IAWA Journal, Vol. 31 (2), 2010: 217–226

TORUS-BEARING PIT MEMBRANES IN OF

Roland Dute1, 5, David Rabaey2, John Allison1 and Steven Jansen3, 4

SUMMARY Torus thickenings of pit membranes are found not only in gymnosperms, but also in certain genera of dicotyledons. One such is Osmanthus. Wood from 17 species of Osmanthus was searched for tori. Fourteen spe- cies from three of the four sections investigated possessed these thick- enings. Ten of the species represent new records. Only the three New Caledonian species of Section Notosmanthus lacked tori. This observation in combination with other factors serves to isolate this section from the remainder of the genus. Key words: Notelaea, Osmanthus, pit membrane, torus.

INTRODUCTION

Osmanthus is a member of the ( Family), a moderate-sized family with circa 24 genera and 615 spp. (Stevens 2001). There are 33 natural species within the genus (Table 1; Xiang et al. 2008), and they show a tropical to temperate distribution (Bailey & Bailey 1976; Denk et al. 2001; Xiang et al. 2008). Generally, individual are found in moist forests (Hsieh et al. 1998; Chou et al. 2000; Denk et al. 2001), frequently on steep slopes (Green 1958). In some instances populations are sub- ject to monsoon conditions and hence to periods of relatively low rainfall (Yang et al. 2008; Hua 2008). Tori are thickenings of intervascular pit membranes found not only in gymnosperm, but also in angiosperm wood, including that of Osmanthus (Dute et al. 2008; Dute et al. 2010). Tori were first observed in O. fragrans, O. heterophyllus, and O. × fortunei (a hybrid between O. fragrans & O. heterophyllus [Green 1958]) by Ohtani and Ishida (1978) and later in O. aurantiacus (subsumed within O. fragrans by Green 1958), O. insularis and O. rigidus (insufficiently known species, Green 1958) by Ohtani (1983). Subsequently, Dute and Rushing (1987, 1988) discovered tori in O. americanus (native to the southeastern U.S.A.) and elucidated the mechanism of torus synthesis. Rabaey et al. (2006) extended the list with the discovery of tori in O. serratulus and O. suavis.

1) Department of Biological Sciences, Auburn University, Life Sciences Building, Auburn, Alabama 36849-5407, U.S.A. 2) Laboratory of Systematics, K.U. Leuven, Kasteelpark Arenberg 31, Box 2437, B-3001 Leuven, Belgium. 3) Institute of Systematic Botany and Ecology, Ulm University, Albert-Einstein-Allee 11, D-89081, Ulm, Germany. 4) Jodrell Laboratory, Royal Botanic Gardens, Kew, TW9 3DS, Richmond, Surrey, U.K. 5) To whom correspondence should be addressed [E-mail: [email protected]].

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Table 1. of Osmanthus as modified from Xianget al. (2008). Binomials in bold identify the species whose wood was observed in the present study. The asterisks denote those species previously found to have tori.

Section 1. Leiolea (Section 4 Osmanthus continued) O. americanus* O. gracilinervis O. marginatus O. hainanensis O. matsumuranus O. henryi O. minor O. heterophyllus* O. scortechinii O. insularis* O. sumatranus O. iriomotensis O. kaoi Section 2. Notosmanthus O. lanceolatus O. austrocaledonicus O. pubipedicellatus O. cymosus O. reticulatus O. monticola O. serrulatus* Section 3. Linocieroides O. urceolatus O. didymopetalus O. venosus O. yunnanensis Section 4. Osmanthus O. × fortunei O. armatus O. attenuatus Section 5. Siphosmanthus O. cooperi O. decorus O. enervius O. delavayi O. fordii O. suavis* O. fragrans* O. × burkwoodii

Comparative wood anatomy (Patel 1978; Baas et al. 1988) showed a close relation- ship among the genera Osmanthus, , Notelaea and . Recent analysis of chloroplast DNA (Wallander & Albert 2000) confirmed the relationship among these four genera along with a fifth,Picconia . Investigations of wood from species of Phillyrea, Nestegis, Notelaea and Picconia showed the presence of tori only in Pic- conia (Dute et al. 2008; Rabaey et al. 2008), a small genus of only two species native to Macaronesia. Tori in species of Osmanthus and other angiosperms are not known to put the plant at a disadvantage under moist conditions, yet the torus is of such a diameter as to oc- clude a pit aperture during aspiration. Because the torus is thicker than the margo, it is less porous and provides greater mechanical support, which may prevent rupture of the pit membrane (Dute & Rushing 1987). Clearly, tori could provide an advantage under conditions of drought stress by lowering the risk of air-seeding, i.e. the induction of cavitation of air sucked through the largest pore of a pit membrane into a functional, water-filled tracheary element. Although six distinct species of Osmanthus have been investigated for tori (Table 1), there are about 33 species in nature (Table 1; Xiang et al. 2008). The aim of this study was to investigate other Osmanthus species for the presence of a torus. Such data would: 1) help with the taxonomic circumscription of this feature in the Oleaceae and 2) provide information for future introduction of new, drought-tolerant species into the horticulture market of the southeastern U.S.A. (Werner 2007).

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Table 2. Herbarium specimens of Osmanthus and Osmarea used in this study. BR = Natio- nal Botanic Garden, Brussels; K = herbarium of the Royal Botanic Gardens, Kew.

Species Herbarium Date of collection Collector(s) no. O. americanus Benth. & Hook. f. BR unknown unknown O. americanus (L.) Gray K Jan 1938 Naruda 2023 O. armatus Diels BR 25 Feb 2009 Rabaey s.n. (liv. col. 19673925) O. austrocaledonicus (Vieill.) Knobl. K unknown White 2200 O. austrocaledonicus (Vieill.) BR unknown MacKee 29532 Knobl. subsp. austrocale- donicus O. austrocaledonicus (Vieill.) K unknown Guillaumin 28014 Knobl. subsp. austrocale- donicus O. austrocaledonicus (Vieill.) K unknown Deplanche 14 Knobl. subsp. austrocale- donicus var. austrocaledonicus O. austrocaledonicus (Vieill.) BR unknown Bamps 6000 Knobl. subsp. badula (Vieill. ex Pancher & Sebert) P.S. Green O. austrocaledonicus (Vieill.) K 1 Nov 1929 Franc 2405 Knobl. subsp. badula (Vieill. ex Pancher & Sebert) P.S. Green O. austrocaledonicus (Vieill.) K 12 April 1968 Bernardi 12767 Knobl. subsp. collinus (Schltr.) P.S. Green O. austrocaledonicus (Vieill.) K 11 October 1963 Green 1294 Knobl. subsp. collinus (Schltr.) P.S. Green O. cymosus (Guillaumin) K 18 October 1982 McKee 40928 P.S. Green O. enervius Masam. & Mori BR unknown Forestry Research Institute 17310 O. fragrans Lour. BR unknown Likkir & Kemp s.n. O. fragrans Lour. K 31 December 1974 Geesink et al. 8002

O. heterophyllus (G. Don) BR unknown Coplan P.S. Green O. insularis Koidz. BR unknown Yokoyama 1132 O. insularis Koidz. K 03 May 1917 Wilson 8367 O. kaoi (Liu & Liao) S.Y. Lu BR unknown Sheng-You Lu 13487 O. marginatus (Champ. ex BR unknown Taam 193 Benth.) Hemsl.

continued on the next page

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(Table 2 continued)

Species Herbarium Date of collection Collector(s) no. O. marginatus (Champ. ex K 03 July 1933 How 71028 Benth.) Hemsl. O. matsumuranus Hayata BR unknown Omarina 17854 O. matsumuranus Hayata K 6 October 1928 Poilane 15828 O. minor P.S. Green BR unknown Taam O. minor P.S. Green K 27 May 1914 W.J.T. 10949 O. monticola (Schltr.) Knobl. BR unknown Bamps 5754 O. monticola (Schltr.) Knobl. K 13 May 1951 Guill. & Baum 11238 O. scortechinii King & Gamble K 14 May 1975 de Wilde & de Wilde- Duyfjes 16912 O. scortechinii King & Gamble K unknown Holttum 10732 O. venosus Pamp. BR unknown Sino-American Ghuizou Botanical Expedition 1693 O. yunnanensis (Franch.) BR 25 Feb 2009 Rabaey s.n. (liv. col. P.S. Green 19392003) Osmarea × burkwoodii Burkwood & Skipwith K 17 April 1969 Burkwood & Skipwith s.n.

MATERIALS AND METHODS

Sources of wood specimens examined in this study are listed in Table 2. Specimens were prepared for light microscopy by two methods. In the first method, small segments were removed from the branches of herbarium specimens with a razor blade. The segments were placed in 95% ethanol under vacuum for one hour; the ethanol was replaced once and the segments left to soak overnight. Next, the segments were infiltrated with and embedded in JB-4 resin. Cross sections of 1.5 to 3 μm in thick- ness were cut on a Sorvall MT-2b ultramicrotome using a glass knife. Sections were heat-fixed to glass slides, stained with benzoate-buffered, aqueous toluidine blue O, embedded in Permount mounting medium (Fisher Scientific, Fair Lawn, New Jersey, U.S.A.), and topped with a coverslip. A Nikon Biophot microscope with a Nikon D-70 digital camera attachment was used to observe and photograph specimens. In the second method for light microscopy, thin sections (10–15 μm) were prepared using a sliding microtome (Reichert, Vienna, Austria). A mixture of 0.5% safranin in 50% ethanol and 1% alcian blue in water (35: 65, v/v) was used as a staining solution. After staining, sections were washed with distilled water, dehydrated with ethanol and treated with the clearing agent Parasolve (Prosan, Merelbeke, Belgium). The sections were embedded in Euparal (Agar Scientific Ltd, Essex, U.K.). LM-observations were carried out with a Dialux 20 (Leitz, Wetzlar, Germany) fitted with an oil immersion objective. Digital images were made with a PixeLINK PL-B622CF camera. Herbarium specimens were prepared for scanning electron microscopy (SEM) also by two different procedures. Branch segments of O. austrocaledonicus and O. scortechinii were split lengthwise exposing radial longitudinal sections. Split segments were affixed

Downloaded from Brill.com10/01/2021 04:40:49AM via free access Dute, Rabaey, Allison & Jansen — Tori in Osmanthus 221 to aluminum stubs with double-stick, carbon-impregnated tape. Segments were then coated with gold-palladium (EMS 550X Sputter Coater, Electron Microscopy Sciences, Hatfield, Pennsylvania, USA) and viewed with a Zeiss EVO 50 at 20 kV (Carl Zeiss NTS GmbH, Oberkochen, Germany). By contrast, branch samples of O. minor and O. × burkwoodii were dehydrated in an ethanol series and then air dried for 12 hours at room temperature. Samples were subsequently split in either the tangential or radial plane and fixed to aluminum stubs with electron conductive carbon cement (Neubauer Chemikalien, Münster, Germany) and coated with platinum using an EMITECH K550 sputter coater (Emitech Ltd., Ashford, U.K.). Samples were observed with a Hitachi S-4700 field-emission scanning electron microscope (Hitachi High Technologies Corp., Tokyo, ) at an accelerating voltage of 2 kV.

RESULTS AND DISCUSSION

Tori are present in intervascular pit membranes of vessel members and vascular tracheids of Osmanthus americanus, O. armatus, O. enervius, O. fragrans, O. heterophyllus, O. insularis, O. kaoi, O. marginatus, O. matsumuranus, O. minor, O. scortechinii, O. veno- sus, O. yunnanensis and O. × burkwoodii (Table 3; Fig. 1 & 2). Osmanthus america- nus, O. fragrans, O. heterophyllus and O. insularis woods already had been found to contain tori (Ohtani & Ishida 1978; Ohtani 1983; Dute & Rushing 1987, 1988), thus serving to confirm the techniques used in this paper. In contrast, tori were absent from pit membranes of the three New Caledonian species (Fig. 3 & 4). SEM of O. scorte- chinii wood showed numerous torus-bearing pit membranes and their associated pit apertures, which varied in shape from circular to elliptical (Fig. 5). Detail of tori in face view showed them to be circular to elliptical in outline often, but not always, with a very irregular surface consisting of a mass of pustules (Fig. 6 & 7 vs. 8). With field emission SEM the border between torus and margo is distinct, and the granular/fibrillar nature of the latter is evident as is the annulus encircling the margo (Fig. 7). Cracks and tears are common in the torus-bearing pit membranes but are located only in the margos (Fig. 6–8). (Contrast this damage with that of the non-torus-bearing membranes in Fig. 9 & 10; see below.) The foregoing observation supports the hypothesis that the thickened torus prevents the tearing of that portion of the pit membrane overlying the pit aperture during membrane deflection (Wheeler 1983; Dute & Rushing 1987). Re- cent observations using field emission SEM and TEM have illustrated that angiosperm pit membranes show a large variation in pit membrane thickness (from 70 nm up to 1900 nm, Jansen et al. 2009). Pit membrane thickness and pore size were found to be significantly correlated, indicating that thick pit membranes are usually less porous and thus provide higher air-seeding thresholds than thin pit membranes. In contrast, SEM of O. austrocaledonicus wood showed non-torus-bearing membranes that were thin enough for the subtending pit aperture to be visualized through the membrane (Fig. 9, arrows). The apertures associated with these membranes were distinctly elliptical (Fig. 9). The pit membranes were frequently damaged. For example, the damage at the central double arrow in Figure 10 was due to the heat of the electron beam. As can be seen, the tears in this figure traverse the centers of the membranes.

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Figures 1–4. LM of intervascular pit membranes in Osmanthus woods. Tori present in Fig. 1 & 2 (arrows), absent from Fig. 3 & 4 (arrows). – 1: O. scortechinii, XS – 2: O. minor, TLS. – 3: O. austrocaledonicus, XS. – 4: O. austrocaledonicus, TLS. — Scale bar = 5 μm for Fig. 1, 2, 4; 10 μm for Fig. 3.

→ Figures 5–10. SEM of intervascular pit membranes in Osmanthus woods, RLS. Fig. 7 & 8 were imaged with a field emission microscope. – 5 & 6: View of pit membranes and apertures inO. scor- techinii. The pustule clusters on the surfaces of the tori are indicated with arrows. – 7: Detail of a pit membrane in the wood of O. minor. The torus (T), margo (M) and annulus (A) are visible as distinct structures. The pustules of the surface of the torus are present but not as obvious

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as in Fig. 6. – 8: Two pit membranes in O. × burkwoodii wood. Tears in the membranes are con- fined to the margo. – 9 & 10: Pit membranes and apertures in wood of O. austrocaledonicus. The pit membranes are so thin that the subtending apertures are visible (arrows). Tears (double arrows) can be found where the membrane overlies the aperture. — Scale bar = 5 μm for Fig. 5; 2 μm for Fig. 6–10.

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Table 3. Anatomical data and origins for wood specimens of Osmanthus used in this study; + = present, – = absent.

Species Tori Vessel Growth Porosity Origin arrangement rings

O. americanus + dendritic + diffuse Mexico, USA (2 specimens) O. armatus + dendritic + diffuse Belgium (native to ) O. austrocaledonicus – radial multiples- + diffuse (8 specimens) dendritic O. cymosus – radial multiples- + diffuse New Caledonia dendritic O. enervius + dendritic + diffuse Taiwan O. fragrans + dendritic + diffuse Thailand (2 specimens) O. heterophyllus + dendritic + diffuse USA (native to East ) O. insularis + dendritic + diffuse Bonin Islands, (2 specimens) Eastern Asia & Japan O. kaoi + dendritic + diffuse Taiwan O. marginatus + dendritic + diffuse China (2 specimens) O. matsumuramus + dendritic + diffuse Taiwan & Laos (2 specimens) O. minor + dendritic + diffuse Taiwan & China (2 specimens) O. monticola – dendritic + diffuse New Caledonia (2 specimens) O. scortechinii + radial multiples- + semi-ring- North Sumatra & Malaysia (2 specimens) dendritic porous O. venous + dendritic + diffuse China O. yunnanensis + dendritic + diffuse Belgium (native to China)

The New Caledonian species of Osmanthus (O. austrocaledonicus, O. cymosus, O. monticola) present a considerable problem as regards classification. Originally placed in Notelaea (q.v. discussion in Green 1963), Johnson (1957) put the first two species in Nestegis (Gymnelaea) but was uncertain what to do with O. cymosus. Green (1958, 1963) placed all three species into Osmanthus but noted that the difference in the in- florescence characters between these three species and other species of Osmanthus is such that the three should be put into a separate section of the genus (Notosmanthus). The uniqueness of these three species extends to their chemistry as well (Jensen et al. 2002; Kubba et al. 2004). The fact that the New Caledonian species lack tori merely confirms their distinctiveness. A comparison was made of general anatomical features among the various woods. The features included presence of tori, vessel arrangement, growth rings and porosity

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(Table 3). The only feature separating the New Caledonian species from the rest was the absence of tori. However, a more detailed comparative analysis of xylem structure is necessary to affirm the placement of these species in a distinct section ofOsmanthus or even in a separate genus (either Nestegis or Notelaea). Osmanthus × burkwoodii has an interesting history. When first named, the hybrid was designated Osmarea × burkwoodii and was considered a cross between and Phillyrea decora (Sax & Abbe 1932; Taylor 1945). Osmanthus and Phil- lyrea are closely related genera (Wallander & Albert 2000), but wood of the former has a torus, wood of the latter does not (Dute et al. 2008; Rabaey et al. 2008). However, Kasapligil (1970) put P. decora into Osmanthus on the bases of venation, petiole venation and type. Thus, the binomial of the hybrid becameOsmanthus × burkwoodii (Green 1972). Osmanthus decorus is of interest in its own right due to its distribution from the Caspian Sea through the northeastern Mediterranean Basin (Xiang et al. 2008), a range distinct from that of other Asian species of Osmanthus.

acknowledgement

This research was supported by an undergraduate Research Grant-In-Aid from the Department of Biological Sciences, Auburn University.

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