IAWA Journal, Vol. 29 (2), 2008: 143–152

VARIABILITY IN APICAL ELONGATION OF FIBRES IN SERICEUS

Joanna Jura-Morawiec1*, Wiesław Włoch2,1, Paweł Kojs2,1 and Muhammad Iqbal3

SUMMARY Morphological variability in wood fibres ofLonchocarpus sericeus (Poir.) Kunth ex DC. (Leguminosae), a tropical hardwood with double- storeyed cambium, was examined in thin tangential and transverse sec- tions as well as in macerations of wood tissue. Occurrence of character- istic protrusions (lateral expansions) was detected on the extended part of the main fibre body. Distance between the adjacent protrusions cor- responded to the height of a storey (horizontal tier) of the cambial initials. Rays were shorter in height than the neighbouring fusiform initials and therefore unable to reach the boundary of the storey. This situation facili- tated the lateral expansion of the adjoining fibres during their apical elon- gation by intrusive growth. The presence of the characteristic protrusions on the fibre body thus indicated that the given fibre was associated with a double-storeyed cambium having rays shorter than the length of fusiform initials. The ultimate shape of fibres was thus correlated to the height of storeys and the height and width of rays. Key words: Wood fibre morphology, intrusive cell growth, vascular cam- bium.

INTRODUCTION

Lonchocarpus sericeus is a leguminous tree, native to and Southern America. Wood anatomy of the Lonchocarpus has been described by Metcalfe and Chalk (1950), Wagenführ and Schreiber (1974), Chudnoff (1984), Richter and Dallwitz (2000) and Gasson et al. (2004). Lonchocarpus sericeus possesses diffuse-porous xylem with paratracheal confluent parenchyma. In transverse sections, bands of light-stained axial parenchyma surrounding the vessels alternate with bands of dark-stained fibres. Like many other tree growing in the canopy and emergent layers of the tropical rainforests, this species is characterized by a double-storeyed arrangement of cambium, where both fusiform cells and rays are arranged in storeyed fashion. The pattern of cambial cell arrangement is also reflected in the arrangement of wood elements (Harris

1) Botanical Garden, Centre for Biological Diversity Conservation of the Polish Academy of Sciences, Prawdziwka 2, 02-973 Warsaw 76, Poland. 2) Department of Biosystematics, University of Opole, Oleska 22, 40-052 Opole, Poland. 3) Department of Botany, Jamia Hamdard (Hamdard University), New Delhi, 110 062, India. *) Corresponding author [E-mail: [email protected]]. Associate Editor: Nigel Chaffey

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1989; Romberger et al. 1993; Carlquist 2001). In the wood of L. sericeus, which consists of vessel members, fibres and axial parenchyma cells in addition to rays, fibres are the only non-storeyed axial elements. Information on the shape of xylem fibres is scanty. Fibres are known to grow by apical intrusive growth (Fahn 1990; Larson 1994; Evert 2006). The rate and duration of cell expansion and secondary wall formation affect the overall cell size and wall thickness of xylem elements (Ridoutt & Sands 1993, 1994). Final dimensions of wood fibres are also affected by the state of maturation of other xylem elements in the vicinity (Honjo et al. 2006). The arrangement and rearrangement of cambial cells influence the pattern of fibre organization in the grain of the wood (Harris 1973, 1989; Hejnowicz & Zagórska-Marek 1974; Włoch et al. 2002; Kojs et al. 2004). In some species, like soyauxii Taub., Ceiba pentandra Gaertn., Dalbergia nigra Fr.All., even the fibres reflect the storeyed arrangement of their precursor cambial cells, thus forming a perfect storeyed structure in the wood (Metcalfe & Chalk 1950; Wagenführ & Schreiber 1974; Richter & Dallwitz 2000; Carlquist 2001). However, no information is available in the literature to determine whether the arrangement of cambial cells also influences the shape of the fibres. The present report on the wood fibres of L. sericeus demonstrates their peculiar shapes and examines if the structure of the cambium has a bearing on the shape of wood fibres in this species.

MATERIALS AND METHODS Sampling and slide preparation The material used in this study, i.e., a piece of wood of Lonchocarpus sericeus (Poir.) Kunth ex DC., was obtained from the collection of wood samples in the Botanical Gar- den, Centre for Biological Diversity Conservation of the Polish Academy of Sciences, Warsaw. Microscopic study of wood fibres was made both from sections and macerations. Small samples of wood (2 × 0.7 × 0.5 mm), deaerated in boiling water and then fixed in 2.5 % glutaraldehyde, were dehydrated in an acetone series and embedded in Epon (Meek 1976). The embedded samples were cut into 3-μm transverse and tangential sec- tions, using a glass knife and microtome, glued to the slides with Hauptʼs adhesive, stained with PAS and toluidine blue and mounted in Euparal, as described by Włoch and Połap (1994). The samples were examined by light microscope. For maceration, a piece of wood (approximately 10 × 5 × 4 cm) was cut into a few tangential slices of less than 0.5 mm thickness. The slices, taken in test tubes containing the Franklin (1945) mixture (1:1, glacial acetic acid : 30% hydrogen peroxide), were placed in boiling water for a few hours for macerating the tissue. The macerated wood elements were separated by shaking the solution vigorously. The solution was then centrifuged and the liquid decanted. The remaining pellet of macerated wood elements was washed with water, centrifuged again, dehydrated in ethylene and placed in isopropanol. Drops of the resultant suspension were placed on slides, stained with safranin T and mounted in Euparal, as described by Włoch (1976).

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Measurement of wood elements The length of axial parenchyma cells, height of a storey, and height and width of rays in parenchyma bands were measured from tangential longitudinal sections using the calibrated ocular micrometre scale in the eyepiece of a light microscope. Macerated material was used for measuring fibre length, distance between two adjacent protrusions of fibres, and width of fibre protrusions of various orders. The first order protrusions were those formed at the ends of the main fibre body, around the boundaries of storeys. During apical growth of fibres the second and third order protrusions could be formed if the growing fibre tip reached the next boundaries of the storeys. The length of the fusiform axial parenchyma cells has been used as an indicator of the length of fusiform initials (Süss 1967). For calculating mean fibre length, 200 unbroken and randomly selected fibres were measured. For the rest of the parameters the mean is based on 50 measurements each. For measuring the mean height of a storey, microphotographs of tangential sections were obtained with a microscope. Boundaries of the storeys were marked by horizontal lines in randomly selected areas of parenchyma bands on these photographs and the distance between the lines was measured.

OBSERVATIONS

The storeyed structure in the wood of Lonchocarpus sericeus, as seen in TLS, involved axial parenchyma cells, rays and vessel members (Fig. 1a). The heights of vessel members, ray bodies and 2–4-celled strands of axial parenchyma were mutually comparable (Fig. 1b–e). Rays were enclosed within the horizontal storeys (Fig. 1a, c). Nonetheless, fibres did not form a storeyed structure. Comparison of sections passing through a parenchyma band (Fig. 2a) and a fibre band (Fig. 2b), revealed that the fibre lumen was bigger at places near the boundaries of the storeys (Fig. 2b, 3a). Normally the tips of rays, as seen in tangential view, were not extended exactly up to the boundary of the storey it belonged to; this became evident also from transverse sections obtained from areas where storeys ended (Fig. 3c, 4b). In transverse sections, ray cells were not visible at the boundary line of the cell storeys (3c); only fibres with a distinctly large lumen diameter were present. In these fibres with larger lumen, pits were visible (Fig. 3c). On the contrary, at the mid-height of storeys, the fibres between rays were distinctly thinner with smaller lumen (Fig. 2b, 3b, 4a). It was interesting to confirm whether the characteristic differences in fibre-lumen diameter, as visible in thin sections, were detectable also in the macerated wood. It was found that the middle part of a macerated fibre cell, corresponding to the length of the cambial fusiform initial from which the given fibre had been derived, formed the main fibre body; this portion of the fibre was usually wider than the rest of the fibre body which was an outcome of subsequent cell elongation by apical intrusive growth (Fig. 5). Apical elongation of the main body of a fibre seemed to have completed after a prolonged course of intrusive growth. It was usual to find more than two protrusions (lateral expansions) on a fibre. The mean distance between two adjacent protrusions, i.e. 182 μm, was comparable with the mean height of a storey (Table 1). The first order

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Figure 1. Paratracheal parenchyma of Lonchocarpus sericeus wood. – a: Tangential section with visible storeyed structure. – b–e: Macerated cells of a storey; b: fusiform cell and 2-celled paren- chyma strands; c: axial parenchyma cells and rays enclosed within the storey as seen in the radial plane; d: macerated broad vessel member; e: narrow vessel member. — p = axial parenchyma cells; r = rays enclosed within the storey; v = vessel members.— Scale bar = 100 μm. protrusions were wider than the second and third order protrusions (Fig. 5). Figure 6 demonstrates the morphological variety of these protrusions. Fibres differed widely in length, some of them being several times longer than the precursor initial cells (Fig. 5; Table 1). Thin extended ends of the fibre cells exhibited a varied degree of intrusive growth on the cell tips. Some of the fibres experienced a monopolar intrusive growth; one of the cell ends in such cases was somewhat roundish, showing no indication of having grown intrusively (Fig. 6c). However, it was not pos- sible in the macerated material to identify which of the two fibre ends was proximal (lower) or distal (upper). A comparison of the length of fibres with that of the fusiform axial parenchyma cells in L. sericeus showed that the longest fibres grew about ten times the size of the cambial fusiform initial from which they had originated. The shortest fibres were about twice as long as the fusiform initials. Most of the fibres were nearly six times longer

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Figure 2. Tangential sections passing through a confluent parenchyma band a( ) and a fibre band b( ) of Lonchocarpus sericeus wood. Ray cells are wider (circular) in section a than in section b. In section b large lumina of fibres can be seen in the boundary region of a storey (white ar- rows). — Scale bar = 100 μm.

Figure 3. Tangential (a) and transverse (b & c) sections passing through a fibre band. – b: Transverse section obtained from mid-height of the storey, and c: section through the boundary region of the storey. Dashed lines 1 and 2 in section a indicate the level from where sections b and c have been obtained. Arrows point to the location of pits on the fibre wall. — r = rays; f = fibres. — Scale bar = 100 μm.

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Figure 4. Transverse sections of Lon- chocarpus sericeus xylem. – a: Section obtained from mid-height of a storey presents clearly visible rays, whereas section b, obtained from the boundary of a storey, is marked with the absence of rays. — p = axial parenchyma cells; r = rays; f = fibres. — Scale bar = 100 μm.

Figure 5. Xylem fibres after maceration. The central wider part of the fibre body corre- sponds to the body of the fusiform cambial cell from which the fibre has been derived (marked with the bracket on the left side). Distance between horizontal broken lines corresponds to the mean height of storeys. The 1st, 2nd and 3rd order protrusions on the fibres are indicated by arrows with corresponding numbers 1, 2 and 3, respectively. An asterisk indicates the monopolar intrusive growth of a fibre.

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a b c d e f g

h i

Figure 6. Partial views of the bodies of macerated fibres with visible protrusions a( –e) and zoom-in of chosen protrusions (f–i). — Scale bar = 50 μm.

Table 1. Dimensions of axial parenchyma cells, rays and fibres in the wood ofLonchocarpus sericeus. The mean for fibre length and other parameters is based on 200 and 50 measure- ments, respectively.

Variables Range (μm) Mean (μm) Axial parenchyma Length 177–250 209 Width 14–28 21 Rays in parenchyma band Height 110–195 159 Width 15–60 43 Fibres Length 480–1920 1294 Width of the main fibre body 8–24 16 Distance between the protrusions 155–220 182 Width of the protrusions First order 12–50 19 Second order 7–17 11 Third order 5–10 9 Height of a storey 165–215 180

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DISCUSSION

The length of wood fibres depends on the length of their precursor cambial initials and the extent of intrusive growth they undergo during differentiation (Bailey 1920; Ghouse & Iqbal 1981; Evert 2006). Fibres experience a diverse degree of extracambial apical elongation by intrusive growth. In general, they are considerably longer than the cambial initials they have originated from (Iqbal & Ghouse 1983; Ajmal et al. 1985). The majority of the fibres ofLonchocarpus sericeus wood are nearly six times longer than the fusiform cambial cells, as observed in the macerated preparations. Elongation of differentiating fibres is normally restricted to their tips (Chalk et al. 1955). The growing tips penetrate between the adjoining cells where the intercellular pectic layer is not yet lignified (Wenham & Cusick 1975; Iqbal 1994; Larson 1994). The extracambial cell elongation may occur at one or both of the ends of a xylem fibre (Süss 1967). The present study demonstrates that wood fibres ofL. sericeus normally undergo intrusive elongation at both ends; rarely they have it restricted to one end only. Fibre morphology provides important clues about the pattern of fibre elongation. The extent of intrusive growth and the ultimate shape of intrusively grown fibre tips also depend on the degree of maturation and the outlines of the surrounding cells. The vessels influence the length of the adjoining fibres by their transverse enlargement during differentiation. According to Honjo et al. (2006), a comparison of the shape of fibres adjacent to and distant from the vessels, has shown that fibres adjacent to vessels (generally called vessel-dependent fibres) are pointed with tapering ends, whereas those away from vessels (vessel-independent fibres) are spindle-shaped. Patel (1995) has reported L-shaped, crooked or V-shaped fibres in the wood of Sophora macrophylla and S. prostrata. The present study has revealed that some xylem fibres ofL. sericeus grow intrusively not only in the axial direction but, on the boundaries of storeys, also in a lateral (tangential) direction, thus resulting in the characteristic protrusions (lateral expansions) of peculiar shapes. The absence of ray cells on the boundary line of the storeys allows the elongating fibre ends to expand not only axially but in tangential direction also. The number, shape and size of protrusions visible on a fibre depend on a) fibre length, b) storey height, and c) the height and width of rays. Short fibres normally have only two protrusions whereas long fibres, which overgrow the length of the cambial cell several times, may have more than two. The magnitude of the distance between two adjacent protrusions depends on the height of a storey of axial cells. The shape of protrusions is affected by the height and width of the rays. Situations where rays are shorter than the height of a storey provide room for development of the lateral protuberances. The protrusions developed in conjunction with multiseriate rays (Braun 1955; Chalk 1955) are quite distinct. In L. sericeus, where ray height is distinctly smaller than the length of the cambial cells, lateral expansion of fibres is convenient because of the absence of ray cells on

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boundaries of the storeys. Some other leguminous of tropical rainforests with a double-storeyed cambium, such as Dalbergia latifolia Roxb. (Ghouse & Yunus 1974), Amphimas ferrugineus Pierre ex Pellegr. (Richter & Dallwitz 2000) and Bergeronia sericea Micheli (Gasson et al. 2004) also possess rays that are shorter in height than the adjoining fusiform initials and, therefore, do not touch the boundary line of the storey in which they are located. On the contrary, in species such as laurentii De Wild., Dialium platysepalum Baker and Mansonia altissima (A. Chev.) A. Chev. (Richter & Dallwitz 2000) and also in L., brevicaudata (Vatke) Dunn, Lonchocarpus latifolius Kunth and Piscidia piscipula Sarg. (Gasson et al. 2004), the rays of the double-storeyed cambium are multiseriate and predominantly as high as the neighbouring fusiform initials. However, these rays are significantly narrower at their tips, thus creating a possibility for the formation of protrusions during the apical intrusive growth of the fibres. Wood fibres of all these species grow several times over the length of the precursor fusiform initials. It will be interesting to re-examine the shape of fibres in all these species with a view to confirm the presence of the protrusions on their apical extensions and establish the relationship, if any, between the protrusions on fibres and the height and width of adjacent rays. In conclusion, the occurrence of wood fibres with visible lateral protrusions almost at equal distances – which correspond to the height of a storey of the cambial initials – might be a characteristic feature of species having a double-storeyed cambial structure with rays shorter than the fusiform initials, as seen in TLS. In those wood samples where only fibres survived, for example in fossil wood, the shape of wood fibres may be of great taxonomic significance. However, these assumptions need to be substantiated by further evidence.

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