in natural forests of the species in southeast Spain and compare its anatomy with other genera of Cupressaceae; 2) compare the wood anatomy of the species in the clade established by molecular phylogeny. Downloaded from Brill.com10/07/2021 12:26:39AM via free access Esteban et al. – Tetraclinis articulata from southeast Spain 25 MATERIAL AND METHODS The material used for this study was collected in the natural forests of Tetraclinis in the Regional Park of Calblanque, Monte de las Cenizas y Peña del Águila, in the province of Murcia (Spain). The five trees felled for the study were all adult individuals over 70 years old repre- sentative of the forest. A disc was extracted 50 cm from the ground and samples were obtained from mature wood in each disc for the observations. Microscope slides were prepared following the usual methods of softening, section- ing, staining and mounting. The descriptions were made in accordance with the IAWA Committee (2004). The samples were observed using light microscopy (Leica DM2500 with a DFC 420 camera) with image processing software IM50 v.5 release 220 and scanning electron microscopy (SEM) mod. JEOL JSM-6380. Observation was conducted on samples without staining and stained with safranin for the lignified cell walls and with Sudan 4 for the resin (Jane 1970). SEM samples were prepared following the method described by Heady and Evans (2000). The biometry was conducted on three slides from each tree, on mature wood from a basal disc obtained 50 cm from the ground, on ring 30 counted from the exterior, using the WinCell image analysis programme. From each slide the following measurements were taken: 25 measurements of axial tracheid length and diameter, ray height (mm and number of cells), and number of pits per cross field in the earlywood; 50 measurements of tracheid pit diameter and largest and smallest cross-field pit diameter in the earlywood; and five measurements of the number of rays per square millimetre measured in five different areas of one square millimetre on the tangential section. Tracheid length was measured following Ladell’s indirect method (Ladell 1959). The study of the most frequent ray height values (µm and number of cells) and number of pits per cross field was performed using frequency histograms, and therefore the most frequent value does not equal the mean value. Statistical calculations were made with the Statgraphics Centurion Ver. 15.2 programme, for a 95% significance level. RESULTS Wood description Transverse section. Sapwood and heartwood markedly different: sapwood with shades of yellow and heartwood reddish brown (Fig. 3). Aromatic with an intense, pleasant odour. Growth rings distinct, with gradual transition from earlywood to latewood (Fig. 4). Tracheids generally rectangular in outline, but also hexagonal in earlywood (Fig. 5). Intercellular spaces absent. On the growth ring boundary tracheid pits are commonly found in the tangential walls (Fig. 6). Axial parenchyma in diffuse arrangement (Fig. 5), marginal in the last row of latewood and tangentially zonate with dark coloured cell content (Fig. 7). Normal resin canals absent, although traumatic axial resin canals were observed in two of the five trees; numerous subsidiary parenchyma cells were associated with traumatic canals (Fig. 8). Downloaded from Brill.com10/07/2021 12:26:39AM via free access 26 IAWA Journal 36 (1), 2015 4 5 6 7 8 Figure 4–8. Tetraclinis articulata. – 4: Growth rings distinct, with gradual transition. – 5: Tracheids with rectangular and hexagonal outline and axial parenchyma diffuse. – 6: Tracheid pits in the tangential walls near the growth ring boundary. – 7: Axial parenchyma diffuse and tan- gentially zonate. – 8: Traumatic axial resin canals: a: transverse section, b: radial section.