Vegetative Anatomy ofthe New Caledonian Endemic Amborella trichopoda: Relationships with the Dliciales and Implications for Vessel Origin1

Sherwin Carlquist and Edward L. Schneider 2

Abstract: Light microscopy was used to study leaf hypodermis, vein scleren­ chyma, stomatal subsidiary cell types, and stem and rootxylem in liquid-preserved material of Amborella trichopoda; oblique borders on tracheid pits, scalariform end walls on tracheids, and porosities in end-wall pit membranes were studied with scanning electron microscopy. Amborella shares stomatal configurations, nodal type (in part), ray types, and porose pit membranes in tracheary elements with llliciaies s.l., but differs from that order in lacking oil cells, vessels, and grouped axial parenchyma cells. These data are consistent with a basal position in angiosperms for Amborella, and for a close relationship with, but not inclusion in, llliciales; inclusion in a monofamilial order is conceivable. Both loss of pit membranes or pit membrane portions on end walls and increase in cell diameter are requisites for origin of vessels. Sarcandra and Illiciaceae show these early stages in origin of vessels; Amborella shows development of porosities in pit membranes. Vessel presence or absence may not be strictly bipolar, because some primitive vessel elements exhibit at least some tracheidlike characteristics and are thus transitional, and because changes in at least two characters define vessel origin. .

THE SINGLE SPECIES OF Amborellaceae Group (1998), who placed a similar roster of (Amborella trichopoda Baill., New Caledonia) is families in an unnamed grouping cited first claimed to be the sister group to the remain­ (and thus basal) in their ordering of angio­ der of angiosperms according to recent stud­ sperms. The phylogenetic significance of ies (Mathews and Donoghue 1999, Parkinson anatomical features of Amborella potentially et al. 1999, Qiu et al. 1999, Soltis et al. 1999). becomes very considerable. These studies each analyzed more than a sin­ In terms of vegetative anatomy, Amborella gle gene site, and this expanded data base as had already attracted attention because of the well as the similarity of the cladistic results vesselless nature of its wood (Tieghem 1900, yielded by these studies have commanded at­ Bailey and Swamy 1948, Bailey 1957). Bailey tention by phylogenists. Branching from the regarded vessellessness as a primitive feature cladogram just above Amborella are Nym­ in woody dicotyledons, whereas recent cla­ phaeales (excluding Nelumbonaceae), and dists, beginning with Young (1981), regarded then an expanded llliciaies (llliciaceae, Schi­ vessellessness as a derived condition in woody sandraceae, Austrobaileyaceae, Trimenia­ dicotyledons (for a discussion, see Baas and ceae). This treatment was foreshadowed by Wheeler 1996). The more recent cladistic the results of the Angiosperm Phylogeny work, cited above, by placing Amborella in a basal position in angiosperms, reopens the possibility that vessellessness is a primitive

1 Manuscript accepted 17 November 2000. feature. If this possibility is valid, the nature 2 Santa Barbara Botanic Garden, 1212 Mission Can­ of tracheids in Amborella becomes worthy of yon Road, Santa Barbara, California 93105. consideration. By means of scanning electron microscopy (SEM), we are attempting to show whether Pacific Science (2001), vol. 55, no. 3:305-312 © 200I by University of Hawai'i Press tracheids ofAmborella are transitional to ves­ All rights reserved sels in any respect, and, by inference, whether

305 306 PACIFIC SCIENCE· July 2001 vessels of llliciaies are essentially relatively stained with a safranin-fast green combina­ little-modified versions of tracheids, as hy­ tion corresponding to Northen's modification pothesized by Bailey (1944) and Carlquist ofFoster's tannic acid-ferric chloride method (1988). Ifvessel origin is a matter of degrees (Johansen 1940). Portions of leaves were ofintermediacy and change in character state cleared in lactic acid and observed with light in several characteristics, one can call into microscopy. The original data presented question the bipolarity of the coding ofvessel below are restricted to features not previously presence in cladistic work. observed or for which conflicting data have Our materials differ from those available to been published elsewhere. earlier authors. Bailey and Swamy (1948) had only dried materials and therefore were un­ able to illustrate and identify some histo­ RESULTS logical features completely. Bailey (1957) had LeafAnatomy wood of a relatively large stem, but was unable to present SEM data. We were able Leaves of Amborella are variously lobed and to study liquid-preserved material of roots, without well-demarcated teeth (Figure 1). stems, and leaves ofAmborella. These materi­ This is reflected in the venation, in which als permit us to answer whether Amborella has easily definable hydathodes are not evident. oil or mucilage cells, the presence of which Freely terminating vein endings are common was denied by Bailey and Swamy (1948), at the margin; there is no marginal vein. although claimed by Perkins (1898), and to Cleared leaf portions and paradermal sections offer reports on other histological features (Figure 2) showed that stomata are densely hitherto undescribed. A useful summary of scattered over the abaxial leaf surface; stomata vegetative anatomy of Amborella was offered overlie veins as well as areoles. by Metcalfe (1987). If Amborella is the sister The variability of stomatal subsidiary cell group to the remainder of dicotyledons, the arrangement is illustrated in Figure 2. Varia­ character state of each feature in this plant tions on a paraeytic type are common, but takes on greater potential significance and other types can be found at lower frequencies. aids in finding the best taxonomic and phylo­ Leaf transections and clearings show that genetic treatment for the genus. adaxial epidermal cells are larger than the abaxial ones (Figure 3). We observed groups of cells with thick gelatinous wall portions MATERIALS AND METHODS (Figure 3, above right); the walls facing the Data on wood are derived from a dried wood center of such cell groups are thickest, sample 7 cm in diameter (voucher: Carlquist whereas distal walls are of normal thickness. 750, RSA), collected on the slopes of the We are uncertain how to define these cell Plateau de Dogny, New Caledonia. Neither groups. Although they could be a definable our samples nor those of other workers in­ cell type for the species, we are inclined to cluded bark, and thus there is no good data record them as an abnormality unless further on the nature of secondary phloem in Am­ studies reveal them to be characteristic of the borella. Fresh material of roots, stems, and species. We did not observe any cells that leaves was furnished by Brett Hall from bear any of the characteristics of mucilage plants cultivated in the Arboretum of the or oil cells. Although the subepidermal layer University of California at Santa Cruz. These of cells on the adaxial side of the leaf is not materials were fixed in 50% aqueous ethanol. clearly definable as a hypodermis on the basis The dried wood sample was sectioned on a of cell size, that layer does have fewer chlo­ sliding microtome. The liquid-preserved ma­ roplasts per cell than do cells of other meso­ terial was sectioned in paraffin after softening phylilayers of the leaf (Figures 3, 4). Inter­ in ethylene diamine (Carlquist 1982a). Some cellular spaces are small (Figures 3,4). sections were observed with a SEM (Bausch Both smaller veins (Figure 4) and larger & Lomb Nanolab). Other sections were veins, such as the midvein, are sheathed with FIGURES 1-4. Amborella trichopoda. 1, Habit of branch bearing fruits; 2, paradermal section, showing stomata with varied subsidiary cells arrangements. 3-4. Transection of lamina of leaf. 3, Portion showing paucity of chloroplasts in hypodermis; and a group of cells with thick wall portions (arrow); 4, portion showing a minor vein sheathed by scIer­ eids with hippocrepiform thickenings. Length of branch in Figure 1, 0.5 m; bars in Figures 2-4, 30 lffil. 308 PACIFIC SCIENCE· July 2001

sclereids said to have hippocrepiform (horse­ sent artifacts of processing and handling, and shoe-shaped) lignified thickenings. These that pit membranes are characteristically thickenings occur on five of the six walls of present. Presence of pit membranes is shown each sclereid and are absent on the wall most clearly in the section ofa scalariform end wall distal from the vein. Such sclereids have been (Figure 9). In Figure 8, pit apertures run reported in the stems ofAmborella also. obliquely to the pit borders, a feature we observed commonly in Amborella wood. Both in stems (Figures 11-13) and in roots Secondary Xylem (Figure 14), porose pit membranes were ob­ Wood of a mature stem shows variations in served. Exposure to the SEM electron beam radial diameter of tracheids: a few layers of caused membrane displacement and damage somewhat narrower than average tracheids that we could watch. Hence, some ofthe tears are succeeded by a few layers of somewhat visible (e.g., Figure 14, right) may represent wider than average tracheids (Figure 5, arrow) artifacts caused by this; minimizing duration Very weakly defined growth rings are pres­ of exposure to the beam at higher magnifica­ ent. The wider "earlywood" tracheids are tions proved the most successful remedy. The the ones that bear scalariforrn pitting (e.g., pit membranes of scalariforrn pits in Ambo­ Figures 10, 13), on radial end walls, although rella tracheids that were observed included Bailey (1957) found that scalariform pitting the following variations: (1) membranes with was distributed randomly within the wood. porosities that are circular and relatively uni­ We also found numerous tracheids in which form in size (Figure 11); (2) membranes radial end walls bear pits intermediate be­ appearing like a finely porose sheet, with a tween circular and typical scalariform shapes few larger holes (Figure 12); (3) membranes (Figure 8). with a wide range of hole sizes, some holes The mature wood pattern of Amborella separated by threadlike membrane remnants features some multiseriate rays up to five cells (Figure 13); and (4) membranes with circular in width (Figure 6, near center), but in our to angular holes, ranging downward in diam­ material uniseriate and biseriate rays pre­ eter below the resolving power of our SEM dominate (Figure 6). Upright to square cells (Figure 14). We believe that these porosities compose rays, for the most part (Figure 7), do not represent artifacts, but additional ob­ but at least a few files of procumbent cells are servations, especially those utilizing other present in radial sections of wider rays. Ray methods, are desirable. cells contain droplets or massive yellowish to We observed secondary wood of relatively brownish deposits (dark cellular contents in young roots (two years' accumulation of sec­ Figures 5-7), but no ethereal oil or mucilage ondary xylem) and found it to be like that of cells are present. Ray cells have lignified sec­ stems in all essentials, except that tracheids ondary walls. In radial sections, we noted that are uniform in diameter. The roots studied many of the pits in ray cells are bordered, as were diarch. illustrated by Bailey (1957). Axial parenchyma is diffuse, as stated by Bailey (1957), and is easily located in our DISCUSSION AND CONCLUSIONS material (Figure 7, arrows), but is not as Relationships and Phylogeny abundant as in most dicotyledonous woods with diffuse axial parenchyma. Strands ofaxial The placement of Amborella as basal in an­ parenchyma are composed of about five cells giosperms by recent workers suggests that that are notably elongate axially. Axial paren­ vessellessness must be regarded as basal in chyma contains the droplets of dark-staining dicotyledons. This possibility tends to be re­ compounds cited above for rays. inforced by the apparent absence of vessels Figures 8-14 depict scalariform end walls in some Nymphaeales and the presence of of tracheary elements. Although we have vessels with rudimentary development of shown two of these without pit membranes pores in pit membranes in other genera (Figures 8, 10), we believe that these repre- (Schneider and Carlquist 1995a,b, 1996a,b, FIGURES 5-9. Wood ofAmborella trichopoda. 5, Transection of mature stem, showing barely discernible growth rings (beginning of earlywood indicated by arrows) and absence of vessels; 6, tangential section of mature wood; one ray wide and multiseriate, and others uniseriate or biseriate; 7, radial section of mature stem, showing dark-staining de­ posits in ray cells and (arrows) two strands of axial parenchyma. 8-9. SEM photographs of wood from young root. 8, Face view of pits on end wall (pit membrane absence considered an artifact), showing offset of angle of pit apertures compared with borders; 9, sectional view of end wall, showing presence of pit membrane in pits. Bars indicate mag­ nifications (Figures 5-7, 60 J.IID; Figures 8-9, 5 Ilm). FIGURES 10-14. SEM photographs of ttacheids from macerations ofwood ofAmborella trichopoda. 10-13. Wood from mature stem. 10, Scalariform end wall of ttacheid (absence of pit membranes considered an artifact), with a second ttacheid in back of it (left), and, to right, ray cells; 11, portions of two scalariform pits ftom end wall, showing circular pores in pit membranes; 12, portions of two scalariform pits from end wall, showing thin meshworklike pit membrane containing a few small circular pores; 13, pit membrane remnants in scalariform end wall pits; 14, portions of three scalariform pits ftom ttacheid ofyoung root secondary xylem, showing pores in pit membranes (large tears considered to be artifacts). Bar in Figure 10, 10 JlIIl; bars in Figures 11-14, 5 JlIIl. Vegetative Anatomy ofAmborella trichopoda . Carlquirt and Schneider 311

Schneider et al. 1995). The next most basal ofvessels and those of the tracheids, as in the group in recent phylogenies, TIliciales s.l., roots of Sarcandra (Carlquist 1987) and Illi­ has vessels. Other features that distinguish cium (Carlquist 1982b) and the other TIliciales Amborella from TIliciales s.1. are the presence s.1. of mucilage or oil cells in TIliciales and the On the basis of these criteria, Amborella occurrence of paratracheal axial parenchyma shows porose pit membranes in end walls (often abaxial, and scanty in any case) in (rather than nonporose end walls); no differ­ Illiciales (data from Carlquist 1982b, 1984, entiation in element diameter other than that 1999, 2001). By virtue of having no vessels, attributable to growth rings is present. axial parenchyma in Amborella is by definition However, stages associated with the origin apotracheal, but there can be grouped apo­ of vessels, including dimorphic diameter of tracheal cells rather than diffuse ones even tracheary elements and change to more po­ in a vesselless dicotyledon (e.g., Winteraceae: rose pit membranes, have been demonstrated Carlquist 1989), and thus the paratracheal in Illicium and Sarcandra (Carlquist 1982b, axial parenchyma of Illiciales does contrast 1987). Vessel elements in these genera show with the diffuse axial parenchyma ofAmborella. the transitions between the morphology of The families of TIliciales s.1. can all be said tracheids in a vesselless wood and the mor­ to have paraeytic stomata, with transitions to phology of tracheids and vessel elements in other types (Carlquist 2001). In this respect, a primitive wood. Dimorphism in tracheary there is a link between Amborella and the TIli­ element diameter and presence of pores in ciales. Nodes are unilacunar with a single trace pit membranes of end walls are features of in Amborella, a condition found also in Illi­ degree rather than "bipolar" characters of cium; the remaining TIliciales have unilacunar presence or absence. Moreover, change in two-trace nodes (Metcalfe 1987), a condition more than one character state is required for that probably is morphologically similar to vessel origin. Herendeen and Miller (2000) the unilacunar one-trace arrangement. advised against use in cladistical analysis of features that cannot be cast into bipolar form, Vessel Origin and Definition and thus "vessels absent" versus "vessels present" is an unreliable contrast because it The vesselless dicotyledons have small po­ conflates more than one character. rosities in end walls of tracheids, end walls that often are scalariform. Such porous pit ACKNOWLEDGMENTS membranes have been illustrated for Bubbia of the Winteraceae (Carlquist 1983) and Tetra­ We express our appreciation for the efforts of centron of the Tetracentraceae or Trocho­ Ray Collett, founding director of the Uni­ dendraceae (Carlquist 1988). Amborella also versity of California, Santa Cruz, Arboretum, has porosities in this size range. The some­ who made certain that live material of Am­ what larger pit membrane holes (Figure 13) borella was brought to the United States, and may be natural and may represent removal of to Brett Hall, who provided material to us larger portions of pit membranes by the con­ from the plants cultivated at that Arboretum. ductive stream over time. Such porose pit Don Zack (Engineering, University of Cali­ membranes may be found in such TIliciales as fornia at Santa Barbara, retired) has tirelessly Illicium -(Carlquist 1992), Schisandraceae helped us obtain the maximal performance (Carlquist 1999), and Austrobaileya (Carlquist possible from our SEM. 2001). Stages in reduction of the pit membrane Literature Cited remnants are well illustrated in Illicium (Carlquist 1992) and show how porose mem­ Angiosperm Phylogeny Group. 1998. An branes likely yielded to clear perforations ordinal classification for the families of through more extensive and efficient lysis of flowering plants. Ann. Mo. Bot. Gard. the membrane. A second criterion for origin 85:531-553. ofvessels is differentiation between diameters Baas, P., and E. A. Wheeler. 1996. Parallel- 312 PACIFIC SCIENCE· July 2001

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