IAWA Journal, Vol. 17 (2),1996: 161-181

INTERVASCULAR PIT MEMBRANE STRUCTURE IN AND WIKSTROEMIA - SYSTEMATIC IMPLICATIONS 1 by Roland R. Dute-, John D. Freeman", Frank Henning- & Logan D. Barnard-

SUMMARY

Intervascular pit membranes were investigated in species of Daphne, Wikstroemia, and other allied genera of the . Results con• firmed a previous study showing that, except for section Mezereum, all sections of Daphne had pit membranes with tori. Taxonomically isolated species D. aurantiaca and D. genkwa had tori, but lacked a G-layer. Tori similar in structure to those of D. aurantiaca and D. genkwa were ob• served in three species from the subgenus Diplomorpha of Wikstroemia. Tori of a slightly different morphology were noted in W kudoi (subg. Daphnimorpha). Tori appeared absent from species of the subgenus Wikstroemia (= Euwikstroemia of Domke), and from the genera Drapetes, Edgeworthia, and Eriosolena. These results suggest a close relationship between Daphne and Wikstroemia. The degree of torus development and the distinctiveness of helical thickenings suggest that smaller tracheary elements serve as a backup water-conducting system to larger vessel elements. Key words: Daphne, Wikstroemia, Thymelaeaceae, pit membrane, torus, wood ultrastructure.

INTRODUCTION

The genus Daphne is unusual in that it is one of four genera ofangiosperms known to have intervascular pit membranes with tori (q.v. discussion in Dute et al. 1990). Ohtani and Ishida (1978a) were the first to comment upon this feature in three species of Daphne common to Japan. Later, Dute et al. (1990) detailed torus development in D. odora . In a survey of five species, Ohtani (1983) observed that the three species with tori belonged to a different section of the genus than the two species without tori. Dute et al. (1990) independently noted that whereas intervascular pit membranes in D. odora and D. cneorum always had tori, those of D. mezereum did not. It was hypothesized

1) Contribution No. 6-955054 from the Alabama Agricultural Experiment Station, Auburn University, Auburn, AL 36849, U,S. A. 2) Department of Botany and Microbiology and Alabama Agricultural Experiment Station, Auburn University, Auburn AL 36849, U.S .A. Correspondence should be addressed to R.R. Dute. 3) College of Agricultural and Environmental Sciences, Cooperative Extension Service, The University of Georgia. 4) Department of Horticulture, Auburn University.

Downloaded from Brill.com10/07/2021 09:32:29PM via free access 162 IAWA Journal, Vol. 17 (2), 1996 that the presence of torus-bearing pit membranes could be correlated with either growth habit (D. odora and D. cneorum are evergreen; D. mezereum, deciduous) or systemat• ics. A later study (Dute et al. 1992) of 22 taxa (21 species and 1 hybrid) of Daphne seemed to confirm the latter possibility. The only species without tori were those with• in the section Mezereum. Nevertheless, because Daphne has 50 to 70 species (Bailey 1949; Brickell & Mathew 1976) the study of Dute et al. (1992) cannot be considered comprehensive. The species so far investigated are listed in Table 1.

Table I. Distribution of common horticultural species within the genus Daphne according to Brickell and Mathew (1976) based on Kessler (1898) . Species with name s in boldface type previously were examined for presence of tori in this laboratory (Duteet al. 1990, 1992).

Daphne (Daphne Sect. Mezereum sect. Daphnanthes subsect. Oleoides contd) D.jezoensis D. rodriguezii D. kamtschatica D. stapfi i D. koreana D. transcaucasica D. mezereum Subsect. Gnidium D. pseudomezereum D.gnidium D. rechingeri Subsect. Collinae Sect. Genkwa D. blagayana D. genkwa D. collina Sect. Laureola D. macrantha D. albowiana D. sericea D. glomerata Subsec t. Cneorum D.laureola D. arbuscula D. pontica D. cneorum Sect. Eriosolena D.juliae D. aurantiaca D. petraea Sect. Daphnanthes D. striata Subsect. Alpinae Subsect. Daphnanthoides D. alpina D. acutiloba D. altaica D. bholua D. caucasica D. grueningiana D. giraldii D. kiusiana D. sophia D. longilobata Subsect. Oleoides D.luzonica D. baksanica D. miyabeana D. euboica D.odora D. gnidioides D. papyracea D.jasminea D. retusa D. kosaninii D. shillong D. linearifolia D. sureil D. mucronata D. taiwaniana D.oleoides D. tangutica

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The genus Daphne is notwell circumscribed.Species of section Eriosolena are some• times included in a separate genus due to their distinct floral characteristics (e.g., Hallier 1922; Leandri 1930; Domke 1934). Other species, mostly of the section Genkwa, have been placed in the genus Wikstroemia, based in part upon opposite leaf arrangement. The purpose of this study is to continue the survey of Daphne species for torus• containing pit membranes. Some species of Wikstroemia and other related genera (Dra• petes, Edgeworthia, Eriosolena) also are examined. Although no one feature provides sufficient grounds on which to base a taxonomic treatment, pit membrane structure in conjunction with floral and vegetative characters furnishes valuable information for understanding the circumscription of Daphne.

MATERIALS AND METHODS

Table 2 lists the species used in this study. Herbarium specimens were provided by Dr. Hiroo Kanai of the Tokyo National Science Museum, Dr. Mikio Ono of the Makino Herbarium of the Tokyo Metropolitan University, Drs. Ghillean T. Prance and G. L1. Lucas of the Royal Botanic Gardens, Kew, and Dr. P. Baas of the Rijksherbarium, Leiden . A single specimen of Daphne genkwa from the Auburn University Herbarium was also studied. Dr. Stephanie Mayer of the University of Chicago supplied air-dried branches from living specimens in her collection. These collections provided exam• ples of species from the closely related genera Daphne, Wikstroemia , Drapetes, and Eriosolena . In addition, living specimens of another genus of the Thymelaeaceae, Edge• worthia, were obtained from Gossler Farms Nursery, Springfield, Oregon, USA and grown in a greenhouse until needed for comparative purposes. For scanning electron microscopy (SEM), split radial sections (from branches 3-5 mm in diameter) of herbarium specimens were attached to aluminum stubs with dou• ble stick carbon tape. Specimens and stubs were sputter-coated with gold-palladium. Split radial sections ofEdgeworthia papyrijera were allowed to air-dry before mount• ing. Specimens were viewed with a Zeiss Digital Scanning Microscope (DSM 940) at voltages of 5, 10, and 15 kY. Preparation of herbarium specimens for transmission electron microscopy (TEM) was according to the method of Dute et al. (1992) in which pieces of wood no more than I mm on a side were soaked in three changes of absolute acetone followed by propylene oxide and embedment in Spurr's resin (Spurr 1969). Transverse, radial and tangential monitor sections of 1.5-2 mm were cut, affixed to glass slides, and stained with toluidine blue 0 and basic fuchsin for light microscopy. Transverse silver sec• tions were cut on a Sorvall MT-2b ultramicrotome and stained with uranyl acetate and lead citrate . Observations were made with a Zeiss 10 transmission electron micro• scope operated at 60 kV. Monitor sections often would not dry flat onto a glass slide but rather contained air bubbles . To eliminate this problem and to provide better resolution, monitor sections were deplasticized using the following method .Transverse sections of Spurr's-embed• ded material were cut to thicknesses of 0.5 to 3 mm and placed into a drop of water on a precleaned circular coverslip (15 mm diameter). The sections then were heat-fixed to

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Table 2. Sources of wood specimens examined in this study.

Taxon Herb./City Collection date Collector(s) number

Daphne Sect. Mezereum D. kamts chaticum TNS 10 Jul1933 Ono s.n. D. koreana TNS 11 Aug 1954 Furuse s.n . Sect. Genkwa D. genkwa Lw < 1827 Siebold & Zuccarini s.n. K 21 Apr 1981 Green 2148 AUA 9 May 1980 Miller & Yinger Sect. Eriosolena D. aurantiaca K 1913 Forrest 1018 D.pendula K 1 Apr 1930 Henderson 23274 Sect. Daphnanthes Subsect. Alpinae D. alpina TNS I Jun 1903 Bouchard s.n. D. altaica TNS 1840-1841 Schrenk 1306 Subsect. Oleoides D. gnidiodes K 26 Jul1960 Khan, Prance & Ratcliffe 255 D.jasminea K Apr 1939 Atchley 1866 D. stapfii K None listed Jacobs 6604 Subsect. Gnidium D. gnidium TNS 6 Oct 1950 Matos & Matos s. n. Subsect. Cneorum D. bholua TNS 9 Mar 1953 Nakao s.n . K 29 May 1971 Colville , Barclay & Syrge 2396 D. petraea K 24 May 1870 Porta s.n. Subsect. Daphnanthoides D. arisanensis TNS 20Aug 1969 Yamazaki, Namba & Tani s.n. D. kiusiana TNS 19 May 1974 Ito 234 D. miyabeana TNS 7-9 Aug 1958 Okuyama 12837 Subsect. Collinae D. collina TNS 26 Jun 1853 Kotschy s.n. Drapetes D. ericoides TNS 24 Jun 1974 Craven 2768 Eriosolena E. wallichii K None listed Hooker 11150 Edgeworthia E. papyrifera Auburn 15 Oct 1992 Dute Wikstroemia Subg . Diplomorpha W. albiflora MAK 16-20 Aug 1960 Hatusima & Sako 25120 W. ganpi MAK Sep 1936 Makino s.n. W. pauciflora MAK Aug 1904 Makino s.n . W. sikokiana MAK 10 Jul1956 Satomi s.n . W. trichotoma MAK 22 Ju11964 Murata 18605 W. yakushimensis MAK 16-20 Aug 1960 Hatusima & Sako 25075 Subg . Daphnimorpha W. kudoi MAK 20Jul1965 Satomi 25126 Subg. Wikstroemia W. pseudoretusa MAK 12 Nov 1970 Ono & Kobayashi s.n. W. retusa MAK 19 Mar 1981 Sugawara 1565 Chicago 21 Sep 1994 Mayer W. indica Chicago 21 Sep 1994 Mayer

Downloaded from Brill.com10/07/2021 09:32:29PM via free access Dute, Freeman, Hennin g & Barnard - Pit membranes in Daphne and Wikstroemia 165 the coverslip. Resin was removed using a Poly/Solv Kit (Polysciences Inc., Warrington, PA, USA, Data Sheet #291). The instructions were followed except that it was found necessary to increase the time in the initial plastic solubilizing mixture from 5 to 10 minutes. After the final treatment, the specimens (still attached to the coverslips) were passed through two changes of absolute ethanol and air-dried. The coverslips were then fixed to aluminum stubs with double-sided carbon tape and sputter-coated. To ensure a conductive surface, silver paint was used to coat the underside and edges of the coverslips where they extended beyond the surface of the aluminum stub. Speci• mens were viewed in a typical manner using the SEM. Although air bubbles were present in the sections at the time that they were dried onto the coverslip, the speci• mens were completely flat after resin removal. Maceration of wood from herbarium specimens was accomplished according to the techniqu e of Wheeler (1983). Wood slivers were placed in a solution cons isting of equal parts of glacial acetic acid and hydrogen peroxide at 50°C for 3 days. Specimens were then washed in distilled water and stained with 1.0% toluidine blue 0 (TBO) prior to observation. Measurements were made from macerations using a light microscope and from ra• dial sections using the scanning electron microscope with a Thomas Optical (Columbus, Georgia, USA) image analysis system running Optimas software . All measurements were based on counts of 25 cells unless otherwise noted.

RESULTS

Daphne aurantiaca and D. genkwa occupy isolated positions within the genus. Wood of these species has not been investigated previously, so detailed descriptions of the water-conducting cells are given below.

Daphne aurantiaca As in other daphnes, wood of D. aurantiaca has both tracheids and vessel elements. The two types of tracheary elements are of similar lengths:tracheids have a mean length of 235.4 urn (range 152.1-293.5 urn); vessel elements a mean of 222.8 urn (range 136.6- 294.7 urn). These tracheids are distinctly shorter than fibre-tracheids (mean 463.0 urn; range 3 14.8-610.3 urn). Tracheids are narrower (19.9 urn mean, 13.7-28.5 urn range) than vessel elements (43.7 urn mean, 28.5-69.9 urn range) (Fig. 1). In macerations, vessel elements stain a much deeper purple (with a slight reddish tinge) with TBO than do the tracheids, so the vessel elements appear darker than the tracheids in black and white photographs (Fig. I). Also, tracheids have bordered pits with circular apertures with only sparse vestures (Fig.2), whereas vessel elements have pits with slit-like apertures containing vestures of variable development (Fig. 2, 3). The most noticeable difference between the two cell types is the presence of distinct helical thickenings in tracheids and their absence from all but the smallest vessel ele• ments (Fig. 1-3). The thickening s are especiall y noticeable with the SEM in radial longitudinal sections (Fig. 2, 4). Among all the Daphne species so far studied in this laboratory, the thickenings in D. aurantia ca project farthest into the tracheid lumen.

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Fig. 1-4. Light and SEM micrographs of Daphne aurantiaca . - 1: Vessel element (left) and tracheid from macerated material. Note the darker stain of the vessel element. Both perfora• tions of this cell are out of the plane of focus. Scale = 30 1JIll. - 2: Comparison of lumen surface s and pit apertures between vessel element and tracheids. Scale = IO 1JIll. - 3: Detailed view of lumen surface of vessel element. Scale = 21JIll. - 4: Detailed view of tracheid lumen with helical thickenings. Scale = 51JIll. - See also the Key to labelling on p. 167.

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Key to labelling of all figures: A = aperture B = boundary between two growth rings G = G-layer of torus H = helical thickening M = margo P = perforation T = tracheid TE = tracheary element TO = torus V = vessel element VE = vesture W = wart WO = wound material

Fig. 5-7. SEM micrographs of Daphne aurantiaca. - 5: Sec• tioned material viewed with SEM showing association of vessel elements and tracheids . Note how the helical thicken• ings constrict the cell lumens ofthe tracheids. Scale = 20 um, - 6 & 7: Surface views of pit membrane . Arrow = radial fi• bril in margo. Scales = I f.l1TI .

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The extent to which the helical thickenings constrict the lumen diameter can best be observed in cross sections of deplasticized wood (Fig. 5). The extent ofthe thickenings is such that in those cells where they remain intact it is often difficult to locate the pit apertures (Fig. 4).

Fig. 8-10. TEM micrographs of Daphne aurantiaca . - 8: Wound material (arrows) impregnat• ing pit membrane and coating torus, pit cavities (and cell lumens). - 9: Detailed view of torus ; no G-Iayer on either surface. - 10: Pit membrane between vessel element (below) and tracheid . Torus thickening (arrow) on lumen surface of latter cell. - All scales =0.5 urn. - See also the Key to labelling on p. 167.

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With the light microscope the interior of vessel elements generally appears smooth as it does at low magnification with the SEM (Fig. 2). Detailed investigations, however, reveal the lumen surface to be coated with small warts (Fig. 3). Cross sections (viewed with both light and SEM) show tracheary elements in radial multiples of varying lengths, and these multiples may cross the growth ring bounda• ries. Vessel elements are closely associated with tracheids (Fig. I, 5). The intertracheid pit membranes are differentiated into torus and margo regions (Fig. 6, 7). The torus is smooth-surface (although one exception was noted) and has a mean diameter of 2.66 JlIl1 (range 2.11-3.17 JlIl1 ; 15 measurements). Sometimes the outline of the torus can be very irregular (Fig. 7). A distinct radial component to the fibrillar margo is noticeable in aspirated pit membranes (Fig. 6), the aspiration being apparent either from the outline of the aperture beneath the torus or by the evident displacement of the membrane. Thin sections of tracheids show no completely aspirated membranes. Some pit cavi• ties and tracheid lumens are lined with a thin layer of material that probably represents a wound response (Dute et al. 1992) (Fig. 8). This material also impregnates the margo of the pit membrane and coats both surfaces of the torus (Fig. 8). Other pit membranes lack this material (Fig. 9). Detailed views of the tori in sectional view (Fig. 9) show that they lack the layer of granular material (a rather unfortunate choice of terms now in the literature) or G-Iayer on the torus surfaces, a feature present in other torus• bearing Daphne species investigated (Dute et al. 1990). Light microscopy indicates that torus thickenings are best developed between tracheids, but they also occur between tracheids and vessel elements with the thicken• ing restricted to the tracheid surface of the pit membrane (Fig. 10). Pit membranes connecting two vessel elements were not observed.

Daphne genkwa Both tracheids and vessel elements of D. genkwa can contain helical thickenings. The distinctness of the thickenings varies from cell to cell; as a general rule, helical thickenings are better developed in narrower cells . Thus, wide vessel elements seem to lack this feature . Measurements from two specimens of D. genkwa show a distinct size overlap in the diameter ranges of vessel elements and tracheids (Table 3). Without careful focusing,

Table 3. Cell sizes in Daphne genkwa.

Specimen Cell type Length Range Width Range (J.IIll) (J.IIll)

Rijksherbarium vessel element 203 111-209 45 11-65 tracheid 188 149-232 14 8-20

Kew Gardens vessel element 200 123-254 42 15-98 tracheid 208 142-310 16 8-22

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Downloaded from Brill.com10/07/2021 09:32:29PM via free access Dute, Freeman, Henning & Barnard - Pit membranes in Daphne and Wikstroemia 171 very narrow vessel elements can be mistaken for tracheids in macerations (Fig. II). Distinguishing these cell types is even more difficult in transverse sections . Therefore, for purposes of this discussion, the tracheids and narrow vessel elements will be re• ferred to as 'tracheary elements' to distinguish them from the wider vessel elements. As mentioned previously (Dute et al. 1992), the apertures are heavily encrusted with vestures as can be seen in both SEM or TEM micrographs. Aperture shape varies from circular to elliptical with the latter associated with (wide) vessel elements. Tracheary elements are associated with vessel elements and, in addition, form ter• minal bands at the latewood boundary (Fig. 12). Transverse sections of deplasticized specimens indicate how the helical thickenings of the tracheary elements decrease the diameter of the cell lumens (Fig. 13). The results of a previous investigation (Dute et al. 1992) indicated that due to depo• sition of wound material , tori distinct from the margo were difficult to observe with the SEM. Therefore, ultrathin sections of two samples were observed with the TEM. Such sections show a variation in the amount of wound deposition on the pit mem• branes from slight (Fig. 14) through moderate (Fig. 15) to heavy (Fig. 16). Torus thickenings are best developed on those pit membranes separating tracheary elements (Fig. 14). Where a tracheary element adjoins a large vessel element, the torus thickening on the vessel element surface of the pit membrane is of variable develop• ment (i.e . from well-developed to absent). Detailed sectional views indicate that the torus surfaces lack the G-Iayer (Fig. 14).

Other Daphne species The following species of Daphne had torus thickenings on intervascular pit mem• branes of both tracheids and vessel elements: D. alpina, D. altaica, D arisanensis, D. bholua, D. collina, D. gnidioides, D. gnidium, D.jasminea, D. kiusiana, D. miyabeana, D. petraea, and D. stapfii. Some specimens were from species (D. alpina, D. altaica, D. collina, D. gnidium, D. kiusiana and D. miyabeana) found to have tori in previous investigations (Dute et al. 1992; Ohtani & Ishida 1978a; Ohtani 1983). The torus in surface view (as seen with SEM) is circular, is larger than its subtending aperture, and often has granules in its center (Fig. 17). Apertures vary from circular to elliptical (Fig. 18). Transverse thin sections of the wood of D. alpina and D. bholua were observed with the TEM. Some pit membranes are aspirated (Fig. 19); some are not (Fig. 20). No

Fig. 11-16. Light, TEM, and SEM micrographs of Daphne genkwa . - II: Cells from macerated tissue. A tracheid (below) alongside two narrow diameter vessel elements. A perforation is visible in the center cell, but none is visible in the upper cell in this plane of focus. Scale = 20 urn. • 12 & 13: SEM of transversely-sectioned wood . - 12: Low magnification showing distribution of large vessel elements and small diameter tracheary elements. Scale = 50 urn. - 13: Detailed view of latewood. Helical thickenings constrict the lumen diameter. Scale = 10 um. - 14, 15, & 16: Pit membranes between tracheary elements as seen with the TEM. Variable amounts of wound material (arrows) are present. A G-Iayer is absent. Scales = 0.5 um,- See also the Key to labelling on p. 167.

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Fig. 17-21. Pit morphology in species of Daphne . - 17: Pit membrane of Daphne gnidioides with a torus. Scale = 11JITl . - 18: Conducting elements in D. gnidioides showing pit membranes with tori and small circular apertures. Scale = 5 1JITl. - 19 & 20: Tori found in D. a/pina and D. bholua . The former is aspirated. Both tori have G-Iayers . Scale = 0.5 1JITl. - 21: Pit mem• branes (unlabelled arrows) in vessel elements of D. koreana. No tori are present. The dark regions in the center of the pit membranes indicate the subtending apertures. Scale = 5 1JITl. - See also the Key to labelling on p. 167.

Downloaded from Brill.com10/07/2021 09:32:29PM via free access Dute, Freeman, Henning & Barnard - Pit membranes in Daphne and Wikstroemia 173 wound material is present in either case . The tori of both species have a granular or G• layer on either surface (Fig. 19,20). In an ontogenetic study of D. odora (Dute et al. 1990), G-Iayer deposition was associated with a microtubule plexus and vesicles. Neither D. koreana nor D. kamtschaticum has tori (Fig. 21). This observation agrees with previous observations on other members of section Mezereum (D. mezereum, D. pseudomezereum, D. jezoensis, Dute et al. 1990, 1992; D. kamtschaticum, D. pseudo• mezereum, Ohtani 1983).

Drapetes ericoides No evidence for tori was found in D. ericoides (Fig. 22).

Edgeworthia papyrifera Helical thickenings in vessel elements of E. papyrifera are visible only at high mag• nification (Fig. 23) and, in some cases, might represent nothing more than small clus• ters of microfibrils. Pit apertures are elliptical with numerous, distinct vestures (Fig. 23). Surface views of pit membranes give no indication of tori.

Fig. 22-25. SEM micrographs of wood from genera related to Daphne. - 22: Intervascular pit membrane of Drapetes ericoides; no torus. Scale = I um. - 23: Vessel element lumen (Edge• worthia papyrifera) with faint helical thickenings . Scale = 5 urn. - 24: Vessel element of Erio• solena wallichii with perforation and faint helical thickenings . Scale = 10 urn. - 25: Intervessel pit membranes (no torus) and apertures with vestures in wood of E. wallichii. Scale = I mm.• See also the Key to labelling on p. 167.

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Eriosolena Like Edgeworthia papyrifera, the vessel elements of Eriosolena wallichii show faint helical thickenings (Fig. 24) as well as elongate, vestured pit apertures and have no tori (Fig. 25). The absence of tori was confirmed using TEM . Among our specimens was Daphne pendula. At one time this species was treated in the genus Daphne within the section Eriosolena (Meisner 1857); it is now considered to be a member of the genus Eriosolena. The wood features of E. pendula are identical to those of E. wallichii.

Fig. 26-30. SEM and TEM micrographs of Wikstroemia pit membranes.- 26: Surface view of pit membrane of W. yakushimensis. Arrow indicates radial fibrillar component of margo. Scale = I urn. - 27: Surface view of pit membrane in W. kudoi. The coarse nature of the margo fibrils probably indicates deposition of wound material. Scale = I 1lJ11.- 28 & 29: Sectional view of torus-bearing pit membranes connecting tracheids in W. pauciflora and W. yakushimensis, respectively. Scales = 0.5 urn & I urn. - 30: Sectional view of torus-bearing pit membrane connecting tracheids in W. kudoi. Considerable wound material (arrows) has been deposited on the torus surface, within and on the margo, and on the surfaces of the pit borders and pit canals . Scale = 0.5 urn. - See also the Key to labelling on p. 167.

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Fig. 31-35. Pit membranes in Wikstroemia species. - 31: Detailed view of torus (w. yakushi• mensis); no G-layer. Scale = 0.2l1J11.- 32:Torus from W kudoi. Note how the internal appearance differs from that in the torus of the previous figure. Scale = 0.5 l1J11. - 33: Torus of unusual morphology (W kudoi) embedded in wound material. Scale =0.5 l1J11 . - 34: Large springwood vessel element with slit-like apertures adjacent to latewood tracheids with round apertures in W kudoi. Scale =20 l1J11 . - 35: Intervascular pit membrane (arrow) of W indica with no torus. The clear area results from the separation of the pit membrane along the middle lamella and has been discussed in a previous study (Dute et aI. 1992). Scale = 0.5 l1J11. - See also the Key to labelling on p. 167.

Wikstroemia Since anatomical descriptions have already been provided for the wood of these species (Hamaya 1959), emphasis will be placed on the structure of the intervascular pit membranes. With SEM, W yakushimensis (Fig. 26), W kudoi (Fig. 27), and possibly W albiflora are the only Wikstroemia species seen to have pit membranes with tori. The outline of the torus thickening in pit membranes of W yakushimensis tends to be quite irregular. The thickness of the torus deposits varies considerably but in some cases appears so

Downloaded from Brill.com10/07/2021 09:32:29PM via free access 176 IAWA Journal, Vol. 17 (2),1996 thin that the underlying pit membrane is exposed (Fig. 26). The margo contains a distinct radial fibrillar component (Fig. 26) and tends to tear at the junction with the torus . The torus is more distinct in pit membranes of ~ kudoi (Fig. 27), but the coarse appearance of the microfibrils of the margo indicates the presence of wound deposits (q.v. Dute et al. 1992). TEM of selected samples shows torus thickenings to be present in ~ albiflora, W yakushimensis, W pauciflora and ~ kudoi (Fig. 28-33) but absent from W retusa, W pseudoretusa, and W indica (Fig. 35). In one species (~ kudoi) , the tori are embed• ded in a matrix of wound-deposited material (Fig. 30). This material also lines the surfaces of the pit borders and the cell lumen. The tori and pit membranes of W pauci• flora sometimes are associated with small amounts of this substance. Tori of ~ albiflora. W pauciflora, and W yakushimensis appear similar to those observed in Daphne aurantiaca and D. genkwa - reasonably homogeneous and with• out G-Iayers (Fig. 31) - although sometimes a middle lamella (or perhaps compound middle lamella) is visible (Fig. 28). Tori from wood of D. kudoi have a different ap• pearance often with an electron-opaque middle lamella and with a fainter staining thickened region on either side (Fig. 30, 32), but exceptions to this morphology also occur (Fig. 33). As a general rule, torus thickenings are well-developed on intertracheid pit mem• branes but are absent on intervessel membranes of large springwood vessel elements in ~ albiflora, W pauciflora, W yakushimensis, and ~ kudoi . In W kudoi the pres• ence of a torus is variable, even between tracheids . The location of tori in these spe• cies is associated with elliptical to round pit apertures in the tracheids versus slit-like apertures in the large vessel elements (Fig. 34). Poorly-developed tori are observed with the light microscope where tracheids are adjacent to the vessel elements. In such cases, the torus thickening probably only occurs on the tracheid side of the pit mem• brane. Tori also exist between small vessel elements in W albiflora and ~ kudoi .

DISCUSSION Daphne species Daphne belongs within the Thymelaeaceae (Mezereum Family). The placement of this family is in question; most authors place it outside the Myrtales (q.v. discussions in Raven 1984 and Van Vliet & Baas 1984). Wood anatomical characters support its placement in the Myrtales (Van Vliet & Baas 1984). The genus Daphne has between 50 and 70 species (Bailey 1949; Brickell & Mathew 1976) and is delimited by the following features: evergreen or deciduous shrubs; leaves mostly alternate, rarely opposite, glabrous to hairy; flowers in axillary or terminal clus• ters; flowers with 4 sepal lobes from the calyx tube; eight stamens in two whorls; short style with capitate stigma; ring-like or cup-shaped nectariferous disk at base of tube or disk lacking (Brickell & Mathew 1976; Gleason & Cronquist 1963; Hutchinson 1967; Ohwi 1965; Walker 1976). The features will be discussed below only when they have direct bearing on the species investigated in this study. As might be imagined, the ordering of the species within the genus has varied considerably over the years.

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Daphne genkwa and related species have an interesting history in the literature. Meisner (1857) put D. genkwa and D. fortunei in section Mezereum because of decid• uous foliage and lateral inflorescences, but unlike D. mezereum (and other daphnes) they have opposite leaves. Bentham and Hooker (1880) put D. genkwa and D. cham• pionii into a separate section (D.fortunei being considered a synonym of D. genkwa). Keissler (1898) retained the section Genkwa, but he considered it closely related to section Mezereum. At first glance this might not make sense based on pit membranes because the section Mezereum lacks tori and D. genkwa has them; however, the ultra• structure of the torus in D. genkwa is rather unlike that of torus-bearing species in other sections of Daphne (Dute et al. 1990, 1992, and this study) . Domke (1934) made considerable changes by placing D. championii in the genus Eriosolena (according to long styles, long filaments and short and upright calyx teeth) and transferring D. genkwa to the genus Wikstroemia. Members of Wikstroemia, as described by Domke (1932, 1934), often have opposite leaves and have the disk at the base of the floral tube di• vided into individual scales or threads (rather than a complete ring). Recent authors (Rehder 1949; Brickell & Mathew 1976) retain section Genkwa within Daphne. It is clear from the foregoing that D. genkwa, if not belonging to the genus Wikstroemia, is closely related to it. This apparent relationship served as the impetus for our survey of tori in woods of Wikstroemia species. Daphne aurantiaca is a special problem. It was named and put into Daphne by Diels (1912) who noted at the time that it represented "a very distinct species, appar• ently not much allied to any other Daphne of the region" (South Central China). It was transferred to Wikstroemia by Domke (1932, 1934) due to its opposite leaves, 5-merous flowers, and distinct scale-like disk (as well as other features). Stapf (1933) , although he noted the distinct nature ofthis species, left it within Daphne. The latter author also disputed the 5-merous nature of the flower stating rather that it was 4-merous. Rehder (1949) also retained this species in Daphne but for some unspecified reason placed it in the section Daphnanthes. Brickell and Mathew (1976) put it in section Eriosolena. Section Eriosolena is set apart from the rest of the genus by floral characteristics (thread• like peduncles and bloom clusters covered by 2-4 wide, deciduous involucralleaves). Meisner (1857) noted that although he made Eriosolena a section of Daphne, it was previously considered a distinct genus by Blume. Despite this, it was retained as a section within Daphne by Bentham and Hooker (1880) and by Keissler (1898). Keissler, however, noted the tenuous connection of Eriosolena to the other sections in that the filamentous floral stalk and involucre cluster are not found elsewhere in this genus . Hallier (1922) , Leandri (1930), and Domke (1934) all classified Eriosolena as a sepa• rate genus. Domke further indicated that bicollateral vascular bundles occur in midribs of the genera Eriosolena and Edgeworthia, but not Daphne (all three within the sub• tribe Daphninae according to him) . Results from the present study on pit membranes can help clarify some of the prob• lems discussed above. The presence of tori in intervascular pit membranes is a con• stant feature for species within the sections Daphnanthes and Laureola of the genus

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Daphne. Species of the section Mezereum (as presently constituted) do not have tori (Ohtani 1983; Dute et aI. 1990, 1992, this study). Both D. genkwa and D. aurantiaca do have tori, but torus structure and location differ from other Daphne species. In these two species, the torus is often poorly-developed (or absent) on the pit surface adjacent to the lumen of a vessel element, whereas in other Daphne species it is well• developed . Also, torus thickenings lack a G-Iayer as found in other Daphne species . In these two respects, D. genkwa and D. aurantiaca are similar to some of the Wikstroemia species investigated in the present study (see later discussion). Finally, D. aurantiaca, because of its tori, does not belong in the section Eriosolena. Furthermore, it is our opinion that Eriosolena itself is best considered as a separate genus .

Wikstroemia species The discovery of tori in Wikstroemia species (sensu lato) not only extends the list of angiospermous species with tori, but also provides information about the Daphne! Wikstroemia species complex. Various authors have considered Wikstroemia either as a rather broad genus with subgenera or have raised the subgenera to genera (q.v. dis• cussions in Domke 1934 and Hamaya 1959). Hamaya, in particular, has carefully con• sidered the matter and feels that Wikstroemia should refer only to the subgenus Wik• stroemia (Euwikstroemia) of Domke (1934). Our results, though preliminary, bear upon this matter because tori were not found in all Wikstroemia species (s.l.). Rather, those species within subgenus Wikstroemia (W retusa and W pseudoretusa, W indica) lack tori, whereas those within subgenus Diplomorpha (sensu Domke) (W albiflora, W pau• ciflora, W yakushimensis) have them. Although we did not embed those samples for TEM investigation, we expect that W ganpi, W trichotoma, and W sikokiana also have tori since they too are classified within Diplomorpha. When Domke (1934) transferred Daphne genkwa to Wikstroemia, he placed it within the subgenus Diplomorpha. Torus ultrastructure confirms the close relationship of D. genkwa and D. aurantiaca to the members of Diplomorpha. These species, unlike the other daphnes, have no G-Iayer. The data from this study could be used to support the elevation of Diplomorpha to generic rank and the inclusion of Daphne genkwa and D. aurantiaca within the new genus . It must be remembered however, that within Daphne itselfthe section Mezereum is devoid of tori (Dute et aI. 1992). In our judgement, such nomenclatural revision should await further studies of taxa in each of the genera involved. Wikstroemia kudoi requires further study. Hamaya (1959) considers it as a species within a distinct genus, Daphnimorpha, closely related to Diplomorpha and distin• guished by pollen grains with a smooth exine. Wikstroemia kudoi has tori, but they are unlike those of the other Wikstroemia species and are somewhat reminiscent of previ• ously studied Daphne species (Dute et aI. 1992).

Functional aspects Helical thickenings are common components of tracheary elements in Daphne and Wikstroemia (s.l.). Ohtani (1983) described both'S' and 'Z' helices in five species of

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Daphne, the former being less common and restricted to larger earlywood vessel ele• ments. Both Sand Z helices were branched (Ohtani & Ishida 1978b). Dute et al. (1990) observed that the narrower tracheary elements had more prominent thickenings. The presence of spiral thickenings also has been noted in Diplomorpha and Daphnimorpha, but not Wikstroemia (sensu stricto) by Hamaya (1959). Here, too, the author noted that when present, helical thickenings were most obvious on the walls of narrow vessel elements, tracheids, or in the tails of wider vessel elements. Such a difference in devel• opment between narrow and wide vessel members has been observed for species in other families (lAWA 1989). Those woods commonly containing helical thickenings more often occur in dry situations (Carlquist & Hoekman 1985). Thus, it is not surprising that some genera of the Thymelaeaceae should exhibit this feature since the family as a whole grows in dry habitats (Rendle 1925). Horticultural species of Daphne prefer sunshine and well• drained soil (Brickell & Mathew 1976). Daphne aurantiaca, with well-developed heli• cal thickenings, grows on limestone cliffs (Diels 1912). Some species of Daphne ex• hibit other xeromorphic adaptations such as leaves with copious wax platelets and stomata sunken relative to groups of tightly-investing, papillose epidermal cells (Keissler 1898; Luxova 1992; and Erskine & Dute unpublished information). Although other roles have been postulated for helical thickenings (such as reduced resistance to water flux - Jeje & Zimmermann 1979), the most likely function is to in• crease the wettable surface of the tracheary element and by so doing decrease the pos• sibility of cavitation (Carlquist 1982). This explanation fits nicely with the fact that helical thickenings are better developed in narrower tracheary elements in Daphne (Dute et al. 1990) as well as Daphnimorpha and Diplomorpha (Hamaya 1959; Dute et al. present study) . A good example of this is in Wikstroemia (Daphnimorpha) kudoi where the helical thickenings of the tracheids rival those in D. aurantiaca in their distinctness. In this study, the tracheids had the best developed tori. Often these tracheids were located in the latewood and did not have aspirated pit membranes in the her• barium specimens. However, where aspiration was observed the torus completely covered the aperture. A previous study (Dute et al. 1990) concluded that the torus prevented pit membrane rupture during membrane displacement. Clearly, the small tracheary elements of this study represent a backup (reserve) system in which the flow rate is very small (already narrow cell diameters are further decreased by helical thickenings), yet such cells possess safety factors that would prevent embolisms or confine any air bubbles that do form. The idea of tracheid s as a system distinct from large vessel members has been expounded at some length by Carlqui st (e.g. Carlquist 1984). From a ontogenetic standpoint, increased distinctness (height) of radial sculptures in smaller diameter tracheary elements (or constricted ends of larger vessel members) can be explained by an increasingly favourable surface area/volume ratio allowing more rapid deposition of wall material during a restricted period of ontogeny prior to cytoplasmic autolysis.

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