Botanical Journal of the Linnean Society (1995), 118: 107-121. With 22 figures Secondary growth and wood histology of Welwitschia SHERWIN CARLQUIST F. L. S.1 AND DAVID A. GO WANS , Santa Barbara Botanic Garden, 1212 Mission Canyon Road, Santa Barbara CA 93105, USA. « Received December 1994, accepted for publication June 1995 New observations regarding secondary growth and vascular tissues in Welwitschia are based on roots (used because of the longitudinal orientation of most vascular tissues). Welwitschia has successive cambia that produce xylem and phloem. Contrary to previous reports, the cambia produce secondary phloem with phloem rays and secondary xylem that has readily definable xylem rays and axial parenchyma. Internal sculpturing (grooves associated with pit apertures) of tracheary elements is figured with SEM; observations confirm die descriptions of Bierhorst (1960). Tori and vesturing are apparently absent on pits of tracheary elements. Abundance of gelatinous secondary walls may be related to water economy to a minor extent. A feature recendy reported for Ephedra wood is newly reported for xylem rays, phloem rays and conjunctive tissue of Welwitschia: presence of minute calcium oxalate crystals lining intercellular spaces. This feature is apparendy limited to the two genera. The ontogenetic origin of vascular strands added by secondary activity can be traced to phelloderm. In this feature, Welwitschia differs from Gnetum, in which the ultimate origin of new cambia can be traced to cortical tissues. T. ISJJ5 The Linnean Society of London ADDITIONAL KEY WORDS:—cambial variants - Ephedra - Gnetum - successive cambia - wood histology. CONTENTS i Introduction 107 Material and methods 108 Anatomical results 109 Plan of root and nature of vascular strands . 109 Tracheary element structure 115 Cambial activity and origin of vascular strands . 116 Conclusions 118 Acknowledgement 120 References 120 INTRODUCTION Much attention has been paid by various workers to the vegetative anatomy of Welwitschia mirabilis Hook., especially concerning vascular anatomy (for a 'Address for correspondence: 4539 Via Huerto, Santa Barbara, CA 93110, U.S.A. 107 0024-4074/95/060107+15 $08.00/0 © 1995 The Linnean Society of London 108 S. CARLQUIST AND D. A. GOWANS review, see Martens, 1971). However, a number of questions have not been answered, a number of interpretations have not been clarified, and even a few features remain to be discovered. Lack of suitable material does not appear to have delayed studies, because cultivated specimens of Welwitschia are numerous and have offered suitable material for decades. Those interested in wood anatomy may have bypassed Welwitschia because of its apparent lack of wood: students of wood anatomy tend to investigate woodier species of plants. In addition, the texture of Welwitschia stems and roots (hard cells scattered in a background of soft tissues) and the sinuous course of vascular strands make study of axial vasculature of Welwitschia difficult. The method of sectioning employed in this paper minimizes problems posed by the texture of the Welwitschia axis. The problem posed by the sinuosity of vascular strands has been minimized by use of root materials only; the vascular tissue of the root is predominantly longitudinally orientated. The fact that the cambia in the vascular strands of Welwitschia are apparentiy short-lived, with the consequent lack of massive accumulations of secondary xylem, has led workers to assume that Welwitschia lacks features of wood histology. Martens (1971: 255), says: "The separation and the very peculiar course of bundles of Welwitschia (hypocotyl, crown, fertile branches) do not permit a useful comparison with this plan [to the wood of Ephedra and Gnetum]". On the contrary, the products of the cambia in Welwitschia are entirely comparable with those of Ephedra and Gnetum. Although root tissue has been selected in the present study, the principles observed surely apply to products of cambia in vascular strands elsewhere in the plant body. The study of Bierhorst (1960) contributes many details on protoxylem, metaxylem and secondary xylem tracheids of Welwitschia. However, as Martens (1971) noted, Bierhorst's drawings suggest presence of tori on pits of tracheary elements whereas other workers claimed tori to be absent in Welwitschia and Gnetum (Martens, 1971), but present in Ephedra (Carlquist 1989, 1992). In fact, tori are present in pits of tracheary elements of some species of Gnetum (Carlquist & Robinson, 1995). Because the present study uses scanning electron microscopy (SEM), which was not available to Bierhorst (1960), confirmation of Bierhorst's results was one of our goals. The meristematic action that results in lateral addition of vascular strands to the axis of Welwitschia has never been clearly established. Martens (1971) asked whether phelloderm is ultimately the source of this meristematic action or whether a special meristem (in either stem or root) is present. Bertrand (1874: 13) claimed never to have found a meristem that ultimately would lead to vascular tissue. Strasburger (1872: 375), De Bary (1884: 616), and Eichler (1889: 126) thought that such a meristem must exist, but did not cite evidence for one. The relatively non-sinuous course of most vascular tissue in the root offers maximal potential clarity for demonstrating the nature of meristematic activity leading to addition of lateral vascular strands. MATERIAL AND METHODS The study of ontogenetic phenomena, which must be traced to thin walled cells, is complicated in Welwitschia by the abundance of tough fibrous structures scattered through a parenchymatous background tissue. Sectioning WELWTTSCH1A WOOD ANATOMY 109 on a sliding microtome is not feasible because of this texture. Sectioning on a rotary microtome is only a little easier, even given softening techniques. A softening technique incorporating ethylene diamine (Carlquist, 1982) proved successful. Transverse, tangential, and radial sections were prepared from the main root of plants of four ages. The youngest and next youngest ('older seedling' in figure captions) of these were cultivated in the greenhouse at Pomona College. The largest root (Fig. 1) was from a specimen collected in Namibia by Dr Lyman Benson about 1965. He dried the plant in its form as collected; this specimen had several cm of cylindrical root below the hypocotyl, and that portion of the root was studied. This dried specimen was kept in storage at Pomona College. The next to largest root was collected in the wild in Namibia (Carlquist 8071, RSA). Roots of all except the largest plant were preserved in formalin acetic alcohol. The dried root of the largest plant was boiled in water and stored in 50% aqueous ethanol prior to sectioning. Sections for light microscopy were stained with a safranin-fast green combination. Some paraffin sections were mounted on SEM aluminum stubs using the same techniques as with glass slides. Prior to examination with an SEM, paraffin was removed from the sections with changes of xylene, and the sections were sputter-coated with gold. Macerations were prepared with Jeffrey's Fluid and stained with safranin. Terminology follows the IAWA Committee on Nomenclature (1964). The large fusiform cells with gelatinous walls in which crystals are embedded are here termed fibrosclereids. They are homologous with the astrosclereids in leaves and bracts of Welwitschia, which Rodin termed 'crystalliferous sclereids' (1958, 1963). The fibrosclereids of the root were termed 'spicular cells' by Pearson (1929) and Parameswaran & Liese (1979) but 'sclerites' by Martens (1971). The term 'conjunctive tissue' is used here for parenchyma added to the root by lateral meristematic action, in accordance with the use of the term in dicotyledons with successive cambia (see Carlquist 1988). The term 'successive cambia' is also applicable to Welwitschia, because cambial activity characterizes the vascular strands of the hypocotyl and root. The term 'secondary xylem' is used to denote xylem produced by action of cambia in each of the vascular strands. ANATOMICAL RESULTS Plan of root and nature of vascular strands Early vascularization of the root was described by Sykes (1910), Rodin (1953) and Butler, Bornman & Evert (1973). The accounts of a one-year seedling by these authors do not illustrate addition of bundles by lateral meristematic action, but the account of Bower (1881) does illustrate some of these additional vascular strands in a root. Hooker (1863) and De Bary (1884) examined older roots and reported five to eight concentric circles of vascular strands. All but the innermost cycle must be attributed to action of lateral meristematic action. One must conclude that Hooker and De Bary had access to relatively small plants, no larger than the collection Carlquist 8071, in which the root is 4 cm in diameter. In the root shown in Figure 1, more than eight circles of vascular strands are present. However, one 110 S. CARLQUIST AND D. A. GOWANS Figures 1-4 Fig. 1. Transverse section (T.S.) of root from large mature Welwitschia plant. Fig. 2, 3. T.S. root of older seedling. Fig. 2. Periphery of root, phellem at top. showing several series of bundles; phloem fibres abundant. Fig. 3. Higher magnification, to show a recently initiated vascular strand (below) and fibrosclereids (blackish) formed from phelloderm cells. Fig. 4. T.S. root from Carlquist 8071, showing thick phellem and the outermost vascular strands. Fig. 1, life size; Figs 2, 4, to scale above Fig. 2 (divisions