lAW A Bulletin n.s., Vol. 5 (1), 1984 13

ANATOMY OF THE SECONDARY PHLOEM IN WINTERACEAE

by

Katherine Esau and Vernon I. Cheadle Department of Biological Sciences, University of California, Santa Barbara, California 93106, U.S.A.

Summary The secondary phloem of nine species in five of representatives of different families were ex- genera of Winteraceae was examined with regard plored with regard to one specific feature of to features that could serve for taxonomic and the tissue or of the sieve element. Thus, we phylogenetic evaluation of the family. The spe- have reported on the kinds of cell divisions oc- cies examined were as follows: Bubbia pauci- curring in the cells contributed by the vascular flora, B. semecarpoides, Drimys lanceolata, D. cambium to the phloem and on the effect of winteri, Exospermum stipitatum, Pseudo win tera these divisions on cell arrangement in the ma- axillaris, Zygogynum baillonii, Z. bicolor, and ture tissue and on the final size and form of the Z. vinkii. The nine species showed the following sieve elements (Esau & Cheadle, 1955). That common characteristics: I) origin from non- paper led to the formulation of the concept of storied vascular cambium with long fusiform secondary partitioning of phloem initials, a initials; 2) ray system consisting of high multi- phenomenon that results in sieve elements seriate and high uniseriate rays; 3) occurrence shorter than the fusiform cambial initials. Of of secondary partitioning in the differentiating the two papers concerned specifically with the phloem so that the sieve elements are much sieve element, one dealt with variations of cell shorter than the tracheids; 4) lack of sharp dif- wall thickening (Esau & Cheadle, 1958), the ferentiation between lateral sieve areas and other with the size of sieve area pores and their those of the sieve plates; 5) predominance of contents (Esau & Cheadle, 1959). Both papers compound sieve plates; 6) short companion provided material for discussions of the rela- cells, often single in a given sieve element; 7) tion between structural and functional speciali- phloem parenchyma cells in strands; 8) lack of sations of the cell as a conduit in translocation. specialised fibres (bast fibres) in the secondary We have surveyed the phloem of vascular plants phloem; 9) presence of nondispersing protein in general with respect to the possible evolutio- body in the sieve element protoplast. Features nary changes in the tissue and its component numbered I, 2, 4-6 are considered to be indi- cells (Esau et aI., 1953). The phloem of the di- cations of low evolutionary level. The signifi- cotyledons alone was also reviewed regarding cance of the other three features (3, 7-9) re- cell types and cell arrangement in more and less quires further evaluation. Among these three is advanced families (Esau, 1979). the secondary partitioning the occurrence of In this paper we describe certain features of which seems to imply that in some taxa the the secondary phloem of 9 species of Wintera- well known sequence of evolutionary shortening ceae (Table I): the kinds of cells and their ar- of cambial initials and their derivatives may be rangement in the tissue, the evidence for second- accelerated on the phloem side. ary partitioning in the differentiation of sieve Key words: Winteraceae phloem, primitive di- elements, the nature and distribution of sieve cotyledons, phloem anatomy, sieve elements. areas, the morphology of the companion cells, and the nature and localisation of sclerenchyma. Introduction This paper resumes our comparative studies Materials and Methods of the phloem of dicotyledons, chiefly of the The species used are listed in Table I together secondary phloem. We have used two approach- with collection data and information on age es in our previous surveys of this tissue. In one, and state of growth of stems. Our collections the phloem of members of one small family are represented by vouchers in the University (Calycanthaceae; Cheadle & Esau, 1958) or of Herbarium at Santa Barbara, California. Four one species of a larger family (Liriodendron tu- additional specimens were kindly supplied in lipifera, Magnoliaceae; Cheadle & Esau, 1964) preserved state by Dr. R.F. Thorne (two collec- were studied in as much detail as the collected tions) and Dr. Sherwin CarJquist (two collec- material provided. In the other, large numbers tions), both at the Rancho Santa Ana Botanic

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Table I. Infonnation about plants used in study.

Collection Name of plant Collection data Age of stem State of years growth 2 3 4 5

CC579 Bubbia paucif/ora (E.G. Baker) Carlquist 15588' unknown2 active Dandy New Caledonia CA227 Bubbia semeearpoides (F. Muell.) VIC 10/13/59 Australia 2 active B.L. Burtt CA390 Drimys laneeoiata (Poiret) Baill. VIC 2/9/60 Australia 15+ active CCto Ibid., cult. VIC 8/21/50 California 8-9 active CC36 Ibid., cult. VIC 12/18/50 California 6+ dormant CC74 Ibid., cult. VIC 4/24/51 California 5-6 active CC5 Drimys winteri J.R. & G. Forst. var. VIC 8/11/50 California 12+ active ehilensiss (DC.) A. Gray cult.

CC89 Ibid., cult. VIC 5/29/51 California 4-5 active CC578 Exospermum stipitatum (Baill.) Carlquist 15590' unknown3 probably active Van Tiegh. New Caledonia CCI6 Pseudowintera axillaris 6 (J.R. & VIC 8/21/50 California 7 dormant G. Forst.) Dandy cult. CC37 Ibid., cult. VIC 12/18/50 California to+ active CC92 Ibid., cult. VIC 5/29/51 California 5-8 active CA454 Zygogynum baillonii Van Tiegh. Thorne 28630' 2 active New Caledonia CA455 Ibid. Thorne 28676' 5-6 dormant New Caledonia CC581 Zygogynum bieolor Van Tiegh. Carlquist 15330' unknown2 active New Caledonia CC580 Zygogynum vinkii Sampson Carlquist 15591' unknown' active New Caledonia

, Collection numbers of Drs. S. Carlquist and R.F. Thorne. 2 Diameter (minus bark) about 14 cm. 3 Diameter (minus bark) about 19 cm. 4 Diameter (minus bark) about 15 cm. S 'var. ehilensis' is not repeated in the paper. 6 May be Pseudowintera eolorata according to W. Vink (pers. comm.).

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Table 2.' Comparative lengths of sieve elements and tracheids.

Collection Name of plant Length in micrometers Ratio number Tracheid Sieve TR/SE element (TR) (SE) 2 3 4 5

CC579 Bubbia pauciflora 3610 471 7.65 CA227 Bubbia semecarpoides 1400 270 5.19 CA390 Drimys lanceolata 1840 330 5.58 CC36 Drimys lanceolata 2880 620 4.65 CC5 Drimys winteri 2270 438 5.18 CC878 Exospermum stipitatum 4420 589 7.50 CC37 Pseudowintera axil/aris 2180 371 5.74 CC454 Zygogynum bail/onii 1600 380 4.21 CC455 Zygogynum bail/onii 1780 400 4.45 CC580 Zygogynum vinkii 3860 450 8.58

, From measurements of ten or more representative cells of each kind in the collections listed (details on collections in Table 1).

Table 3.' Some dimensions of sieve elements in Drimys and Pseudowintera.

Collection Plant species Cell diameter Cell Lumen Wall Percent number /lm area area' area3 Lumen Wall TAN RAD /lm' /lm' J.Lffi' area area 2 3 4 5 6 7 8 9

CCI0 Drimys lanceolata 14.0 11.4 159.6 112.8 46.8 70.7 29.3 CC36 Drimys lanceolata 14.6 10.4 151.8 105.8 46.0 69.7 30.3 CC74 Drimys lanceolata 13.9 12.1 168.2 120.2 48.0 71.5 28.5 CC5 Drimys winteri 25.6 21.3 545.3 455.5 89.8 83.5 16.5 CC89 Drimys winteri 17.4 16.9 294.1 229.5 64.6 78.0 22.0 CC16 Pseudowintera axil/aris 16.0 16.6 265.6 204.4 61.2 76.9 23.1 CC37 Pseudowintera axil/aris 18.8 18.1 340.3 270.5 69.8 79.5 20.5 CC92 Pseudowintera axil/aris 18.4 14.0 257.6 196.8 60.8 76.4 23.6

, From measurements of tangential and radial diameters, 10 of each for each collection listed (de- tails on collections in Table I). , Thickness of wall approximately 1 /lm along both diameters. 3 Column 5 minus column 6.

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Garden, Claremont, California. Dr. W. Vink, at Some measurements characterise the sieve the Leiden Rijksherbarium, generously assisted plates (see data for species of Drimys and Pseu- with the identification and citation of names of dowintera under Results). The degree of incli- the species listed in Table I. nation of sieve plates was determined by refer- Smith (1969), motivated primarily by data ence to outlines of sieve elements in tangential on chromosome numbers (Ehrendorfer et aI., sections (e.g., Figs. 8D & 9H). The fraction of 1968), raised the Tasmannia section of Drimys length of the wall bearing the sieve plate over to generic rank and included in it all the Old the diameter of the sieve element beneath the World species of Drimys. The New World spe- sieve plate served to classify the sieve plates as cies were retained in Drimys. The dictionary by follows: 1.0 transverse, 1.1-2.0 slightly oblique, Willis (1973, p. 1131), revised by Airy Shaw, 2.1-5.0 oblique, 5.1+ very oblique. The same does not list Tasmannia as a separate genus, sieve plates were used to count the numbers of and we have followed that classification. But sieve areas per sieve plate. we did merge D. aromatica (our collection num- Pore sizes were obtained for 20 pores from ber CA390 in Table I) with D. lanceolata fol- either radial or transverse sections, sometimes lowing Vink (1970) and the previous confirma- for 20 pores from each kind of section. Two tion by Smith (1969, p. 287). diameters perpendicular to one another were Pieces of stem were placed into CRAF III measured in each pore. In transverse sections (Sass, 1951, p. 18) immediately after collection the diameters measured were the tangential and in the field. Most of the material was sectioned radial. If simple and compound sieve plates in unembedded condition on a sliding micro- were present in nearly equal numbers, measure- tome with or without preceding cautious soft- ments were obtained for 20 pores from each of ening by hydrofluoric acid or by other means. the two kinds of sieve plates. Some material was embedded in celloidin or Under Results, we describe first the three paraffin for sectioning. The staining was done species for which material collected and data in a combination of a tannic acid-iron chloride obtained were most abundant: Drimys winteri, mixture followed by resorcin blue. The proce- D. lanceolata, and Pseudowintera axillaris. dures of sectioning, attaching the sections to slides, staining and finishing the slides are de- scribed in Cheadle et al. (1953). Results Some features of the sieve elements were measured. The comparative lengths of tracheids Drimys winteri. and sieve elements for all 9 species were obtain- The 4-5 (CC89) and 12 (CC5) years-old ed by measuring ten or more representative stems of Drimys winteri have superficial peri- cells of each kind in the collections listed in derm. The cortical parenchyma beneath the pe- Table 2. This item served to determine whether riderm shows adjustments to the increase of secondary partitioning and the consequent re- stem circumference. The intercellular spaces duction in potential length of the sieve element are tangentially elongated and the tissue shows occurred regularly in the species examined. As results of tangential stretching and anticlinal is well known, tracheids and vessel members subdivision of cells (Fig. lOA, above). In radial are approximately of the same lengths as the sections the cortical parenchyma cells are ar- cambial initials. If the sieve elements are consis- ranged in long vertical columns (Fig. II A). At tently shorter than the tracheids, the observa- various depths, some cortical cells become scle- tion proves that the precursors of sieve elements rified (Fig. lA, stippled areas). Near the phloem, become subdivided (secondary partitioning). In sclerification extends into nonfunctional pri- some collections the occurrence of secondary mary phloem and interfascicular regions so that partitioning could be ascertained by examina- groups of sclereids become associated with tion of the cambial region (species of Drimys strands of protophloem fibres (Figs. I A, black and Pseudowintera). dots, lOA, II A). Drimys and Pseudowintera also served for In the stems used, the primary phloem was checking some additional cell dimensions based no longer functional, that is, nonconducting. on measurements of radial and tangential dia- The groups of protophloem fibres served as a meters, 10 of each for each collection (Table 3). marker of the outermost limit of the phloem, Nacreous wall was not discernible in the species but inwards, the delimitation between primary studied and the thickness of the cell wall was and secondary phloem was not clear because approximately 1.0 /lm along both diameters. both primary and the outermost secondary This value was used in the calculations of lumen sieve elements were partly collapsed, and the and wall areas as seen in transections (Table 3, ray tissue was much distorted by the enlarge- columns 6 and 7). ment of the nUmerous oil cells.

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The most prominent feature of D. winteri does in the mature phloem. Figures 2B, C, 100 phloem are the oil cells (Fig. lA, B), and their show the characteristic mixture of multiseriate presence has a profound effect on the confor- and uniseriate rays (one of each is marked 'r' mation of the tissue. The majority of oil cells left and in the middle in Fig. 100). Both kinds develop from phloem parenchyma cells (Fig. of rays are considerably extended vertically ex- 20), but some also differentiate from ray cells cept that the recently initiated rays have short (Fig. 3F, J). Despite the abundance of oil cells, profiles. Figure 100 shows this early stage for their distribution in the tissue is not entirely three uniseriate rays (small arrows at right). uniform. Blocks of axial tissue, in which most The multiseriate rays frequently have uniseriate of the sieve elements are located, are relatively extensions resembling uniseriate rays. The ex- free of oil cells and stand ou t like aggregates of tensions may be on either upper or lower or small, compactly arranged cells (Fig. 2A) alter- both margins of the ray. nating with wider tissue blocks containing In the wood (Fig. 10E), the ray system of numerous oil cells (Fig. lOA, B). In addition to Drimys is interpreted to be of the primitive axial cells, the narrow-celled tissue includes heterogeneous type I, in which the cells of the uniseriate rays (Fig. 2A). The wider blocks of uniseriate rays and of the uniseriate extensions tissue contain axial cells, uniseriate and multi- of the multiseriate rays are vertically elongated seriate rays (the latter at 'r' in Fig. lOB), and (upright cells), whereas the multiseriate rays or most of the oil cells. The rays are considerably ray parts consist of nearly isodiametric or ra- obscured by the large oil cells, and the uniseriate dially elongated cells, that is, procumbent cells rays are more easily identifiable in the narrow- (Bailey, 1944). In the phloem of D. winteri, the celled blocks of tissue than in the intervening difference between the component cells of the tissue. The first oil cells become discernible at uniseriate and multiseriate rays is less pro- the beginning of cell differentiation among nounced than in the xylem (Figs. 2B, C, 100), newly formed derivatives of the vascular cam- but the cells of the multiseriate ray regions bium (Fig. 2C). match those in the xylem in being nearly isoT The nonstoried vascular cambium (Fig. 2B) diametric or radially elongated (Figs. 10C, IIA, consists of long fusiform initials and short ray 'r' at lower right). initials in assemblages typical of rays. In Figure The absence of truly upright cells in phloem 2C, the derivatives of the fusiform initials are rays of D. winteri is in part conditioned by the seen partitioned by anticlinal walls (dashed increase in bark circumference. The rays are lines). The corresponding cell walls in the pho- subjected to tangentially directed stresses, their tomicrograph in Figure 100 (arrowheads) are cells become wider and divide anticlinally. Their seen to be exceedingly thin. Some of the fusi- form changes materially from that characteris- form cells in Figure 2C are partitioned into tic of the cambial state. In D. winteri, moreover, rather short cells - the future phloem paren- the initially vertically elongated cells undergo chyma cells and their derivatives, the oil cells. transverse divisions (Fig. 3G, right, pair of cells F our of the latter are already more or less en- in third position from above), a phenomenon larged (shaded cells). A similar stage of oil cell previously recorded for this species by Stras- development is seen to the left in Figure 100. burger (1891, pp. 165-166). That author also (This half of the section was farther from the noted sclereids in the wide rays of D. winteri, cambial initials than the half to the right.) a feature not recorded in our material. Occa- Figure 20 shows the secondary phloem in ma- sional ray cells contain phenolic material. ture stage with numerous oil cells among phloem The conformation of the axial system is parenchyma cells and one in the ray to the left largely determined by anticlinal divisions in the below. precursors of phloem cells, the phloem mother Since the oil cells begin to enlarge in a tissue cells. Some fusiform derivatives of the cambium that has barely I?assed the cambial stage (Fig. are subdivided by transverse or slightly oblique 2C), their growth affects the neighbouring cells anticlinal walls into phloem parenchyma by displacing and compressing them or prevent- strands. Others divide by more or less inclined ing them from attaining the average width of and, in part, nearly vertical cell walls' into pre- such cells in the tissue (Fig. 2A, C, D). Both pa- cursors of sieve elements and their associated renchyma cells and sieve elements are affected parenchymatic cells. As a result of the anti- (Figs. 20, 3E). If an oil cell is differentiating clinal divisions (Figs. 2C, 100), the sieve ele- next to a ray, it may push the ray cells apart ments are shorter than the tracheids (Table 2). and become partly embedded in the ray (Fig. The phloem mother cells thus exhibit the phe- 2A, right and left above). nomenon of secondary partitioning. The ray system appears more clearly in the The an ticlinal divisions cause a disturbance cambial region, still devoid of oil cells, than it in the radial seriation characteristic of the im-

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mediate derivatives of the periclinally dividing organised into sieve plates. According to our cambial cells. This effect can be observed close measurements of sieve areas in the functional to the initial region of the cambium (Fig. 2A). phloem with a light microscope, the_ diameters Since no secondary partitioning occurs in the of pores, including the callose lining, vary from axial precursors of the xylem and the new tra- 1 JlIll to less than 0.5 p.m and the pores inter- cheids show little or no increase in size in the grade in size within this narrow range (Fig. 3B- tangential plane, the radial seriation of cells in D). Moreover, sieve areas of different degrees the secondary xylem is maintained during tis- of differentiation vary widely in size and clear- sue maturation. A minor disturbance in radial ness of delimitation (Fig. 3A-D). The smallest alignment of transectional ou tIines of cells in sieve areas consist of a few pores and are not in some of the rows of tracheids is caused by the any way delimited from the rest of the wall, overlapping of the beveled ends of tracheids the largest are sharply circumscribed by a thick- (Fig. IDA, below). ening of the surrounding wall like pit areas; and Whereas the phloem parenchyma cells origi- these two extremes are interconnected by a ser- nate during the secondary partitioning process, ies of transitional types. -the companion cells appear later, as derivatives Despite the lack of a clear distinction be- of sieve element precursors (Fig. 2D, arrow). tween more and less specialised sieve areas, we They arise by divisions in various planes of concluded that wall areas with the larger pit- orientation, frequently periclinal (Fig. 2A). like sieve areas, having also larger pores, consti- The companion cells are shorter than the sieve tute sieve plates. Such sieve plates occur on elements and occur singly or in twos (Figs. 3 I, transverse or nearly transverse end walls and on 12A). Because the cells are short, not many more or less inclined radial walls. Those on the transverse sections of a given sieve element in- radial walls are much compounded and elong- clude its companion cell or cells_ In 30 serial ated and may cover the entire length of the transections including 20 sieve elements, two wall. They appear in face views in radial sec- sieve elements seemed to lack companion cells. tions (Figs. 3A, dashed outlines, 3B, 12A, part The identity of one of these two presumed of plate only) and in sectional views in tangen- sieve elements was somewhat uncertain and the tial sections (Figs. 3A, knobby walls, 12B). The other was not seen end to end in the available lateral sieve areas occur on both radial (Fig. 3C, sections. If any sieve elements of D. winteri D) and tangential (Fig. 3A, below and middle, lack companion cells, the phenomenon is prob- righ t of dots) walls. ably of rare occurrence. The sieve elements The typical localisation of the elongated sieve contain nondispersing protein bodies of about plates on the radial walls is frequently obscured 4 JlIll in diameter- in D. winteri because of the displacement of The phloem parenchyma cells are in strands, cells by the enlarging oil cells. The pressure of which may be difficult to distinguish from the the expanding oil cells appears to cause the uniseriate rays in tangential sections. The cells nearest sieve element to twist and to expose vary in length and are usually wider than the the face view of the sieve plate in a tangential companion cells in the same sections (Fig. 2A). section (Fig. 3A, arrow upper right). When the sieve elements and companion cells The sieve plates are compound on both trans- die and collapse the parenchyma cells remain verse and radial walls. The mean number of intact (Fig. 3H). Many parenchyma cells con- sieve areas per plate was found to be 11.8, with tain accumulations of phenolic material. Fibres none of the sieve plates simple. Only 10% of the are not present in the secondary phloem of D. sieve plates were transverse or slightly oblique, winteri. 65% were oblique, and 25% were very oblique. Some dimensions of the sieve elements in the transverse plane are given in Table 3 for Drimys lanceolata. three species of Winteraceae. The sieve elements In all three California collections of Drimys in the different collections of D. lanceolata and lanceolata (CC 10, CC36, CC74) the periderm Pseudowintera axillaris had dimensions similar is superficial. Beneath the scaly cork is a phel- to those in D. winteri except that in the latter loderm composed of narrow cells arranged in one of the two collections had much wider radial series partly disturbed by anticlinal walls sieve elements than the other. All Winteraceae (Fig. 13A). The cortical cells beneath the peri- listed in Table 3 were collected in the San derm show results of tangential elongation and Francisco Bay Area of California (Table I). anticlinal divisions induced by the increase in In view of the absence of vessels in the xy- stem circumference. The parenchymatic cortex lem of Winteraceae it is important to determine extends to the protophloem fibres, which ap- whether this family has sieve tubes, with mem- pear in transections as very narrow cells (Fig. bers interconnected by specialised sieve areas 13A, asterisks) associated with groups of corti-

Downloaded from Brill.com10/07/2021 05:17:02PM via free access IAWA Bulletin n.s., Vol. 5 (1),1984 19 cal sclereids (Figs. IC, 13A, large arrow). Groups they are compared with the D. winteri collec- of sclereids occur here and there in the peri- tion having the narrower sieve elements. Al- pheral parts of the cortex (Fig. I C, stippled though the cell walls lack the nacreous layer areas). and are as thin as those in D. winteri, the wall The California collections of D. lanceolata area per cross section of sieve element is smal- reveal no oil cells in the phloem (Figs. 4A, 13). ler than that in D. winteri, obviously because The axial system forms blocks of tissue that are of the smaller cell diameters (Table 3). Because penetrated by uniseriate and biseriate rays of secondary partitioning, the sieve elements (Figs. 4A, 13A, B), and alternate with multise- are shorter than the cambial cells and tracheids riate rays (Fig. I C, D). The latter increase in (Table 2) and have some slightly oblique and width toward the periphery of the stem, con- nearly transverse end walls (Figs. 5D, 12H, I, tain sclereids (Fig. I C), and frequently become 13C, black arrowheads to the right). The sieve torn in sectioning. The two lacunae I in Figure elements contain non dispersing protein bodies 13A were formerly occupied by such rays. This about 4 p.m in diameter. relative arrangement of axial and ray systems in As in D. winteri, the sieve areas of D. lanceo- D. lanceolata is conspicuously different from lata intergrade in the size of pores and degree the more uniform alternation of axial blocks of delimitation from the surrounding wall areas, and multiseriate rays in D. winteri. the most highly differentiated ones forming The rays are very high (Fig. lD) and the groups with the attributes of sieve plates. The multiseriate rays often show uniseriate exten- latter occur on slightly oblique to oblique end sions. These extensions and the peripheral cells walls and on nearly vertical radial walls. In the in the multiseriate rays (upright cells; Fig. 5C) absence of oil cells, those on the radial walls tend to be vertically more or less elongated. As are not displaced so as to appear in face views seen in tangential sections, the multiseriate rays in tangential sections. Lateral sieve areas occur in D. lanceolata are better delimited than are on longitudinal walls of various orientations the rays in D. winteri because of the presence (on tangential walls in Fig. 12H, I). of upright bordering cells and the absence of Thirty percent of sieve plates were found to oil cells. The uniseriate rays are often difficult be transverse or slightly oblique, 55 percent to distinguish from phloem parenchyma strands oblique, and 15 percent very oblique. Twenty- and in both many cells contain phenolic com- five percent of the sieve plates were simple, 75 pounds. percent compound. The mean number of sieve In the collections CC 10 and CC36, sclerifi- areas per sieve plate was 4.1 when all sieve cation of the expanding multiseriate rays occurs plates were counted, 5.1 when only the com- in mature phloem close to the cambial region pound sieve plates were counted. (Fig. I C). The interior ray parenchyma cells are The Australian collection of D. lanceolata converted into heavily pitted sclereids forming (CA390) consisted of a rather meagre sample masses extending through almost the entire and the staining reaction of the phloem tissue cross and tangential sections of the rays. The was unusual. Many phloem parenchyma and uniseriate extensions and one or more layers of ray cells were extremely chromatic, whereas the peripheral cells remain parenchymatic. the sieve elements (excepting those containing The axial system of D. lanceolata resembles callose) and the immediately associated phloem that of D. winteri except in the absence of oil parenchyma and companion cells remained es- cells. The sieve elements vary in number in the sentially unstained (Fig. 14). The callose and different radial series (Fig. l3B, cells with cir- the sclereids were stained blue. cles) and bear sieve areas on cell walls of any The more than 15 year-old stem (Table I) still orientation (Fig. 4A, walls marked with knobs). has the first periderm consisting ofcork and phe\- The phloem mother cells undergo secondary loderm. The cortex contains numerous groups partitioning (Fig. 5C, dashed lines; Table 2) so of sclereids located at different depths. Some that in transections, the radial seriation is less occur also in the phelloderm. The sclereid lumi- precise than it is in the cambial region (Figs. na are filled with phenolic material. The cortex 4A, 13B). The companion cells are mostly contains numerous oil cells. It is poorly delim- single and short (Fig. 5D). The phloem paren- ited from the phloem. No protophloem fibres chyma cells occur in strands and are wider than were detected along the periphery of the phloem the companion cells (Fig. 4A). Many contain in the stem listed in Table I. Additional sec- phenolic material (Fig. 13C). No fibres are tions of younger stems from herbarium mate- present and sclereids are lacking in the axial rial of collection CA390 showed such fibres in system of all three California collections. combination with othersclerenchyma cells. The According to Table 3, the sieve elemen ts are protophloem fibres intergrade in length and less wide than those of D. winteri even when form with adjacent sclerenchyma cells.

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The secondary phloem has the same basic suIt of meristematic activity in the parenchyma features as that in the other D. lanceolata: a outside the zone of sclereids (Fig. ISA, curved combination of high muItiseriate and high uni- arrow), the subsequent pulling apart of the seriate rays; blocks of axial system traversed by sclereids, and the closure of the gaps by in- uniseriate rays (Fig. 4B); sieve elements shorter growing parenchyma cells. These phenomena than the tracheids (Fig. SA, B; Table 2); short are evidently associated with the circumferen- companion cells (Fig. SA); variation in inclina- tial expansion of the vascular cylinder. The ex- tion of sieve plates; and the intergrading be- pansion of the sc1ereid zone seems to displace tween sieve plates and lateral sieve areas in de- and even disrupt the groups of protophloem fi- gree of differentiation. bres. As seen in Figure ISA, two fibres above The differences between the Australian and the left straight open arrow, may have been de- Californian collections may be related to dif- tached from the group labeled fb. Occasional ferences in ages of the available stems (Table I). single or small groups of sclereids occur in the The features distinguishingD.lanceolata CA390 outer cortex and at some depth in the region of from the same species collected in California are the nonfunctional phloem (Fig. I G, black the following: high phenolic content of ray and spots). Crystal deposition may· occur in cells phloem parenchyma cells (Fig. 14); massive ac- adjacent to the sclereids (Fig. ISA, cr). Scatter- cumulations of sclereids in the muItiseriate rays ed oil cells occur in the cortex (Fig. ISA, aster- (Figs. IE, F, 14A); occurrence of numerous oil isk in one, lower left). cells in cortex and some in the older phloem; The primary phloem is nonfunctional but scattered sclereids in the axial phloem (Fig. SF). most of its sieve elements and companion cells The pattern formed by the alternation of mul- are only partially crushed. The primary phloem tiseriate rays and blocks of axial system in the imperceptibly merges with the oldest, nonfunc- Australian collection is more regular than in tional part of the secondary phloem in which the Californian collections (compare Figs. IC the sieve elements and companion cells also are and E). Age disparity in stems may be a factor only partially collapsed (Fig. ISB, curved ar- in the anatomical differences. rows). As seen in transections, wide muItiseriate Pseudowintera axillaris. rays alternate with wide complexes of axial tis- In all three collections of Pseudowintera sue penetrated by uniseriate rays (Figs. IG, 4C, axillaris (CC 16, CC37, CC92), stems over five D). The latter are not sharply defined and may years old (Table I) still have the cortex and best be identified by reference to the uniseriate most of the epidermis. The latter is covered rays in the xylem (Fig. ISA). The multiseriate with a thick cu tic1e and, in places, the cell lu- rays are spaced farther apart than in Drimys mina are almost occluded with wall material re- (compare A and G in Fig. I). sem bling the cuticle in density and staining The muItiseriate rays are high (Fig. IH) and reaction. It probably is cutinised wall substance. some have uniseriate extensions. Many marginal Wherever some periderm had been formed, be- cells are vertically extended and help to delimit neath the epidermis, the phellem cells also are the rays. The middle layers of ray cells become partially occluded with that wall substance. No sclerified close to the cambium (Figs. IG, SE, phelloderm is discernible in anyone of the ISA). Occasional crystal-containing cells are three collections. The outermost cortical layers, found near the ray sclereids (Fig. ISA). The however, consist of tigh tly packed cells, smaller uniseriate rays are not as high as the multiseriate than those at greater depth in the cortex. In ones and are not well differentiated from the deeper layers, the cortical cells show the usual phloem parenchyma strands, especially when conformation of a tissue that had been subject- both cell complexes contain phenolic material. ed to tangential stretching. The cells are in tan- Oil cells occur in uniseriate (Fig. 4C, D) and gential rows within which groups of two to multiseriate rays (Fig. SE). four cells appear to have been formed by anti- The axial system consists of sieve elements, clinal divisions of single cells. companion cells, phloem parenchyma cells, and Sclereids develop in the innermost cortex, in oil cells (Figs. 4C, D, 15). The latter are less the adjacent primary phloem, and in the inter- numerous than in D. winteri. A few sclereids fascicular regions, so that the protophloem fi- occur in the outer part of the axial system (Fig. bres (Fig. I G, dots; ISA, fb at lower left) are IG). completely embedded in sclerenchyma. The The phloem mother cells undergo secondary sclereids form a zone four to six cells deep con- partitioning (Fig. SG; Table 2). The width and tinuous around the circumference of the stem, other dimensions of sieve elements that were except for occasional breaks occupied by pa- recorded are within the same range as those renchyma. These breaks appear to arise as a re- found in Drimys (Table 3). The sieve elements

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contain the non dispersing protein bodies with a renchyma cells that have replaced the primary diameter of about 4 p.m. phloem and thus they identify the functional The percent of inclination types of sieve part of the phloem in Figure 6C as secondary plates are as follows: transverse or slightly obli- tissue. que (Figs. 5J, 12C) 55; oblique (Fig. 5J, below) As judged by the older stem of B. pauciflora, 25; very oblique (Fig. 5H, I) 20. The occurrence the clear ray pattern of a young stem is obscured of secondary partitioning is well supported by during subsequent secondary growth. The rela- the high percent of transverse or slightly oblique tive volumes of rays and axial tissue change in end walls. Thirty-five percent of the sieve plates favour of the rays (Fig. 7A) because of tangen- were simple, 65 percent compound. The pores tial expansion of original rays and interpolation in the sieve plates appeared to be somewhat of new multiseriate rays in the axial system. narrower than those in Drimys. There was suf- Correspondingly, at this stage of development, ficient contrast between the most and the least tangential sections reveal the multiseriate rays highly differentiated sieve areas (Fig. 12D, E) forming a dense pattern, with some quite nar- that the presence of sieve plates was unques- row blocks of axial tissue between neighbouring tionable. But as in Drimys, the lateral sieve rays (Fig. 6B). Furthermore, the continued areas in tergrade with the sieve areas of the sieve growth in stem circumference brings about an plates in their main features. increase in size of parenchyma cells in ray and The companion cells are short. They may be axial tissues, followed by anticlinal divisions of single in one position or double (Fig. 5 I, J). the enlarged cells so that the distinction between Phloem parenchyma cells are in strands (Fig. ray and axial tissues becomes blurred (see inter- 5G, H). Some have phenolic contents. rupted outlines ofrays in Fig. 7A). The axial system contains sieve elements, Bubbia pauciflora and B. semecarpoides. companion cells, phloem parenchyma cells In the growing two-year-old stem of B. seme- (Fig. 6C), and, in older phloem (Fig. 7A), also carpoides (CA227) cork is present in variable sclereids. The comparative lengths of tracheids amounts around the circumference but is still and sieve elements (Table 2) indicate that sec- covered by the epidermis. The parenchymatic ondary partitioning occurs during phloem dif- cortex contains single and groups of sclereids ferentiation. The sieve elements show various (Fig. 6A, black spots), cells with phenolic ma- degrees of inclination of walls bearing sieve terial, and cells with starch but free of phenolic areas, and the companion cells are shorter than accumulations. One to three cells away from the sieve elements. A few oil cells were seen in the sclerenchyma delimiting the vascular bund- the axial (one in Fig. 6C) and ray systems. The les of the young stem, the cortical sclereids sclereids originate by sclerification of phloem form a layer several cells deep, interrupted here parenchyma and ray cells. In the primary and there by parenchyma cells (Figs. 6A, liB). phloem sclereids appear in compact groups de- These sclereids are isodiametric or somewhat fining the outer limits of the phloem (Fig. 6A, elongated as seen in either transverse or longi- C). These sclereids are elongated and promi- tudinal sections. nently pitted. In the much older stem of B. pauciflora (CC In B. pauciflora, sclereids are abundant in 579) the cortex is still present, the cork is mas- the secondary phloem. Closer to the cambium sive in places, and sclereids, single or in groups, they occur mainly in the axial tissue (Fig. 6B), are scattered throughout the cortex (Fig. 7A). but in the older phloem they are present also No continuous layer of sclerenchyma is present in the multiseriate rays (Fig. 7A). The axial in perivascular position, possibly having been sclereids are rather short and narrow and may fragmented by stresses resulting from the cir- be arranged in vertical files. The sclereids in cumferential expansion of the stem. rays are short and wide. Protophloem fibres The B. semeca,rpoides stem is young enough were recorded in B. pauciflora (Fig. 7 A, curved to illustrate the origin of the first rays. In tran- open arrows). Both species contain nondisper- sections, the vascular system appears in the sing protein bodies. In B. semecarpoides the form of strands (axial regions) separated from bodies are about 4 p.m in diameter, in B. pauci- one another by multiseriate rays which are ini- flora near 8 p.m. tiated in the interfascicular regions of the stem as the latter completes primary growth. As sec- Exospermum stipitatum. ondary growth begins, uniseriate rays appear in The stem of Exospermum stipitatum (CC the axial regions. In Figure II B, the uniseriate 578), which is about 19 cm in width, shows rays are indicated by open arrows at the cam- massive cork but no rhytidome (Fig. 7B). The bial level and in Figure 6C by dots along the cortex has numerous sclereids scattered singly cell walls. These rays reach the groups of scle- or in groups. Protophloem fibres are detectable

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(Fig. 7B, curved arrows) at the outennost limit from parenchyma cells and consequently short of the phloem. The secondary phloem consists in all dimensions - a stone cell type of sclereid. of relatively wide blocks of axial tissue which A somewhat interrupted layer of sclereids, sev- include poorly defined uniseriate rays and al- eral cells deep, occurs just outside the vascular ternate with four to five cells-wide multiseriate region (Fig. 9A, stippled area). These sclereids rays (Fig. 7B, r). The latter are spaced far apart are also relatively short and have the same form (Fig. 7C), much more so than the rays in the as viewed in transverse and longitudinal sec- old stem of Bubbia pauciflora (Fig. 6B). tions. In places, sclerification reaches the non- The axial system contains sieve elements, functional primary phloem so that protophloem companion cells, and phloem parenchyma cells. fibres may be in contact with the layer of corti- The sieve plates occur on transverse, variously cal sclerenchyma (Fig, 9A, arrowheads). The inclined, and nearly vertical walls (Fig. 8A, D), protophloem fibres have very thick walls (Fig. and are commonly compound. The degree of 9C, asterisk) and, in macerations, appear as differentiation of sieve areas and lack of sharp long narrow cells pointed at both ends, or distinction between lateral sieve areas and pointed at one end and blunt at the other. those of the sieve plates are similar to such fea- The vascular system consists of rather wide tures in other Winteraceae studied. Figure 8 axial blocks penetrated by uniseriate rays and gives examples of compound sieve plates on ra- alternating with multiseriate rays. The latter dial (E) and transverse (I) walls and of more increase in width toward the cortex, particular- (F) and less (G, H) highly differentiated lateral ly through enlargement of cells (Figs. 9A, C). sieve areas. The dashed outlines indicate sieve The rays are high (Fig. 9B). Cells in the unise- areas that appear to be in slight depressions be- riate and multiseriate rays become sclerified cause of the thickening in the surrounding cell where they cross regions of sclerification in the wall. The comparative lengths of tracheids (Fig. axial system (shown for a multiseriate ray in 8C) and sieve elements (Fig. 8D; Table 2), and Fig. 9C). Some ray cells contain phenolic ma- the frequent occurrence of transverse and terial. sligh tly inclined end walls with sieve plates The secondary phloem has a narrow layer of (Fig. 8A) clearly indicate occurrence of second- functional phloem, which appears poorly pre- ary partitioning. The sieve elements contain served and radially compressed (Fig. 9E). Most non dispersing protein bodies that are close to of the phloem is nonfunctional and shows mas- 8 J.Ul1 in diameter. sive sclerification, a feature that must have con- The companion cells are much shorter than tributed to compression of the younger phloem the sieve elements and occur one or two to a during sectioning. The axial sclereids vary in cell (Fig. 8B). If two are present, they may be width, length, and wall thickness (Fig. 9D) and contiguous or some distance apart. The phloem are related to phloem parenchyma cells in fonn parenchyma cells occur in strands and are free (Fig. 9F), Some are of the stone cell type (Fig. of phenolic accumulations. (The fluid used for 9G); these are indicated in black in the axial preservation of this species may have been dif- phloem in Figure 9A. ferent from that used for our own collections.) The functional part of the phloem contains Oil "ells are occasionally discernible. sieve elements, companion cells, and phloem Sclereids are present in both axial and ray parenchyma cells. Oil cells were not encounter- tissues (Fig. 7B, C, at arrow), singly or in groups. ed. Phloem parenchyma cells are in strands and Closer to the cambium, where the tangential some show phenolic accumulations. The com- section in Fig. 7C was obtained, sclereids occur panion cells are short and occur singly or in mainly in the axial tissue. Among the cells in twos along the same wall (Fig. 9H). Figure 8J, left above, those between the two The occurrence of secondary partitioning solid arrows are ray cells. Three of the ray cells was determined by the relative lengths of tra- are sclerified. The other sclereids in Figure 8J cheids and sieve elements (Fig. 9J, K; Table 2) are from the axial tissue. Most of them are and many of the latter have transverse or slight- slightly elongated. In the axial system one or ly inclined end walls bearing sieve plates (Fig. more members of a parenchyma strand may be- 9H, K). Long, much compounded sieve plates com'e sclerified while others remain parenchy- occur on longitudinal walls (Fig. 91, L). Sieve matic (Fig. 8J, single rows of cells). areas of the sieve plates intergrade with lateral sieve areas. Zygogynum bail/onii (CA454) is Zygogynum baillonii. Z. bicolor and Z. vinkii. represented by a two-year old stem. In cellular The 5-6 year old stem of Zygogynum bail- detail it agrees with Z. bail/onii (CA455), in- lonii (CA455) has a periderm ·12 cells in depth cluding the short lengths of sieve elements as but no rhytidome. The cortex contains scattered sclereids (Fig. 9A), mostly in groups, derived (text continued on page 39)

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0 0 00 0 00 '0 A o .0 - BOo 0 o

G

F

Common abbreviations in legends and symbols in drawings: Companion cell (s), cc; multiseriate ray(s), mr; phloem parenchyma cell(s), ppc; sc1ereid(s), sc; sieve area(s), sa; sieve element(s), se; sieve plate(s), sp; uniseria~e ray(s), ur; knobby, or beaded wall part, sa in section; group of dots, sa in face view; dashed outline, sa surrounded by somewhat thickened wall (sa appears depressed).

Fig. I. A, B, Drimys winteri CC5; C, D, D. lanceolata CC36; E, F, D. lanceolata CA390 (syn. aro- matica); G, H, Pseudowintera axillaris CC37. - Outlines of some features of tissue organisation in stems in transverse (A, C, E, G) and tangential (B, D, F, H) sections. Details: ca (A, C, E, G), cam- bium with secondary phloem above it; r or shading consisting of dots or hatchings (A, B, D, G, H), multiseriate rays; cross-hatched shading (E, F), sc in mr; dots and rods (E, F), sc in axial system; stippling (A, C, G), sc in cortex and phloem ; groups of dots at arrowheads (A, C, G), protophloem fibres; areas in black (G), sc in axial and ray systems; brackets (D, H), arrangement of mr; outlines of mr outside brackets (D , H) are continuations of the mr in brackets similarly marked a or b. A,B,E- H, x 15;C,D,x 17.

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Fig. 2. Drimys winteri CC5. - A, transection of secondary phloem with some cambium below; B-D, tangential sections of cambium (B), differentiating phloem (C), and mature phloem (D). Details: in mature phloem (A, D), se lumina are clear and walls with sa, knobby; cc are stippled (alSo marked with arrow in D); nuclei are shown in ppc; dots along the walls denote ray initials and ray cells; oil cells are shaded; cr, crushed phloem cells. In C, dashed lines, walls formed in secondary partitioning of axial cambial cells. A, x 154; B-D, x 71.

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. I I • ---' J

Fig. 3. Drimys winteri CC5. - Phloem cells from tangential (single dots) and radial (single squares) sections. Details: p, phloem parenchyma cells; r, ray cells; se, sieve elements; shading, oil cells. In A, open arrows point to individual sections; the solid arrow above, right, points to change in orienta- tion of sp from sectional view above, to face view below. Part of compound sp in B, lateral sa in C and D. E, se next to the enlarged oil cell is compressed. F, a pair of sections of an mr with an oil cell. G, parts of se with various parenchyma cells; the one in the middle may be the first cell of a new ray (r). H, I, se associated with dead (H) and functional (I) cc. J, a pair of sections of a ur with two oil cells in one section. A, x 203; B-D, x 524; E, x 11 5; F, J, x 79; G, H, I, x 191.

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E B

f G ; Ii

Fig. 5. A, B, F, Drimys lanceolata CA390; C, D, D. lanceolata CC36; E, G-J, Pseudowintera axil- laris CC37. - Groups of phloem cells except tracheid in B shown as two halves. Differentiating phloem in C, G, with dashed lines indicating secondary partitioning. Ray in E from radial section, all other sections are tangential. Details: r, ur; mr to the left in C; nuclei indicate cc, also curved arrows in A; straight arrows in A, J, delimit lengths of se; sc and ppc in F; oil cells are shaded and sc shown with secondary walls in E. A-C, x 88; D, x 161; E, x 46; F, x 235; G-I, x 112; J, x 185.

-Fig. 4. A, Drimys lanceolata CC36; B, D. lanceolata CA390; C, D, Pseudowintera axillaris CC37. - Transections of phloem, with cambium below in A, C, D. Two successive sections in C, D. Details: lumina clear in se, stippled in cc; nuclei in ppc; dots along walls, ray cells; shading, oil cells (none in A, B). At C at in, apparent interruption of ray which is closed in D; cell outlined in dashes in C identified as an oil cell in D. A, B, x 240; C, D, x 206.

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.... - .... -~ ? -.'

Fig. 6. A, C, Bubbia semecarpoides CA227; B, B. pauciflora CC579. - Transverse (A, C) and tan- gential (B) sections from two-year (A, C) and an older stem. A, vascular tissue divided in blocks by mr, cambium at ca, oldest phloem hatched, sclerenchyma stippled next to the phloem, blackened farther out. In ' B, mr are shaded and sc in axial system and at margins of some rays blackened. C, cambium below, secondary phloem with stippled cc, single dots in se, no marks in ppc, ray cells with dots along walls; primary phloem above replaced by sclerenchyma; large cells right and left, parts of mr merging with cortical cells .. bove. A, B, x 18; C, x 184.

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. - . ~ .... ~...... -- ....~ - ~~.:f.. .-• :.....-..6:.'- :~~::;,:-'" ~a _I &

t , '" .. .. ' . '. t o ... . ~ :) , '. ,1/114 , ;. , ;' 4': • \ ' . : ,,'.,' :. .,i ~ ' " ~' '. ",?.' ' ... " /";> I . :.. " '.... t ' J. ( , ... . "" ... , .. , r . .,'t'·" , "w,·'''; I";" . r4 r r c , :: ~ ',. : l '~, : • .: ; ~'. 'I ' ,', B " f· "', ',: ';". ~ I • \:

) , ~'.. ' : ; .",'\ I '. l : ~ • • A ; r o •• ~ r! ~ ~ • r .~ . ,

Fig. 7. A, Bubbia pauciflora CC579; B, C, Exospermum stipitatum CC578. - Outlines of tissue organisation in secondary phloem in transverse (A, B) and tangential (C) sections from stems 14 cm (A) and 19 cm (B, C) in diameter. Multiseriate rays (r) are approximately delimited from the axial tissue by dashes in A, B, and indicated by shading in C. Dilatation growth has partly obscured the limits of rays in A, B. The section in C was located near the cambium. Sc in cortex, rays, and axial tissue are shown in black; periderm areas are shaded in A, B. Curved arrows in A, B, point to proto- phloem fibres. Arrow in C marks sc at margin of mr. A, x 13 ; B, C, x IS.

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o

A c o

...

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F G H

Fig. 8. Exospermum stipitatum CC578. - Groups and single cells of phloem except four parts of single tracheid in C used for comparison with two pairs of se in D. From transverse (I), tangential (A, D, G, H, J), and radial (B, E, F) sections. A, cells immediately above the two sc (with secondary .walls) are part of mr. B, parts of se with cc marked with outlines of nuclei. E, I, compound sp; F, G, H, lateral sa, surrounded by thickened wall in F; J, sc and associated ray cells (black arrows) and ppc (outlines of nuclei). Sc occur in rays and as members of parenchyma strands. Open arrows point to individual sections. A, B, x 201; C, D, x 61; E, F, x 306; G, x 329; H, x 679; I, x 720; J, x 117.

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~F~ ~~ ' 1I'5J K

l

B

Fig. 9. Zygagynum bail/anii CA465. - Phloem tissues and cells from transverse (A, C-E, G), tan- gential (or close to) sections (B, F, H, I, K, L) and single complete tracheid in four parts (1) used for comparison with se in K. A, outline of tissue organisation in stem delimiting the cambium (ca), mr (r), axial phloem regions, and cortex uppermost. Stippled and blackened areas, sc as exemplified in F and G. Groups of black dots in A (arrowheads) and asterisk in C, protophloem fibres. B, mr (shaded), the one farthest left shown to one third of its height. C, mainly secondary phloem with mr to the right; details of tissues above cambium (ca) are shown in D, sc from stippled area in C, and E, functional phloem from near the cambium in C. In E, se blank, cc stippled, ppc with nuclei, ray cells with dots along walls. H, sieve elements with companion cells. Sp in surface (1) and sec- tional (L) views. A, B, x 20; C, x 42; D- G, x 100; H, I, L, x 104; J, K, x 43.

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Legends to Figures 10-15: Fig. 10. Drimys winteri CC5. - A, transection of stem with tissues from below upward: secondary xylem; cambium (ca); functional secondary phloem extending about 2 cm from the cambium; non- functional secondary phloem; nonfunctional primary phloem with protophloem fibres (f) next to sc, which are derived from cortical and interfascicular parenchyma. Oil cells (asterisk in one) ob- scure the rays. Some cortical parenchyma above the sc. B, tangential section through functional phloem. The three blocks of narrow cells, many with dark contents, consist of axial tissue and ur. Blocks between the denser tissue contain some mr (r) and additional axial tissue with numerous oil cells (asterisk). C, nearly radial section including part of mr (r). D, tangential section through cam- bial region showing anticlinal walls (arrowheads) formed during secondary partitioning. A few mr alternate with more numerous ur (complete profile at r in the middle below). To the left above, some differentiating oil cells. Small arrows to the right, short profiles of ur. E, tangential section through secondary xylem showing tracheids in the axial system alternating with ur and mr. No tracheid is short enough to be visible in its entire vertical extent. All, x 54. Fig. 11. A, Drimys winteri CC89; B, Bubbia semecarpoides CC227. - A, radial section of stem inclu- ding, from below upward: secondary xylem (x); cambium (ca); secondary phloem, mostly axial with numerous oil cells (asterisk) and ray tissue (r)at right; nonfunctional primary phloem with protophloem fibres (f); cortex with sc and one oil cell (asterisk). B, transection of stem showing from left to right: epidermis; cortex containing a layer of sc and extending to fibres marked with a large arrow; primary phloem represented by groups of fibres (large arrow) separated from each other by interfascicular pa- renchyma; secondary phloem (beneath the fibre bundles) consisting of axial tissue, ur (open arrows), and mr (r); poorly defined cambial region; secondary xylem; and a few pith cells. A, x 91; B, x 52. Fig. 12. A, F, Drimys winteri CC5; B, G, D. winteri CC89; C-E, Pseudowinteraaxillaris CC37; H, I, D.lanceolata CC36. - Radial, A, D, E, transverse, F, and tangential, B, C, G-I sections. A,partofse with compound sp (arrow) in face view; cc shows a nucleus. B, much inclined compound sp at arrow. C, slightly inclined compound sp at arrows. D, compound sp and, E, lateral sa in face views. F, some se (circles) are associated with densely stained cc; large oil cell below. G, enlarged view of part of section similar to that in Figure lOB; white arrows point to two ends ofa complete profile ofse with inclined sp; ppc and oil cells (asterisk) on both sides of the se-containing block; part of mr to the left. H, I, arrows point to inclined sieve plates in three of which individual sa, thickened by callose deposits, are discernible; lateral sa in face views are visible on longitudinal walls upward from arrow in Hand downward from uppermost arrow in I. A, x 550; B-E, x 624; F, x 450; G, x 156; H, I, x 325. Fig. 13. Drimys lanceolata CC36. - Transverse, A, B, and radial, C, sections of phloem from stem. A, tissues from right to left: secondary xylem; cambium (ca); functional secondary phloem extend- ing to the level of the open arrow marked r; nonfunctional secondary phloem merging with non- functional primary phloem, which ends at the level of two groups of protophloem fibres (asterisks); cortex with sc (large arrow) next to phloem; periderm with narrow cells, some scaling off. Letters r mark narrow rays, one and two rows wide. The much wider mr contain sc (cf. Fig. 14A) which may become dislodged in sectioning, leaving large lacunae (I below and above in A). B, functional secondary phloem with se indicated by circles and the two rays by r's. Cambium is to the right. a few xylem cells at x, cambium (ca) and phloem below. Two black arrowheads to the right point to sp. Cells with more or less chromatic contents in axial phloem are ppc. Small part of ur (r) to the left, clearest in the cambial region. A, x 91; B, x 520; C, x 143. Fig. 14. Drimys lanceolata CA390. - Tangential, A, and radial, B, sections of secondary phloem from stem. A, parts of three mr with sc; in axial tissue, se and associated ppc are unstained. Other ppc and ur (r) cells are filled with phenolic material. B, a skimming section of a ur (r) is superim- posed over some axial phloem. The latter exposes the highly chromatic strands of ppc interspersed with a few sc. The se and associated cells (white spaces) are not discernible. Both, x 130. Fig. 15. Pseudowintera axillaris CC37. Transverse sections of part of stem, A, and of secondary phloem, B. A, tissues from right to left are: cambium (ca); secondary phloem extending almost to the band of sc (open arrow near fb); nonfunctional primary phloem with protophloem fibres at fb; band of sc, partly cortical, partly stelar; cortex. Details: asterisks, oil cells in cortex (lower left) and phloem; open arrows, sc (in mr to the right); curved arrow left, above, subdivided cortical cells; cr, crystal-containing cells. B, tissue to the right is nearest the cambium. Details: asterisks, oil cells; curved arrows, crushed phloem cells; arrowheads, sp with callose; circles, se; r, ur. A, x 78; B, x 390.

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Fig. 10

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Fig. II

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Fig. 12

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Fig. 13

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Fig. 14

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Fig. 15

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(text continued from page 22) An inadequately documented attempt to in- vestigate the fine structure of the secondary contrasted with the tracheids (Table 2). The phloem of Winteraceae by Behnke and Kiritsis transections of the stem show a relation of the (1983) has resulted in a misleading interpreta- first multiseriate rays to the interfascicular re- tion of the evolutionary status of Winteraceae gions similar to that in Bubbia semecarpoides phloem. Using radial sections (instead of tan- (Fig.6A). gential as was necessary) the authors reported The two collections of Z. bicolor (CC581) that the phloem of Winteraceae originates from and Z. vinkii (CC580) were technically unsatis- a storied cambium (p. 5) and that the sieve factory for a detailed study. In Z. vinkii, the plates are predominantly simple and only slight- degree of differentiation of sieve plates and the ly inclined. Thus Winteraceae have been en- intergrading of their sieve areas with the lateral dowed with two highly advanced anatomical sieve areas are similar to those in Z. baillonii characters, an implication completely contrary (CA455). Companion cells are short. Axial to our observations. Furthermore, the authors' sclereids are elongated and have very thick cell choice of 'primitive features' at the ultrastruc- walls. Small isodiametric sclereids were noted turallevel (p. II) is meagrely supported. in rays. In Z. bicolor, sclereids are more numer- The nine species of Winteraceae examined ous in rays than in the axial system, in which by us consistently show several common fea- these cells tend to be in groups. In Z. bicolor tures in the axial system of the secondary and Z. vinkii, a few oil cells were seen in the phloem: I) occurrence of secondary partition- functional phloem region. The comparative ing in the differentiating phloem; 2) lack of lengths of tracheids and sieve elements indicate sharp differentiation of sieve areas between occurrence of secondary partitioning (Table 2). those of the sieve plates and those commonly Rugose non dispersing protein bodies occur in referred to as lateral sieve areas; 3) short com- the sieve elements. In the collections of Z. bail- panion cells, often single in a given sieve ele- lonii they are near 4 }lm in diameter, in Z. bi- ment; 4) phloem parenchyma cells in strands; color, about 8 }J.ID. 5) lack of specialised type of fibre (bast fibre) in the secondary phloem; 6) presence of non- dispersing protein body (cf. Esau, 1978) in the Discussion sieve element protoplast. The phloem of Winteraceae has received scant The secondary partitioning, first given major attention in the literature. Metcalfe and Chalk attention by Esau and Cheadle (1955) and ad- (1950, p. 29) briefly described the bark of Dri- ditionally illustrated in Calycanthaceae by mys winteri, mainly with regard to the peri- Cheadle and Esau (1958), was recorded by derm and sclerenchyma. Strasburger (1891, pp. Zahur (1959) in 200 species (47 percent) among 161-166) concluded that D. winteri had com- 424 species in 83 families. This phenomenon panion cells rather than albuminous cells of causes a reduction in the potential length of gymnospermous type. He recorded the differ- the sieve element and the appearance of trans- ences in ray structure between xylem and verse or slightly oblique sieve plates, even in phloem and reported on the origin of the secre- derivatives of cambia with long fusiform initials tory cells (oil cells) from phloem parenchyma as in Winteraceae. The systematic and phylo- cells. DeBary (1877, p. 542) recognised the pri- genetic significance of the phenomenon is ob- mary phloem fibres (protophloem) in the peri- scure, for partitioning occurs in families at va- pheral stelar sclerenchyma. Van Tieghem (1900) rious levels of evolutionary advancement as de- reported such fibres under the term 'pericyclic termined by congeries of characters (Cronquist, fibres' in D. winteri, Exospermum stipitatum, 1981). Zahur (1959) suggested that the 'second- and Zygogynum baillonii, and apparently saw ary septation' (his term) is a derived character them in other species as well. He also examined and that it has appeared de novo in phloem the sclerenchyma in the bark and pith in six development of angiosperms. Carlquist (1961, genera of Winteraceae (five if Wintera is classi- p. 77) has questioned this in terpreta tion, spe- fied as Drimys). Santos (1960) observed scle- cifically on the basis that secondary partition- reids but no fibres in the secondary phloem of ing occurs in a considerable number of fami- D. lanceolata. lies which are not advanced in many other According to Behnke (1972, 1981), Wintera- characters. The common occurrence of sec- ceae belong to dicotyledons having starch- ondary partitioning in the generally primitive depositing plastids in the sieve elements. Behnke Winteraceae lends support to Carlquist's scep- and Kiritsis (1983) and Spanner and Moattari ticism. (1978) observed P-protein in sieve elements of The most certain way to identify secondary Drimys winteri. partitioning is to study the cambial region in a

Downloaded from Brill.com10/07/2021 05:17:02PM via free access 40 IAWA Bulletin n.s., Vol. 5 (I), 1984 growing stem. Such cambium was available in containing parenchyma cells are sparse. Some our collections of Drimys winteri, D. lanceolata were noted in Drimys lanceolata. and Pseudowintera axil/aris. In all other taxa, a Sclerenchyma is a common component of comparison of sieve elements with xylem tra- phloem tissue and is frequently represented by cheids gave us the necessary information, for fibres (Esau, 1969, 1979). Typical pointed (fu- mature tracheids closely agree with the fusi- siform) fibres with thick cell walls and few pits form cambial initials in length and form. The are not present in the secondary phloem of sieve elements of Winteraceae are at least a Winteraceae. Most species examined, however, fourth the length of the tracheids, and no sieve contained sclereids. Several of the species ex- element seen was more than a third the length amined (Drimys lanceolata, Bubbia pauciflora, of the shortest tracheid. Exospermum stipitatum, Zygogynum bail/onii) The lack of sharp differentiation between had sclereids in axial tissue and in multiseriate the least and the most well-developed sieve areas rays. Sclereids were particularly massive in rays is a primitive character and places the Wintera- of D. lanceolata (CA390), whereas in Zygogy- ceae alongside Austrobaileya (cf. Esau, 1979) num sclerification was more prominent in the and certain Pomoideae (cf. Evert, 1960). The axial system and included uniseriate rays. Dri- attributes of the most highly differentiated sieve mys lanceolata from California and Pseudo win- areas of the Winteraceae are those usually ascri- tera axil/aris showed sc1ereids in the multiseriate bed to sieve areas of the sieve plates, even though rays. No sclereids occurred in the secondary the pores are only about I pm wide: relatively phloem in our collections of D. winteri. large size, clear delimitation from the rest of In the axial system, sclereids originate main- the wall, and well defined pores and callose de- ly by sclerification of phloem parenchyma cells posits. The wall parts bearing such sieve areas and may be as long as the neighbouring paren- may be transverse or variously inclined end chyma cells or longer. The form of some of walls or major portions of longitudinal walls. them indicates that the cells undergo a minor Using the presently widely accepted termino- amount of intrusive growth producing slight logy, we consider the sieve elements of the protrusions between adjacent cells and curving Winteraceae to be sieve tube members. We hold at the ends. It is doubtful, however, that the the same view regarding the sieve elements of longish sclereids attain their length by intrusive Austrobaileya and the Pomoideae. The occur- growth. Instead, they may be derived from pre- rence in the primitive family Win teraceae of a cursors of phloem parenchyma cells in which low level of differentiation of sieve areas and of fewer transverse divisions occurred during the an unbroken intergradation between lateral formation of a parenchyma strand. Isodiametric sieve areas and those in sieve plates suggests sclereids of the stone cell type may be mixed that such a condition itself is primitive for sieve with elongated types, as in Zygogynum. ScJe- elements. This conclusion is in harmony with reid walls vary in thickness and are pitted. the widely accepted view that in a highly evolv- We were able to identify protophloem fibres ed sieve element the sieve plates differ greatly in the outermost part of the phloem as groups from sieve areas elsewhere. of thick-walled, small, isodiametric cells in The small companion cells are often single in transverse sections and as equally thick-walled, a given sieve element, although there may be long slender cells in longitudinal sections and two closely associated or spatially separated macerations. Such fibres were located in their companion cells. Using his survey of 423 spe- proper positions in all species except in the cies of dicotyledons, Zahur (1959) suggested older stem of Drimys lanceolata (CA390), prob- the following evolu tionary sequence in the sieve ably because of dislocations associated with di- element-companion cell association: 1) one or latation growth and subsequent general sc1eri- more single companion cells much shorter than ficatipn in that stem. In Bubbia semecarpoides the sieve element; 2) one or more single compa- the presumed early fibres were embedded in nion cells as long as the sieve element; 3) com- masses of scJereids among which no cell stood panion cells in a strand extending the length of out as a possible fibre. Longitudinal sections, the sieve element. Winteraceae is in the least however, revealed long fibre-like cells among advanced position in this sequence. shorter scJereids. In Winteraceae, phloem parenchyma cells ap- The scJerenchyma appearing in a perivascu- pear as strands, which may be difficult to dif- lar position in Winteraceae shows an inconsis- ferentiate from uniseriate rays. Cells in both tent relation to the protophloem fibres in the kinds of assemblages often contain chromatic different species. In Drimys winteri and D. lan- phenolic materials. Parenchyma cells closely as- ceolata the perivascular scJerenchyma appears sociated with sieve elements and companion in discrete groups, some of which are in con- cells are usually free of these materials. Crystal- tact with the fibres. Pseudowintera axil/aris,

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Bubbia semecarpoides, and Zygogynum bai/- rays with differences in cell form in the two lonii show a more or less continuous layer of kinds ofrays (Bailey, 1944). perivascular sclerenchyma. In P. axillaris the Since phloem and xylem arise from the same protophloem fibres are embedded in this scle- cambial initials their tissue organisation is fun- renchyma, whereas in the two other species the damentally similar. Accordingly, the phloem of fibres occur more deeply in the stem and are Winteraceae contains high multiseriate and high free from outer sclerenchyma. We consider that uniseriate rays. But the methods of growth of the perivascular sclerenchyma arises entirely in the secondary xylem and secondary phloem are the cortex in some species and partly in the so different that structural divergencies arise cortex and partly in the primary phloem region during the differentiation of the two kinds of in others. tissue. On the xylem side, the adjustment to Van Tieghem's (1900) survey of sclerenchy- the increase in stem circumference depends en- rna yielded data somewhat at variance from tirely on increases in number and tangential ours. Sclerification is affected by the age of diameters of cells in the cambial initial region stems. Since all stems used by Van Tieghem in- (Bailey, 1923). Subsequently the development cluded pith they must have been relatively of secondary walls in the axial component of young. Stems in our collections varied greatly the xylem fixes the ray pattern essentially in in age and only those of Bubbia semecarpoides the same form as it was in the cambium. In and Zygogynum baillonii included pith. Van contrast, the phloem rays are exposed to tan- Tieghem combined the survey of sclerenchyma gential stresses resulting from growth in cir- with that of crystals (calcium oxalate). The cumference of the entire bark, to which they processing for microscopy of our material was respond by tangential enlargement and radial not designed to preserve crystals. Only Pseudo- anticlinal divisions. wintera axillaris showed some crystal-containing Slightly oblique tangential sections passing cells in which the crystals were represented by through xylem, cambium, and phloem graphi- outlines of their cytoplasmic sheaths. cally illustrate that the cell form in the phloem Van Tieghem observed no sclerenchyma cells ray is initially the same as that in the xylem and no crystals in Drimys winteri but mentioned ray, but farther from the xylem the cellular their occurrence in some other Drimys species. conformation in the phloem ray changes. The In a small contribution to bark anatomy of D. typical feature of the heterogeneous type I ray piperita, Rao (1977) described sclereids in the system, the presence of upright cells in the uni- cortex of this species and distinguished between seriate rays, and in the margins and vertical ex- the short brachysclereids and the elongated tensions of the multiseriate rays, becomes ob- idiosc1ereids. In Wintera (presumably Drimys scured by an increase in cell width. In addition, lanceolata), Bubbia and Belliolum, Van Tieghem according to Strasburger (1891, pp. 165-166) found sclereids, singly or in groups, each in- and our present observations, in Drimys winteri cluding a solitary crystal. In Exospermum and the initially upright cells divide transversely Zygogynum sclereids were present in cortex and produce shorter cells. This phenomenon and pith, bu t the crystals occurred separately occurs as soon as the rays emerge from the in thin-walled cells. The secondary phloem in cambium, before secondary partitioning is ini- these two genera showed some long, late matu- tiated. In contrast to D. winteri, D. lanceolata ring sclerenchyma cells referred to as fibres by and Pseudowintera axillaris show no transverse the author. The late maturation suggests that divisions in the young rays, and the cells in the cells may have been fibre-sclereids. their uniseriate rays and parts of the multiseriate All species of Winteraceae examined have rays are more elongated than the cells in similar non dispersing protein bodies in the sieve ele- position in D. winteri. Elongated but wide ray ments. The bodies were found to be approxi- cells are found also in Zygogynum baillonii mately 4 J.lID in diameter in the two species of and Exospermum stipitatum. Bubbia paucij70ra Drimys, Bubbia semecarpoides, Pseudowintera shows no vertically extended ray cells in our axillaris and Zygogynum baillonii, and close to material. 8 J.lID in Bubbia paucij7ora, Exospermum stipi- In our collections the phloem rays of Pseu- tatum, Zygogynum bicolor and Z. vinkii. The dowintera axillaris (Fig. lH) did not materially species with the larger bodies were repesented differ from those of Drimys winteri and D. lan- by old stems 14-19 cm in diameter. ceolata (Fig. lB, D), whereas in the wood used The principal primitive anatomic features of by Bailey (1944) the rays of P. axillaris were the xylem of Winteraceae are the absence of two to three times wider than those in the vessels and the presence of ray system of the phloem of the two Drimys species. The dispari- heterogeneous Type 1. This system is character- ty between the two sets of observations may ised by high multiseriate and high uniseriate have been caused by differences in age of the

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materials collected. As is well known, the ray tion. But he also was aware that more data on structure changes with the age of the stem. distribution of oil cells in Winteraceae are need- Barghoorn (1940) showed that the primitive ed for an evaluation of the possible systematic rays are much extended longitudinally in the value of these cells. early secondary xylem, but are dissected into lower rays during the subsequent enlargement Acknowledgements of the stem. Other more or less extensive modi- This research was supported in part by Natio- fications of ray structure occur in successive nal Science Foundation Grant DEB 79 18833. stages of secondary growth (Barghoorn, 1941). We are grateful to Dr. W. Vink of the Leiden With regard to the phloem rays in Winteraceae, Rijksherbarium for checking the identification the most consistent feature of systematic signi- of our specimens. ficance observed in this study was the occur- rence of the combination of high multiseriate References and high uniseriate rays. Bailey, I.W. 1923. The cambium and its deriva- The secretory cells noted by Strasburger tive tissues. IV. The increase in girth of the (1891) are mentioned by Metcalfe and Chalk cambium. Amer. J. Bot. \.0: 499-509. (1950, p. 26) as being generally present in lea- - 1944. The comparative morphology of the ves and occurring in the phloem of stems of Winteraceae. III. Wood. J. Am. Arbor. 25: Drimys winteri. Bailey and Nast (1945) made a 97-103. summary statement that the leaves of Wintera- - & C.G. Nast. 1945. The comparative mor- ceae have 'numerous ethereal oil cells.' West phology of Winteraceae. VII. Summary and (1969) referred to presence of 'idioblastic oil conclusions. J. Am. Arbor. 26: 37-47. cells' as a 'unifying characteristic for the woody Barghoom, E.S. 1940. The ontogenetic develop- ranalian complex.' He included D. winteri and ment and phylogenetic specialization of Pseudowintera axillaris in his study and used rays in the xylem of dicotyledons. l. The shoot tips and developing and mature stems primitive ray structure. Amer. J. Bot. 27: and leaves. Phloem and vascular cambium were 918-928. not investigated. Van Tieghem (1900) briefly - 1941. The ontogenetic development and mentioned the presence of oil cells in the cor- phylogenetic specialization in the xylem of tex, secondary phloem, and pith of stems of D. dicotyledons. II. Modification of the multi- winteri, Exospermum stipitatum, and Zygogy- seriate and uniseriate rays. Amer. J. Bot. 28: num baillonii, but did not imply that they were 273-282. absent in species that were not specifically Behnke, H.-D. 1972. Sieve-tube plastids in rela- mentioned in this connection. In D. winteri oil tion to angiosperm systematics- an attempt cells were also present in the cortex and second- towards a classification by ultrastructural ary phloem of the root. analysis. Bot. Rev. 38: 155-197. In our material, oil cells were particularly - 1981. Sieve element characters. Nordic J. abundant in D. winteri phloem, in which they Bot. I: 381-400. were found in both axial and ray systems. They - & U. Kiritsis. 1983. Ultrastructure of dif- were less abundant in Bubbia semeearpoides, ferentiating sieve elements in primitive an- Exospermum stipitatum, Pseudowintera axilla- giosperms. II. Winteraceae. Protoplasm a (in ris, Zygogynum bie%r, and Z. vinkii. We think press). that our failure to find oil cells in species stud- Carlquist, S. 1961. Comparative plant anatomy. ied, but not named in the list above, does not Holt, Rinehart & Winston, New York. necessarily mean that they never occur in those - 1983. Wood anatomy of Bubbia (Wintera- species. For example, Z. baillonii collected by ceae), with comments on origin of vessels in Van Tieghem (1900) showed oil cells, whereas dicotyledons. Amer. J. Bot. 70: 578-590. none was seen in Z. balllonii in our collections. Cheadle, V.I. & K. Esau.1958. Secondary phloem Perhaps the developmentof oil cells is promoted of the Calycanthaceae. Univ. Calif. Pub!. by some environmental conditions and repres- Bot. 29: 397-510. sed by others. - & - 1964. Secondary phloem of Lirioden- Carlquist (1983) recorded oil cells in the xy- dron tulipifera. Univ. Calif. Pub!. Bot. 36: lem rays of Bubbia, Exospermum and Zygogy- 143-252. num, but not in Bellio/um. Patel (1974) report- - , E.M. Gifford & K. Esau. 1953. A staining ed their absence in xylem rays of Pseudo win- combination for phloem and contiguous tera. Carlquist (1983) pointed out that the tissues. Stain Techno!. 28: 49-53. three genera having oil cells in xylem rays also Cronquist, A. 1981. An integrated system of show similarities in morphology of the carpel, classification of flowering plants. Columbia form of the stamen, and geographic distribu- Univ. Press, New York.

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DeBary, A. 1877. Vergleichende Anatomie der Rao, A.N. 1977. Bark and sclereid structure in Vegetationsorgane der Phanerogamen und Drimys piperita Hook. Curro Sci. 46: 529. Fame. Wilhelm Engelmann, Leipzig. Santos, C.F. de Oliveira. 1960. Estudo do escle- Ehrendorfer, F., F. Krendl, E. Habeler & W. renchyma no floema secundario de algumas Sauer. 1968. Chromosome numbers and dicotiledoneas lenhosas. Rev. AgricuIt. 35: evolution in primitive angiosperms. Taxon 199-206. 17: 337-353. Sass, J.E. 195 I. Botanical microtechnique. 2nd Esau, K. 1969. The Phloem. Handbuch der Ed. The Iowa State College Press, Ames, Pflanzenanatomie. Band 5. Teil 2. Gebr. Iowa. Borntraeger, Berlin, Stuttgart. Smith, A. C. 1969. A reconsideration of the - 1978. The protein inclusions in sieve ele- genus Tasmannia (Winteraceae). Taxon 18: ments of cotton (Gossypium hirsutum L.). 286-290. J. Ultrastruct. Res. 63: 224-235. Spanner, D.C. & F. Moattari. 1978. The signifi- - 1979. Phloem. Chapter 12, pp. 181-189 & cance of P-protein and endoplasmic reticu- plates 11-18 in C.R. Metcalfe & L. Chalk, lum in sieve elements in the light of evolu- Anatomy of the dicotyledons. 2nd Ed. Vol. tionary origins. Ann. Bot. 42: 1469-1472. I. Systematic anatomy of leaf and stem, Strasburger, E. 1891. Ueber den Bau und die with a brief history of the subject. Claren- Verrichtungen der Leitungsbahnen in den don Press, Oxford. Pflanzen. Histologische Beitriige. Heft III. - & V.I. Cheadle. 1955. Significance of cell di- Gustav Fischer, Jena. vision in differentiating secondary phloem. TIeghem, Ph. van. 1900. Dicotyledones du groupe Acta Bot. Neerl. 4: 346-357. de Homoxylt~es. J. de Bot. 14: 259-297, - & - 1958. Wall thickening in sieve ele- 330-361. ments. Proc. Natl. Acad. Sci. USA44: 546- Vink, W. 1970. The Winteraceae of the Old 553. World. I. Pseudowintera and Drimys. Mor- - & - 1959. Size of pores and their con- phology and taxonomy. Blumea 18: 225- tents in sieve elements of dicotyledons. 354. Proc. Natl. Acad. Sci. USA 45: 156-162. West, W.C. 1969. Ontogeny of oil cells in the - ,-& E.M. Gifford Jr. 1953. Comparative woody Ranales. Bull. Torrey Bot. Club 96: structure and possible trends of specializa- 329-344. tion of the phloem. Amer. J. Bot. 40: 9-19. Willis, J .C. 1973. A dictionary of the flowering Evert, R.F. 1960. Phloem structure in Pyrus plants and ferns. 7th Ed. Revised by H.K. communis L. and its seasonal changes. Univ. Airy Shaw. Univ. Press, Cambridge. Calif. Publ. Bot. 32: 127-194. Zahur, M.S. 1959. Comparative study of Sec- Metcalfe, C.R. & L. Chalk. 1950. Anatomy of ondary phloem of 423 species of woody di- the dicotyledons. Clarendon Press, Oxford. cotyledons belonging to 85 families. Cornell Patel, R.N. 1974. Wood anatomy of the dicoty- Univ. Agr. Exp. Sta. Mem. 358. ledons indigenous to New Zealand. 4. Win- teraceae. New Zeal. J. Bot. 12: 19-32.

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