Vessels of Illicium (Illiciaceae): Range of Pit Membrane Remnant Presence in Perforations and Other Vessel Details

VESSELS OF ILLICIUM (ILLICIACEAE): RANGE OF PIT MEMBRANE REMNANT PRESENCE IN PERFORATIONS AND OTHER VESSEL DETAILS

Sherwin Carlquist1 and Edward L. Schneider

Santa Barbara Botanic Garden, 1212 Mission Canyon Road, Santa Barbara, California 93105, U.S.A.

Scanning electron microscope (SEM) examination of vessels from radial sections of lllicium wood showed a wide range of pit membrane remnant presence within any given species and within the seven species studied here. Earlier studies showed that dried specimens offer a reliable indicator of pit membrane presence. In all species, wide variation occurs, from intact pit membranes to perforations virtually free of pit membrane remnants. lllicium parviflorwn has the largest number for the genus of perforation plates, with little or no pit membrane presence, although, in some plates, appreciable pit membrane presence was evident. Species differ in the forms taken by the pit membrane remnants: threads running axially in the perforations are common, but weblike conformations or pit membranes perforated by small circular to oval pores are other commonly encountered conditions. Artifacts attributable to handling and other factors are analyzed to obtain an image of pit membrane presence. Pit membrane remnant presence is consistent with the presence of other strongly primitive features of lllicium and its near-basal position in phylogenetic trees based on molecular data. Unusually narrow and sparse helical thickenings are figured with SEM for the genus for the first time; these thickenings occur in three species from habitats in which winter freezing occurs.

Keywords: basal angiosperms, ecological wood anatomy, Illiciales, perforation plates, primitive wood features, vessel evolution, wood evolution, xylem.

Introduction identify rips and other distortions in pit membrane remnants as artifacts from handling, but these are easily separated from Presence of pit membrane remnants in perforations of vessels the characteristic patterns of pit membrane remnant presence. in wood of primitive dicotyledons has been noted and figured In an attempt to explore whether drying of wood samples, by, most notably, Meylan and Butterfield (1978), Ohtani and pickling of wood samples, or other treatments had any effect Ishida (1978), Butterfield and Meylan (1982), and Metcalfe in modifying presence of pit membrane remnants, we studied and Chalk (1983). Although pit membrane remnants are usu woods of lllicium floridanum Ellis stems and roots represent ally present in scalariform perforation plates with numerous ing different provenances and different methods of preserva perforation plates, they have been reported in simple perfo tion (Schneider and Carlquist, in press). We concluded that ration plates of Knightia of the Proteaceae (Butterfield and none of these factors modifies the pit membrane remnants ap Meylan 1982). Likewise, pit membrane remnants occur not preciably; therefore, a wider survey of the genus lllicium was just in perforation plates with numerous, closely spaced bars undertaken and is reported here. Although lllicium contains but also in some with relative large perforations, as is shown 42 species (Smith 1947), many of these are from remote areas by Bruniaceae (Carlquist 1992). A survey of this occurrence of difficult access, and wood samples available in xylaria are of pit membrane remnants was undertaken earlier (Carlquist relatively few. Herbarium specimens were not very helpful in 1992) in an attempt to explore the extent of this phenomenon supplementing our assemblage of wood samples because twigs in dicotyledons. The conclusion reached there is consonant of lllicium on herbarium specimens are of such limited di with the distributions Meylan and Butterfield (1978) found in ameter that sectioning is difficult and reliability of patterns is woods of New Zealand dicotyledons, namely, that presence of uncertain. Pit membranes are not illustrated for lllicium tash- pit membrane remnants is a feature of vessels in primitive iroi Maxim, because a series is figured for that species in Carl dicotyledons. These remnants are either vestiges of pit mem quist (1992); those figures as well as those of /. floridanum branes present in vessel-like tracheids of dicotyledons crossing (Schneider and Carlquist, in press) can be considered addenda from vesselless to vessel-bearing, or, conceivably, they might to the illustrations for the seven species in this article. Nev be regressions in which the ability for complete lysis of the pit ertheless, the patterns assembled in the two earlier articles as membranes during vessel maturation is lost. well as this article seem to coalesce into variations on basic There has been no published comment refuting these pit patterns, and none of the species of lllicium yielded structural membrane remnants as artifacts; the discoveries cited above features not seen in one or more of the other species. have been credited as natural occurrences. To be sure, one can Although we present these patterns as probable primitive features retained in an ancient family of angiosperms, there 1 Author for correspondence. are interesting implications with regard to wood physiology \\.inu<:npt received February 201)2: revised manuscript received May 2002. and developmental anatomy, particularly at the ultra structural

755 756 INTERNATIONAL JOURNAL OF PLANT SCIENCES level. In addition, our scanning electron miscroscopy (SEM) Results preparations proved convenient for reporting vessel features of lllicium other than pit membrane remnants. Therefore, we Pit Membrane Remnants mention morphology of bars of perforation plates and figure helical thickenings in vessels of several species and attempt to The pit membrane remnants of lllicium anisatum (figs. offer a correlation between the presence and prominence of 1.1-1.6) contain a range of features commonly seen in lllicium. these structures and the environment of the areas where the In a few perforation plates (using the definition of end walls species are native. of vessel elements with pits wider than those of lateral wall pitting), pit membranes are nearly intact (fig. 1.11. In figure 1.1, some tears are apparent. Such tears are undoubtedly ar tifacts, but they may be formed at the sites of small circular Material and Methods or oval pores that commonly occur in such intact membranes (see also fig. 4.20, in which tearing is minimal). Some perfo ration plates have weblike or meshworklike pit membranes (figs. 1.2, 1.3, 1.4). Occurrence of porosities is often less near Wood samples for sectioning were obtained from xylaria ends of perforations (fig. 1.2, rightl than in central portions (see the acknowledgments). These collections are as follows: of perforations that retain relatively extensive pit membrane lllicium anisatum L. (USw-16844), lllicium cambodianum remnants. Porosities are mainly circular to oval in shape (figs. Hance, lllicium cauliflorum Merr. (supplied as /. sp.), and ll 1.2, 1.3). In perforation plates where pit membrane remnants licium griffithii Hook f. 8c Thorns (SJRw SJRw-21944). Wood are more limited (figs. 1.5, 1.6), the remnants tend to take the from herbarium specimens was available for lllicium ekmanii form of threads running axially (parallel to the long axis of A. C. Smith (Liogier 14800, NY) and lllicium majus Hook f. the vessel element). Transitions between the weblike and &C Thorns (R. C. Ching 7202, NY). Locality data for species threadlike plates are illustrated in figures 1.3 and 1.4. of lllicium are given by Smith (1947). Wood from stems and The perforations of lllicium cambodianum vessel elements roots of living plants of lllicium parviflorum Michaux culti (figs. 2.7-2.10) range from porose (fig. 2.7) to threadlike (figs. vated on the University of Florida campus, Gainesville, was 2.8-2.10). Some tearing is evident in figure 2.7 (lower right). collected by William L. Stern and pickled in 50% ethanol. The sparsity of pit membrane remnants in figures 2.9 (top Dried wood specimens were boiled in water, stored in 50% perforation) and 2.10 (center) is probably indicative of artifact ethanol, and sectioned on a sliding microtome. The radial sec formation. Greater tension on some perforations during han tions obtained were dried between clean glass slides, mounted dling or processing than on others may account for breakage on aluminum stubs, sputter-coated, and examined with a of threads (note that perforation at the top [fig. 2.9] and in Bausch and Lomb Nanolab 200 SEM. Imaging of pit the center [fig. 2.10] are wider than adjacent perforations). membrane remnants when studied with SEM provides prob Threadlike pit membrane remnants predominate in this spec lems because electron return from concave objects such as pits imen of /. cambodianum, and formation of axially oriented is much lower than from convex objects. Consequently, our strands is suggested by the relatively large and elongate pores images cannot provide the ultimate in resolution that is avail in pit membranes of figure 1.7. able in SEM photographs of various other plant objects such In lllicium ekmanii (figs. 2.1 1, 2.12), the number of per as primordia, pollen grains, trichomes, and so forth. foration plates that could be observed was limited because of Identifications provided by the xylaria are accepted, al the use of twig material with small diameter. However, two though Smith (1947) disagrees with identifications of some distinctive types are illustrated: weblike (fig. 2.11) and thread specimens referred to as /. cambodianum. We have tentatively like (fig. 2.12). identified a specimen supplied as u/. sp." by USw as /. cau- The vessels of lllicium cauliflorum are exceptionally long, liflorum because that is the only species ever collected in Sa with slender bars; few vessel elements with fewer than 100 rawak (Smith 1947), which is the source of the xylarium wood bars per perforation plate were observed. Most of the perfo sample provided. rations were clear of pit membrane remnants (fig. 3.13). Debris There is no standard terminology for various appearances is often visible in perforation plates of /. cauliforum (fig. 3.13), and conformations of pit membrane remnants in perforation as well as perforation plates of the other species. Intact mem plates. Consequently, simple descriptive adjectives are used. branes with relatively coarse pores (fig. 3.14) or with relatively Although perforation plates lacking in pit membrane remnants small pores (figs. 3.15, 3.16), although uncommon, were are common in all of the lllicium woods we have studied, we observed. have illustrated only a few such plates (e.g., figs. 3.13, 4.25, Pit membrane remnants in the perforations of lllicium majus 5.29), and the illustrations should not be taken to represent (figs. 3.16-3.19) are often restricted to the ends of the per modal conditions in a specimen. Where both perforation plates forations. The four perforation plates illustrated give an idea of adjacent vessel element tips are present, the preservation of of the varied appearances that may be seen in the lateral ends pit membrane remnants is more likely and such entirely intact of the perforations: laminar with small pores (fig. 3.16), large perforation plates were preferred; where one cell of the pair holes transitional to webs (fig. 3.17), porose or weblike (fig. is sectioned away, however, the pit membrane may remain, in 3.18), and pores transitional to strands (fig. 3.19). whole or in part (figs. 2.9, 2.11, 3.17). Helical thickenings in Of all species of lllicium examined, pit membrane remnants vessels were figured on the basis of light microscopy for 1. are most abundantly represented in lllicium griffithii. Laminar parviflorum (Carlquist 1982). intact pit membranes, with minimal lysis in the form of circular Fig. 1 SEM photographs of portions of perforation plates from radial sections of lllicium amsatum wood. Fig. 1.1, "Imperforate perforation plate" in which small pores are present in pit membranes; torn or ripped appearances are judged to be artifacts. Fig. 1.2, Ends (rips) of three perforations showing areas ol pit membrane without porosities (at extreme right in perforations) and with thick weblike texture elsewhere. Fig. 1.3, Loose weblike pit membrane remnants in upper perforations, contrasting with more threadlike remnants in the lower perforation. Fig. 1.4, Pit membrane remnants intermediate between weblike and threadlike appearances. Fig. 1.5, Four perforations in which pit membranes take the form of anastomosing threads running in an axial direction. Fig. 1.6, Five perforations thai contain sparse threadlike pit membrane remnants running in an axial direction. Scale bars = 3 j*m. v\fc »W\\%\\ 8 H

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Fig. 2 SEM photographs of portions of perforation plates from radial sections of lllicium wood. Figs. 2.7-2.10, lllicium cambodianum. Fig. 2.7, Perforations with maximal retention of pit membrane remnants. Fig. 2.8, Threadlike pit membrane remnants running in an axial direction. Fig. 2.9, Threadlike pit membrane remnants with a small amount of anastomosis in lower perforation; absence of remnants in upper perforation is judged to be an artifact; borders of perforations evident. Fig. 2.10, Anastomosing threadlike pit membrane remnants well preserved in upper and lower perforations but less evident in central perforation because of tearing in handling. Figs. 2.11, 2.12, lllicium ekmanii. Fig. 2.11, Weblike pit membrane remnants. Fig. 2.12, Sparse threadlike pit membrane remnants, some broken or distorted. Scale bars = 3 f*m.

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Fig. 3 SEM photographs of portions of perforation plates from radial sections of Illicium wood. Figs. 3.13—3.15, llliciiim cauliflorum. Fig. 3.13, Tip of perforation plate showing one forked bar, absence of pit membrane remnants (some debris present). Fig. 3.14, Perforation in which pores in pit membrane are large. Fig. 3.15, Perforation in which pores in pit membrane are relatively small but various in size. Figs. 3.16-3.19, Illicium mains, pit membrane remnants in ends of perforations. Fig. 3.16, Laminar pit membrane remnants bearing small pores. Fig. 3.17, Weblikc pit membrane remnants; borders of perforations evident. Fig. 3.18, Pit membrane remnants showing transition between porose and weblike pattern. Fig. 3.19, Pit membrane remnants transitional, between weblike and threadlike. Scale bars = 3 /tm in all except fig. 3.13, in which bar = 10 pirn.

759 "60 INTERNATIONAL JOURNAL OF PLANT SCIENCES

or oval pores, were observed in a few perforation plates (fig. Porose sheets of pit membrane material occur in some per 4.20). A greater degree of pore formation, consisting mostly foration plates of Ulicium, but more commonly, the remnants of axially elongate holes, is evident in the perforations shown take the form of threads or webs. The threads run axially in figure 4.21; perforation plates with such extensive retention (parallel to the long axis of the vessel). The commonness of of pit membranes are not abundant. Pores of various sizes as the axially oriented threads in Ulicium contrasts with forms well as large holes occur in the perforations shown in figure of membrane remnants typical of perforation plates in other 4.22. In figure 4.23, the pores are more nearly uniform in size, genera of dicotyledons (Meylan and Butterfield 1978; Carl and there is a tendency because of the axial elongation of the quist 1992). In some species of Ulicium, pit membrane rem pores for one to think of these pit membrane remnants as nants are more extensive (e.g., Ulicium griffithii), in others less transitional to the axially oriented threads so frequently seen so (e.g., Ulicium parviflorum). The distinctive patterns ob in pit membrane remnants of Ulicium. A meshwork consisting served in Ulicium are essentially natural phenomena in our mostly of relatively large holes is featured in the perforations opinion, in agreement with the comments of other authors who (fig. 4.24). Minimal retention of pit membrane remnants char have reported pit membrane remnants in perforation plates in acterizes the perforations of figure 4.25, in which a single dicotyledons. Artifacts do occur, chiefly in the form of tearing thread (upper left) as well as shrunken knoblike pit membrane or ripping of pit membrane portions, and are easily separated bits along the edges of the perforations may be seen. from natural occurrences in our opinion. These artifacts are The illustrations selected for perforations of Ulicium par- mostly attributable to handling and processing of sections. The uiflorum Michaux (figs. 5.26-5.29) are not the most abundant basic patterns of the pit membrane remnants aside from these conditions because this species shows less retention of pit artifacts reflect a natural lysis of the pit membrane as the vessel membrane remnants in perforations (fig. 5.29) than do the element matures, probably exaggerated somewhat by the ac other species. At upper and lower ends of perforation plates tion of the conductive stream. of Ulicium in which pit membrane remnants are present, there We believe that porose pit membrane remnants represent an may be one or two potential perforations in which pit mem initial stage in conversion of an end wall into a perforation branes are entirely intact (fig. 5.26); this is common in all plate, as evidenced in Trochodendron (Carlquist and Schneider species. The occurrence of the three holes in the uppermost pit 2002). Conceivably, evolutionary loss of vessels might also membrane of figure 5.26 appears natural. Likewise, although feature such a stage. However, the pit membrane remnants in some tearing is present, most of the holes and pores of the pit Ulicium vessel elements seem to represent a terminal stage in membranes in figure 5.27 are not artifacts and do not have the transition between tracheids and vessel elements. The oc characteristics of artifacts. Pit membrane portions are re currence of pit membrane remnants was considered a vestige stricted to the ends (tips) of the perforations (fig. 5.28); in this of tracheid-like structure earlier (Carlquist 1992) because of figure, a few coarse threads or strands running in an axial the systematic distribution of these remnants in dicotyledons direction can be seen. and the presence of other primitive vessel features in the genera in which these remnants are present. Two families of Illiciales have also been reported to have pit membrane remnants in perforations: Austrobaileyaceae (Carlquist 2001) and Schis- Helical Thickenings andraceae (Carlquist 1999). The pit membrane remnants in Two species were selected for illustration of helical thick these families are less extensive than those of Illiciaceae, enings by means of SEM. In /. anisatum (fig. 5.30), helical presumably because scandent dicotyledons show accelerated thickenings are inconspicuous and tend not to be continuous; specialization of perforation plates as compared with their rather, ends of thickenings fade into the wall surface, as in /. closest relatives (Carlquist 1975). Pit membranes remnants are parviflorum. In /. majus, on the contrary, thickenings are more not restricted to dicotyledons; they are widespread in ferns pronounced and continue down the vessel wall without dis (Carlquist and Schneider 2001) and characterize vessels in continuities (fig. 5.31). primitive monocotyledons such as Araceae (Carlquist and Schneider 1998; Schneider and Carlquist 1998). Further ex ploration of vessels in vascular plants with SEM will aid in Discussion and Conclusion refining our understanding of the phylogenetic significance of pit membrane remnants. SEM photographs of pit membrane remnants of Ulicium With respect to morphology of perforation plates, Ulicium cubense and Ulicium tashiroi (Carlquist 1992), together with is typical of primitive dicotyledon families: bars are numerous those of Ulicium floridanum (Schneider and Carlquist, in press) (averaging more than 100 in Ulicium cauliflorum). A few and the seven species of this study, present a coherent and forked bars are present (Carlquist 1982), and anastomosis of distinctive pattern. Because none of the species we studied bars may be found in some species, such as /. parviflorum (fig. show a unique pattern in presence of pit membrane remnants, 5.29), /. cubense A. C. Smith (Carlquist 1982), and other spe we believe that similar patterns probably characterize the entire cies. Markedly aberrant plates that resembled transitional pat genus (42 spp.). Retention of pit membrane remnants varies terns of pitting were figured for Ulicium anisatum (Carlquist from extensive sheets occluding what would be perforations, 1982, figs. 13, 14). to barely discernible remnants along the edges of the perfo Helical thickenings are reported on lateral walls of vessels rations. In most perforation plates, some remnants crossing in three Ulicium species. These thickenings are not typical for portions of the perforations are present. Pit membrane rem dicotyledons in that they are few and sparse. Interestingly, the nants are particularly common at lateral tips of perforations. trend in Ulicium follows that of other families (Carlquist •VI * *V' ;*

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Fig. 4 St.VI photographs of portions of perforations plates from radial sections of lllicium griffitbn wood. Fig. 4.20, "Imperforate perforations" in which the pit membranes contain numerous small pores. Fig. 4.21, Fnds of perforations in which pit membranes contain larger pores. Fig. 4.22, Perforations in which some small pores are present, but in which large pores (some possibly the result or coalescence) are also present. Fig. 4.23, Perforations in which pit membrane remnants show a pattern intermediate between porose and coarsely threadlike. Fig. 4.24, Pit membrane remnants composed of anastomosing threads. Fig. 4.25, Ends of perforations showing one threadlike pit membrane remnant (upper left), plus a few irregularities, representing minimal pit membrane presence, along edges of perforations. Scale bars = 3 p.m. Fig. 5 SEM photographs of vessel details from radial sections of Illicium wood. Figs. 5.26-5.29, Portions of perforation plates, Illicium parviflorum. Fig. 5.26, Perforations from end of perforation plate; three pores present in uppermost perforation, hut lower "perforations" contain intact pit membranes. Fig. 5.27, Ends of perforations showing various degrees of pore presence and various sizes of pores in pit membranes. Fig. 5.28, Threadlike pit membrane remnants, plus vestiges of pit membranes in ends of perforations (tears indicative of artifact formation). Fig. 5.29, Portion of perforation plate with anastomosing bars. Figs. 5.30, 5.31, Lumen surface of vessel walls to show presence of helical thickenings. Fig. 5.30, Illicium anisatum; thickenings are inconspicuous and fade into wall surface. Fig. 5.31, Illicium majus; thickenings are prominent but sparse. Scale bars = 3 jtm except in figs. 5.30 and 5.31, in which bars = 10 jim. CARLQUIST & SCHNEIDER—ILLICIVM VESSELS 763

1975): helical thickenings are more frequent and more pro Acknowledgments nounced in species of colder regions. The species of lllicium in which helical thickenings are reported occur in localities We thank Dennis Stevenson and Lisa Campbell of the New where frost typically occurs in winter, whereas lllicium species York Botanical Garden for wood from herbarium specimens. for which helices have not been reported occur in localities Sectioning blocks from xylarium specimens were provided where frost is absent or infrequent, lllicium anisatum occurs through the kindness of Regis Miller (U.S. Forest Products in montane southern Japan; /. floridanum ranges from north Laboratory) and Stanley Yankowski (U.S. National Museum ern Florida to Louisiana; lllicium majus occurs at lower ele of Natural History). William L. Stern of the Department of vations in southern China (230 m) but higher elevations (to Botany, University of Florida, kindly provided the material of 2000 m) in Tonkin and Myanmar (Smith 1947). lllicium parviflorum.

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