Pollen wall development in Eucommia ulmoides (Eucommiaceae)

Review of Palaeobotany and Palynology, 70 ( 1992): 297-323

297

Elsevier Science Publishers B.V., Amsterdam

Pollen wall development in Eucommia ulmoides (Eucommiaceae) J o h n R. Rowley a, J o h n J. Skvarla b and J o h n M. Pettitt c aBotany Department, University of Stockholm, S-106 91 Sweden bOklahoma Biological Survey and Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019, USA CDepartment of Pathology and Immunology, Monash University, Prahran, Vic. 3181, Australia

(Received March 6, ! 99 !; revised and accepted July 25, 199 i)

ABSi'RACT Rowley, J.R., Skvarla, J.J. and Pettitt, J.M., 1992. Pollen wall development in Eucommia ulmoides (Eucommiaceae). Rev. Palaeobot., Palynol., 70: 297-323. In Eucommiathe foot layer plays a prominent part in microspore wall development. Bacules ( > 100 nm) are rods originating within the foot layer. Small bacules (diameter ca. 10 nm) form at the same time as the foot layer. The tectum is considered to be made up of these microbacular rods. Spinules are continuous with the bacules. An endexine is differentiated during a middle to late microspore stage. Except for the pore the furrow includes tectum and foot layer on the endexine. Since the furrow consists of a region of reduced foot layer and the reduction is gradual near the polar ends of furrows, assessment of furrow length depends to some extent upon variations in exine infolding. The pore is well defined, but, because it is crossed by lamellations of the endexine and foot layer and often overlaid by tongues and bridges of foot layer plus tectum (including spinules) it is obscured from view using either light microscopy or scanning electron microscopy.

Introduction

There is but one species of Eucommia and it forms a family of its own, Eucommiaceae Van Tiegh. The family was referred to the Rosales by Engler and Diels (1936) while Hamamelidales was suggested by Solereder (1899), Hutchinson (1926) and Lemesle (1946). The family was assigned to the Urticales by Oliver (1891), Harms (1930), Wettstein (1935), Tippo (1940) and Varossieau (1942). Takhtajan (1969, 1980)suggested recognizing the family as forming the separate order, Eucommiales, established by Nemejc (1956). According to Takhtajan (1980), the order is evidently related to Urticales and had a common origin with them from the Hamamelidales. Thorne (1989) places Eucommia in an order of its own under the Hamamelidales but rejects a close relationship with Urticales. Oliver (1891) was the first to point out the possibility of an ulmaceous affinity,

because of the general similarity and common type of fruit. The Ulmaceae, the most primitive family of the Urticales, had in all probability a common origin with Eucommiales. In contrast, Cronquist (1968, 1981) felt that morphologically, the Eucommiaceae were closest to the Urticales. However, he cited the embryological work of Eckardt (1963) as suggesting that the ovulate nature of Eucommiaceae is more primitive than the Urticales while features such as unitegmic and not fully crassinucleate ovules, and absence of stipules showed considerable advancement. He concluded that a distinct order, Eucommiales, was necessary to accommodate Eucommia. He also suggested an evolutionary "link" between Hamamelidales and Urticales with the likelihood of an origin from the first order. Hutchinson (1969: p. 166) has written that "It is a remarkable fact that like tbe ancient Gingko biloba, the Maiden-hair tree, this Eucommia

298

ulmoides Oliver has never been found in a wild state, but owes its preservation to Chinese cultivation. The bark furnishes a drug valued by the Chinese and used by them from very early times". Interpretation of the exine morphology is particularly di~cult using the light microscope, e.g., Erdtman (1948, 1952, 1969), Pokrovskaja (1950), Ikuse (1955, 1956) and Kuprianova (1965). The special nature of the germinal apertures of Eucommia pollen grains have made them noteworthy. Like Cercidiphyllum and Euptelea pleiosperma (Erdtman 1969) the three meridional furrows of Eucommia are reported to be of unequal length and to have rounded ends. Pollen of Oliniaceae (Patel et al., 1983) consistently has furrows with one short arm with respect to pore position. We have attempted by studying development and using electron microscopy to add to the descriptions of the exine, its apertures and the intine. Materials and methods

TEM preparation Buds and mature flowers of Eucommia ulmoides Oliver were collected from the Hortus Botanical Garden, Leiden, by the late Jan Muller. Anthers were fixed in 6% glutaraldehyde (GA) in 0.15 M sodium cacodylate-HCl buffer (pH7.6). This material was processed for TEM in the following ways: (I) Cross sections of anthers were transferred to a mixture of 0.1% Alcian Blue (Gurr, 8GX) in !% GA in 0.1 M cacodylate-HCl buffer (12 h, pH 6.9, 20C) followed by 2 h in 0.1% OsO4. Samples were dehydrated with an acetone series and infiltrated and embedded in Mollenhauer's (1964) EponAraldite mixture No. !.

J.R. ROWLEY ET AL.

(2) Shed mature pollen was put into 0.1% OsO,, for 12 h and processed as in No. 1. (3) Shed mature pollen grains were dehydrated in acetic acid and heated to 100°C in Erdtman's (1960) acetolysis mixture for 4 min. The residue was washed, dehydrated in acetone and embedded as in No. 1. LM and TEM sections were stained as follows: (1) Thick sections (ca. ! lam) for light microscopy were stained with a 0.05% toluidine blue solution in benzoate buffer (pH 4.5). (2) For general contrast with TEM thin sections were stained with 1% aqueous uranyl acetate for 15 min followed by lead citrate for 15 min.

SEM preparation Pollen grains were removed from plastic embeddings through use of sodium methoxide (Skvarla et al., 1988; Polysciences Inc., Cat. No. 6712). The isolated pollen was dried using the Peldri II critical point drying alternative as outlined by Chissoe et al. (1990). Dry pollen grains were then mounted on SEM specimen stubs and sputter coated with gold/palladium (60/40). Micrographs were taken with Zeiss EM-9S, EM10A and JEOL 2000 transmission electron microscopes (TEM) and JEOL 880 scanning electron microscope (SEM). Results

L Microspore tetrad period ( A ) Proexine template There was a variety of cell surface glycocalyx coatings prior to initiation of the proexine. These were apparently transitory and differed on adjacent microspores and portions of single cells (Plate I, 1).

PLATE I TEM sections were stained with uranyl acetate and lead citrate unless otherwise stated. l. Microsporetetrads prior to formationof proexine. The plasma membraneglycocalyxwas variable in form between cells and in individual microspores. Such variation is likely to be related to asynchrony in differentiationbetween microspores. The lightly stained cells, for example, have higher volumedensities of amoeboid plastids (see Plate II,2) than the more stain acceptingcells. Tetrads are arranged tetrahedrally. Bar: 10 ~tm.

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Some of them looked like probacules and protecturn (Plate II,2). The early exine template of Eucommia differed from the transitory glycocalyces seen in Plate I,! and Plate II,2.

(B) Development of the exine template Since neither a tectum nor bacules were boldly defined through early development it may be useful to consider micrographs of mature stages to see why we refer to both microbacules, spinules/ bacules and a tectum. In Plate XV,45 and 46 that portion of a spinule between the tectum and foot layer can reasonably be called a bacule. These bacules are much wider than the multitude of small rods (microbacules) in Plate XV,48-51 between tectum and foot layer. The slender rods (10-15 nm in diameter) we term microbacules were not detected until there were receptors for the foot layer. They are seen directly on the plasma membrane in Plate III,7 and on the forming foot layer in Plate V,l I. Apparently, components for foot layer and promicrobacules form at about the same time. Precursors (exine receptors) for the foot layer were seen on surfaces of microspores having no other glyocalyx elements contrasted (Plate III,3). On end these receptors have a contrasted core

bounded by a weakly stained zone (Plate III,3,4). Profoot-layer receptors accumulate exine material (sporopollenin) and enlarge both laterally and in height (Plate III,4-8). Receptors are seen subsequently only at the surface of some proexine components (Plate III,5,7, Plate VIII,20). Spinules were evident when the foot layer had begun to be contiguous (Plate V,l l). By the late microspore tetrad period spinules, while small, were frequent (Plate V,12). The foot layer began to be continuous locally in the stage represented by Plate III,8 and Plate IV, 10. The foot layer was contiguous in interaperture sectors during the late microspore time (Plate V, i 2). Development of the foot layer was, however, asynchronous on individual microspores (Plate V,12). In aperture regions there was a tectum and disjunct foot layer (Plate V,13). Aperture regions were detectable at early stages of foot layer development (Plate III,9 and Plate IV,10).

II. Early free microspore period As separation of tetrads was completed the foot layer became fairly uniform in thickness except for the colpoid furrows (Plate VI,14,15). Near the polar ends of furrows the foot layer was thin and

PLATE II 2. Same material as in Plate l,i. The glycocalyx at this early tetrad stage was unlike the proexine of Eucommia (see Plate III,3-8. Glycocalyx (G), callose (C), plastid (P), amoeboid plastid (Ap), Starch (Ch), lipid (L), nucleus (N), dictyosome (D), autophagic vesicle (Av.) Bar: 5 l~m. PLATE III (see p.302) 3-9. The early exine receptor stages. Initially (fig.3) receptors (arrowheads) were widely spaced structures 40-70 nm in diameter located on the plasma membrane. Later, receptors appeared in short stacks (arrows, rigA). Thereafter, receptors became obscured through accumulation of exine forming material (which we will refer to as sporopollenin). Structural evidence for the presence of receptors can be seen at the surface of forming components of the exine until the late microspore period. Structures of receptor size are marked by arrows in fig.5. Figures 6-9 illustrate the progressive buildup of proexine components. The proexine components are dark centrally and have a weakly stained outer zone (arrows, fig.6). The protectum and microbacules are weakly stained rods (arrowheads, figs.6, 7) 10-15 nm in diameter. Exine receptors are marked by ~rrows in fig.7. Following the stage in fig.8 the proexine will become coherent except for the pore and furrow. Figure 9 shows section profile of a profurrow (between stars) at the stage in fig.8. Bars, figs.3-8:0.5 ttm; fig.9: l ~tm. PLATE IV (see p.303) 10. Middle microspore tetrad. The small and discrete exine components are mostly located on a future furrow (between stars). Several prospinules are marked by arrows. Spinules are on the developing foot layer (F). The fuzz (arrowheads) at the surface of the foot layer is the future system of microbacules and rectum. Bar: l ~tm.

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frequently disjunct (Plate V, 13, Plate VI, 14). Near the equator the foot layer in furrows was variable but often thick (Plate VI, I 5). Spinules were 100200 nm in diameter. The tangential sections in Plate VI, 14 show that spinules can be located close together (ca 200 nm) and also show that their spacing is unequal (Plate XIV,40).

IlL Vacuolate period of microspore formation During this period of wall and cell enlargement the foot layer undergoes great changes in form. It becomes extremely irregular (Plate VII,16) and shows rod-shaped substructure. Spinules and microbacules are elevated (Plate VII,16). Later during this period the foot layer shows substructures that are rounded at its top and bottom margins (Plate VII,17, Plate VIII,! 8-20). In the equatorial region of furrows there are large globular structures on the often greatly elevated tectum (Plate VII, 17, Plate VIII, 19,20).

J.R. ROWLEY ET AL.

IV. Middle to late microspore period The inner part of the foot layer becomes solid appearing with a uniform proximal surface in close contact with the plasma membrane (Plate IX,21). The radial units of the foot layer except near the distal surface become fused. Rod-shaped foot layer substructures are exposed in the outer portion longer than elsewhere. Distally some of these are spinules/bacules (Plate IX,21). The tectum and associated slender rod structures do not strongly accept stain until late in microspore development (Plate IX,22). The section in Plate IX,23 was prepared by fixation No. 2 using osmium tetroxide. With only osmium but no section staining there was an indication of endexine differentiation. The tectal region in this figure was, like younger stages, poorly contrasted (e.g. Plate V,l.~, Plate IX,21). The entire foot layer was solid in appearance in interapertural sectors during the late microspore period (Plate IX,22).

PLATE V !!. Period of thickening and amalgamation of foot layer (F) components. Receptors can be seen on end (arrowheads) and in side view (arrows). Spinule (S). Bar: 0.5 ~tm. 12. Late tetrad. Exine formation is asynchronous between microspores of a tetrad and even on individual microspores. Areas having small bead-like structures (stars) are future furrows. The relatively large globoid structures (arrows) are located over furrows in the equatorial region. Spinules are not easily seen at this low magnification but several are marked by arrowheads for comparison with the large globular structures marked by arrows. Bar: 5 ~tm. 13. A furrow (star) in transverse section. The foot layer (F) forms the most obvious part of the furrow margin. Over the furrow the foot layer is not yet coherent. The tectum (arrowheads) is very weakly stainable and adjacent to the foot layer on and near the furrow. Bar: 0.5 lam. PLATE Vl (see p.306) 14,15. Early free microspore period (prior to vacuolation). In interaperture sectors the foot layer (F) is ca. 0.5 ~tm in thickness. Spinules (arrows) appear widely spaced in transverse sections but in the oblique sections (triangles) they are seen to be separated by as little as 200-300 n m In the microspore in fig.14 the furrows (stars) are in sections cut well away from the equatorial zone. The furrows (stars) in lower section were located near the equatorial plane. The furrow marked by a filled star in fig.15 is near to a pore. Both the foot layer (F) and tectum (arrowheads) occur over the furrow. Developing oncus (O). Bars: 1 lam. PLATE VII (see p.307) 16. Intermediate in development between Plate VI, 15 and Plate VII, 17. The receptors are rod shaped by this interval of development. Receptors on end (arrowheads). Receptors in side views (arrows). The distal portion of large exine rods will be spinules (S). Subunits of the foot layer in end view (u). Bar: 0.5 l~m (500 nm). 17. Vacuolate microspore period. This was a period of cell enlargement and a second interval of exine thickening. The exine ceased to be more or less solid in appearance. There was separation between rod-shaped components of the exine and both surfaces of the foot layer became irregular. Some rods protruded outward resulting in elevation of the tectum and supratectai components (arrowheads). These large supratectal components (arrowheads) are on or near furrows. The pericytoplasmic spaces (stars) are under or near furrows. Cytoplasmic vesicles (V). Bar: 5 ~m.

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V. Late microspore to microspore mitosis period There were large cytoplasmic invaginations a n d formation of onci (Plate X,24). A pericytoplasmic space or preintine is apparent in Plate X,24 a n d an initial intine in Plate XI,25. The exine increased in thickness to about 1.5 ~tm a n d an endexine zone became stain differentiated (Plates X, XI) by the time of microspore mitosis. The tectum extended without interruption over the furrow. In the furrow in the equatorial region the tectum remained elevated and coated with large globular structures (Plate XI,25). Elsewhere in the furrow the tectum was near the foot layer (Plates X, XI). In interapertural sectors the tectum was uniformly elevated above the foot layer (Plates X, Xl).

VI. Mature pollen grains The aperture system is colporate. In cross sectional profile some furrows showed a very gradual reduction in foot layer thickness toward the furrow

J.R. ROWLEY El" AL.

floor (Plate XII,26). In others the foot layer reduction was a b r u p t a n d the furrow cross section profile was a V-shape (Plate XII,27). S o m e furrows had a b r o a d flat floor (Plate XII,28). The S E M ' s in Plate XIV,41,43 and 44a indicate that furrows m a y taper t o w a r d their polar ends or have r o u n d e d ends. N e a r the e q u a t o r there can be r e m n a n t s of supratectal globular structures (Plate XII,29) and/ or bridges or tongues o f foot layer (Plate XII,30-33). Pores were rarely seen in surface views with light microscopy. They could be seen quite frequently using either phase or N o m a r s k i differential interference contrast by focusing on apertures a r r a n g e d on edge rather t h a n in surface views. In t h a t way they are seen in planes like Plate XII,33 a n d Plate XIII,34. Sections o f relatively unobscured pores were very rare (Plate XII,33, Plate XIII,34). M o s t pores were filled by lamellations (Plate XIII,35) a n d / o r covered by a bridge of foot layer plus tectum (Plate XIII,37,38, Plate XIV,42). T h e encroachment of foot layer plus tectum on pores was specially intruding during the microspore mitosis

PLATE VIII 18-20. Vacuolate microspore stage (see Plate VII,17). Tectum (T) is elevated in figs.18-20. Rod structures in figs.18, 19 are marked by white arrows. They extend outward from the foot layer (F) and in some sections join the tectum (T). Arrowheads mark rods in end views. Figure 20 is near or on a furrow. In fig.20 the supratec~.el components show dark receptor structures (arrowheads). Bars: 0.5 ~m. PLATE IX (see p.310) 21.

Intermediate stage between Plate VIII,18-20 and Plate IX,22,23. Enlargement of the foot layer (F) is near completion in this material. The spinule (S) can be traced as a separate component below the generalized outer surface of the foot layer. Tectum (arrow). Bar: 0.5 m. 22,23. Middle microspore period. The foot layer is ca. 1 um thick in the interaperture zone and generally solid in appearance although the outer surface of the foot layer remains uneven. Spinules are ca. 100 nm in diameter and 0.2 to 0.5 om in height. Microbacules and tectum consist of substructures about 10-15 nm in diameter. Many of these substructures, like spinules, extend above the level of the tectum (T). Figure 23 shows two bacules/spinules and many microbacules; the inner low contrast portion of the exine will differentiate into endexine. Stain: fig.22, 0.1% phosphotungstic acid in 10% chromic acid; fig.23, osmium tetroxide only. Bars: fig.22:0.5 I~m; fig23:100 nm. PLATE X (see p.311) 24. Late microspore to microspore mitosis period. The foot layer is ca. 1.5 ~tm in thickness. The exine has an inner zone of low contrast that is ca. 0.3 ~tm thick. The tectum and spinules are readily seen at this stage. The section passes close to two pores (stars). The pore zone is crossed by lamellations, some from the inner (endexine, En) layer of the exine. The pore zone marked by an open star is overlapped by the foot layer (F). This could appear from the surface as a bridge crossing the furrow (Plate XIV,42-44). The exines marked by an asterisk and triangle represent aborted microspores. One of these (asterisk) aborted at an early stage (compare with Plate V, ll), in the other (triangle) development is typical for a late microspore. Exine oncus (O). lntine oncus (!0). Bar: l ~tm.

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and early pollen grain intervals (Plate X,24, Plate XIII,36,38). An oncus forms in association with the exine in the early free microspore interval; onci or developing onci are seen as pericytoplasmic spaces in Plate VI, l 5). An oncus forms in conjunction with intine formation during the microspore mitosis period (Plate X,24, Plate XI,25, Plate XIII,36,38). During most of the pollen grain period there is an exineoncus and intine-oncus, as the exine and intine associated onci are referred to by Rodriguez-Garcia and Fernandez (1988). In mature pollen the exine-oncus exists only as a remnant (Plate XII,32) contributing to compaction of lamellations in the pore (compare Plate X,24 with the section of the

J.R. ROWLEY ET AL.

mature exine in Plate XIII,35). The oncus associated with the intine is large in mature pollen (Plate XII,32,39a,b). The circular shape of these onci can be appreciated in Plate XIV,39b. The photomicrographs in Plate XIV,39 were made from a thick section from the same plastic embedding as Plate XII,32. For Plate XIV,41-44 the resin had been removed from that same embedding. The grains in these SEM's are expanded and show a pore region that is either more or less flush (Plate XIV,44a) or raised above the neighboring interapertural surfaces Plate XIV,43,44b. Aperture shape can be variable (Plate XIV,41,43,44a). Some apertures taper in width while others have parallel margins. The grain in Plate XIV,44a has an aper-

PLATE XI 25. Same stage as fig.24. The section parallels a furrow in the equatorial region. The foot layer (F) and endexine (En) are both decorated by globules. The large globules on the foot layer and tectum (arrowheads) are characteristic for the equatorial portion of the furrow at this stage (see Plate XIII,36). The spinule marked by an arrow was cut transversely. Intine (I). Bar: I lam. PLATE XII (see p.314) 26-33. Sections across the farrow and pore on mature pollen. Figures 26-28 are near the polar end of furrows. In fig.26 the furrow region includes tectum (arrows) foot layer (F), low contrast endexine (En) and intine (I). Cytoplasm (Cy). The exine in fig.27 was acetolyzed. The furrow includes both foot layer and endexine. Figure 28 is a section of a fixed polien grain with a relatively broad furrow. Tectum (arrows). Foot layer (arrowheads). Endexine (En). Intine (I). Figures 29, 30 are near the equator and the pore. In fig.29 there are remnants (wavy arrows) of the relatively massive components associated with the surface of the foot layer and tectum (see Plate XI,25). In fig.30 there are tongues of foot layer (arrows) over the furrow). The endexine (En) is darkly contrasted in figs.30, 31. The sections in figs.30, 31 are near the pore. In fig.31 the foot layer (arrowhead) is thin and the endexine (En) relatively thick in the furrow; the tectum is marked by arrows. In fig.32 the foot layer (F) is relatively slender but continuous (arrowheads) in the region of the pore. Remnant of exine oncus (star). lntine oncus (It)). In fig.33 the pore is crossed by lamellations of the endexine (En). Tongues of the foot layer (arrowheads) extend to margin of the pore. Intine oncus (It)). Bars: 1 ~tm. PLATE XIII (see p.315) 34-38. Sections of the pore region. 34. Longitudinal section of pollen grain. Pore in acetolyzed mature exine. The foot layer (F) and endexine (En) are differentially contrasted. Lamellations extend from both foot layer and endexine. These lamellations and material interbedded with them are resistant to acetolysis. Pores, like this one, without overlapping foot layer were rarely seen. Bar: I ~m. 35. Equatorial section of pollen grain. This pore region in a fixed grain is interlaced by lamellations from both the foot layer (F) and endexine (En). The tectum (arrows) is adjacent to the foot layer. Bar: l I~m. 36. Section of microspore mitosis stage that parallels a furrow and passes through the pore region (star, located in os below pore), exine oncus (O), and intine oncus (IO). The furrow extends from the pore region to the right margin of the figure and includes endexine (En), foot layer (F), tectum (arrows) with supratectal components. In this section the foot layer (arrowheads) also extends over the pore region. The components around the asterisk are part of a furrow on an adjacent grain. Bar: ! I~m. 37. Acetolyzed mature pollen. Pore region (star, located in os below pore) is overlaid by foot layer (arrowheads). The tectum is adjacent to the foot layer over the pore (arrows) and surface of the furrow (asterisk). Bar: l I~m. 38. Microspore mitosis stage. The foot layer (F) extends over the greater part of the pore region (star, located in os below pore). Globular structures are common equatorially during microspore stages at the inner surface of the endexine (En: see also Plate XI,25) and supratectaUy. Exine oncus (O). Intine oncus (I0). Bar: ! ~m.

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ture with rounded ends and a flat floor, like in Plate XII,28. The materials on the equatorial areas of the furrows in Plate XIV,43,44 are likely to be supratectal globular structures (Plate XIII,35,38). The tectal bridge over the pore zone in Plate XIV,42 includes spinules and, therefore, also tecturn. There was equal infolding of the three colpi in polar view (Plate XIV,40). The spacing of spinules is unequal (Plate XIV,40), as can also be seen in tangential sections (Plate VI,14). A tectum covers all of the Eucommia pollen grain except the pore where, however, it can occur on bridges and tongues of foot layer. The tectum was in close contact (separation only ca. 30 nm) with the foot layer over the colpus. There were rare alveolations between foot layer and tectum on the colpus (Plate XII,26, Plate XV,52). Over the rest of the foot layer tectal alveolation was the rule in mature pollen. During the vacuolate microspore interval the tectum was variously elevated (Plates VII, VIII) but thereafter the tectum was separated uniformly by only ca. 100 nm (Plates X, XI) until the pollen grain period. Thus, the alveolation seen in Plate XII and Plate XIII,34 and emphasized in Plate XV,45,46 and 48-52 represents dynamic change in exine form. If attachment of the tectum to spinules/bacules is stable, the attachment height might represent a programmed elevation for the tectum (e.g. Plate XV,45,46). Oblique sections like Plate XV,47 fail to show clear contact between tectum or microbacules and spinules. Considered together, Plate XV,46-52 show that small rods extend between the foot layer and tectum. On end these microbacules are circular and 10-15nm in diameter

J.R. ROWLEY ET AL.

(Plate XV,47,51). The tectum was not a contiguous sheet but was made of disjunct structures having a circular profile (Plate XV,47). The obliquely sectioned microbacules have diameters similar to components of the tectum (Plate XV,47). Microbacules are well illustrated in Plate XV,51). There are short microbacules under the tectum on furrows (Plate XV,52). The pore can be seen to be free of the tectum in Plate XII,30,33, Plate XIII,34,35. The tectum at the margin of pores was 50 nm (0.05 pm) or less in thickness. Discussion

Gunnar Erdtman was greatly interested in the unusual germinal aperture system in pollen of Eucommia largely because the three apertures are not always of the same length. Among pollen of extant plants the frequent occurrence of apertures of unequal length in tricolpate grains appears limited to Euptelea, Eucommia and Cercidiphyllum (Erdtman, 1948, 1952, 1969). Pollen grains of these genera also have apertures with rounded ends; otherwise, as Erdtman (1952) noted, they have few morphological features in common. The significance of tricolpate or tricolporoidate (Eucommia) apertures of unequal length is not understood, but such differences in length will have a lasting influence on the nomenclature of fossil pollen. The Early Jurassic to Cretaceous sporomorph Tricolpites troedssonii n. spm. was named Eucommiidities because of the morphological similarity of the two long and often one short apertures with rounded ends of Eucommia ulmoides (Erdtman, 1948). We

PLATE XIV 39-44. Photomicrographs and SEMs of mature pollen: same material as in use for fig.32. 39a,b. The shape of the oncus of the pore region is shown in two views. The equatorial plane of focus in fig.39a is similar to the TEM in fig.32. The oncus is nearly circular in oblique surface views (fig.39b). Bar: 20 pm. 0. An acetolyzed mature pollen grain. The three interfurrow sectors and furrows are folded inward and they appear equal. Spacing of spinules is unequal. Bar: 5 pm. 41. Some furrows tapered poleward (arrow), others had parallel margins with rounded ends (see also fig.44a). Bar I0 pro. 42a,b. The furrow over the pore is bridged (arrows) by foot layer plus tectum which includes spinules. Bars: 5 pm. 43. The pore as judged by the location of the raised material (arrow) appears to be located acentrally in this furrow. Bar: 5 pro. 44a,b. The raised material on equ"torial portions of the furrows is typical of globular components seen throughout development (e.g., Plate V,12, Plate VII,17, Plate XI,25, Plate XII,29). Bars: 5 pm. Preparation for the SEM samples used for Plate XIV,41-44: Epon-Araldite resin was removed with sodium methoxide. The pollen was washed in methanol and transferred to an alternative to critical point drying (Peldri II).

POLLEN WALL DEVELOPMENT IN EUCOMMIA ULMOIDES

PLATE XIV

3 !7

318

J.R. ROWLEY ET AL.

now know that the Eucommiidites pollen exines described from the lower Jurassic of Sweden are gymnospermous (Cycadalean) (Couper, 1958; Van Konijnenburg, 1971; Pedersen et al., 1989). In our study, differentiation of a template for colpi was evident from early tetrad time onward. The pore became easily recognizable after endexine, oncus and material interbedded with endexine lamellae were inserted. In spite of its bold appearance in TEM sections of the colporate system it can only rarely be detected in surface views with LM. Erdtman (1952) referred to it as "oidate". The aperture is, however, colporate. The form of the colpus is also difficult to analyze using the light microscope because the sides may taper either gradually to the furrow floor or steeply. The furrow floor may be slightly rounded--concave, V-shaped, or fiat. These variations coupled with modification in form due to variable dehydration and exine infolding make the position of the furrow ends difficult to ascertain. Paying special attention to the apertures we have looked at pollen of both acetolyzed exines (Plate XIV,40) and fixed, expanded grains (Plate XIV,41-44) using multiple methods (SEM, TEM, Nomarski and phase microscopy). We find no evidence for apertures of unequal length in individual grains. The pore appears off-center (Plate XIV,43), this assessment is influenced by the arrangement of tongues and bridges of foot layer across the pore zone. Globular structures on the foot layer and tectum also mask

or obstruct the pore (Plate XIV,43,44). The difficulty is that the pore may not be directly under the foot layer tongues and bridges and globular structures. In TEM thin sections these overlying exine components appear off-center with respect to pores (Plate X,24, Plate XIII,37,38). The Leiden collections of Eucommia from Jan Muller (to JRR) were sent to Stockholm. In 1970, while they were being processed for electron microscopy, Gunnar Erdtman studied acetolyzed grains from these collections and made the drawings in Fig.l. These working sketches show that pollen grains used for our work had the same apertural

Fig.l. Sketches made by Gunnar Erdtman (May 13, 1970) from an acetolyzed portion of the same collection used in our study. From these sketches it is c!.?ar Professor Erdtman saw evidence for one relatively short furrow. His interpretation indicates that pollen of our collection was similar to other collections he had studied (Erdtman, 1952: fig.95).

PLATE XV 45-52. The tectum and bacules in mature pollen. 45. The tectum (arrows) is, as is typical for our Eucommia material, near the foot layer (F) in furrows (star) and variously elevated elsewhere. That portion of a spinule (S) proximal to the tectum can be called a bacule (B). Endexine (En). Bar: 1 p.m.

46. 47.

48. 49. 50. 51. 52.

Includes portions of three spinules and the relatively massive tectum of the mature pollen. Bar: 100 nm. An oblique section passing through the foot layer (arrowheads), Tectum (T), and forest of rods (bacules) between those layers. The tectum is made up of structures that are circular (arrows) in cross sections. Spinule/bacule (B). Obliquely sectioned foot layer (arrowheads). Microbacules (circular frames). Bar: 100 nm. Arrows mark two of the many microbacules extending from the tectum (T) to the foot layer (F). Bar: 100 nm. Microbacules (arrows) are seen in two alveolate regions. Supratectal rods (arrt, wheads). Tectum (T). Foot layer (F). Bar 100 nm. The tectum is extensively alveolate (asterisk) in this section. Tectal height position before alveolation may be indicated by tectal position on the spinule (S)/bacule (B). Bar: 0.5 lam. The tectum is only slightly alveolated in this section. Many microbacules (arrows) are evident. In cross section microbacules are circular (arrowhead). Bar: 0.5 pm. In furrows the tectum is near the foot layer and bacules are very short (arrows). Alveoli however, do occur between foot layer and tectum (asterisk). Bar: 0.5 lam.

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characteristics Erdtman had seen and recorded earlier, e.g., from China (Erdtman, 1952). They also show a bit of how Gunnar Erdtman worked. He had a special interest in Eucommia but in general he made drawings of the grains in each slide examined. All of these interpretative records were retained in hand lettered folders forming a treasure for pollen morphologists. It would appear from these sketches that one aperture was shorter, especially with respect to one arm (Fig. 1). Unequal apertures in Eucommia are reported by Zavada and Dilcher (1986) based upon SEM and TEM observations and also by Zhang et al. (1988), working on local collections in Beijing. Zhang et al. (1988) using, LM, SEM and TEM consider apertures tricolporoidate although they sometimes saw the outline of ora. They report that "Colpi are narrow, unequal in length, often two long and one short or two short and one long, sometimes rather irregularly arranged, with indistinct and thin colpus membranes". In his study of embryology and microsporogenesis of Eucommia ulmoides, Eckardt (1963) observed that different stages of meiosis can be recovered from upper and lower parts of an anther. Eckardt's photomicrograph of shed pollen shows grains similar to our Plate XIV,39a,b. Apertures are described as tricolporoidate. Division of microspore mother cells was found to be simultaneous, resulting in tetrahedral tetrads. The interapertural exine of Eucommia, while unusual, has parallels with other unrelated groups. In Eremolepis punctulata (Eremolepidaceae) and several other species of mistletoe there is a very slender tectum supported by microbacules (Feuer and Kuijt, 1978). The tectum is generally separated in these taxa by only 100 nm from a thick foot layer on an endexine of irregular thickness. Zavada and Dilcher (1986) include Eucommia as part of comparative work concerning pollen morphology in the Hamamelidae. Aided by both TEM and SEM micrographs their description includes several points in agreement with ours, namely a rather thick endexine that does not thicken in the apertural regions and exine sculpturing that is spinulose. With regard to the endexine, in our material there is a modest increase in thickness associated with apertures (see Plate

J.R. ROWLEY ET AL.

XII,30,31,33). In other respects observations from our collection differ from Zavada and Dilcher. We find the aperture system to be tricolporate rather than tricolpate with all colpi essentially equal. We interpret the narrow colpus in the figure of Zavada and Dilcher (1986, p.43) as the consequence of dehydration during preparation; infolding in the apertures was minimized by drying pollen through use of a drying agent. We also are at variance with the sculpturing of Eucommia pollen reported by Zavada and Dilcher. They have characterized sculpturing as minutely spinulose, rugulate and also verrucate. Minutely spinulose corresponds to the more recognized description of nanospinules as discussed by Erdtman (1969) for spinules less than 0.5 ~tm high as seen by electron microscopy, making it appropriate for description of the Eucommia pollen surface. However, we find no evidence to support the rugulate ("covered with creases", Erdtman, 1969, p.45) or verrucate ("blunt processes, broader than high", Erdtman, 1969, p.42) sculpturing descriptions. Our interpretation is that the sculpturing (texture) of the exine surfaces, apart from spinules, consists of the ends of the approximately 10 nm wide microbacules. A final point of discord is with Zavada and Dilcher's (1986) interpretation of exine structure. Our pollen is not atectate but has a tectum of variable elevation supported by microbacules and relatively large spinules/bacules. Results of Zhang et al. (1988) show tectum, microbacules and spinule/bacules above a zone we and Zhang refer to as foot layer. In contrast to that described by Zavada and Dilcher (1986) our pollen has a foot layer which is, in fact, the dominant layer of the exine. The sketch in the upper portion of Fig.2 represents an interpretation based upon Gunnar Erdtman's extremely skillful use of an excellent light microscope. He labeled it "Speculation only!" but it was not an invention, he simply could not be sure. At the top he sketched a slender tectum supported by infratectal radially-oriented material. At the bottom of the figure he showed an alternative interpretation without these features. In our sections (e.g., Plate XV,45-52) the separation of the tectum and foot layer (the bacular arcade) is about 0.3 lam in height in strongly alveolated regions (Plate XV,45) and can be appreciated with

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the LM. With sectioned exines (even relatively thick frozen sections) the tectum, bacular arcade and spinule-bacules are easily resolved using phaseor differential interference-contrast microscopy. The microbacules (Erdtman's "infratectal radially oriented material") require electron microscopy to be seen with any degree of confidence. The same is true for the pore system of the compound apertures of Eucommia. The ora can be seen (often best in side views) with phase contrast and differential interference contrast. Erdtman (1952, fig.95) showed the ora in side views; they were described as having a faintly delimited oroid opening that appears in his drawing to be 2-4 ~tm wide. That width is about the same as the pore (gap in the tectum) in oar Plate XII,29,30,33, Plate XIII,34,35. The ora in the above cited drawing of Erdtman appear to be 15 and 17 ~tm in length and oriented longitudinally (that is a little more than the width of Plate XIII,34). In our collections ora are seen with LM as weakly defined gaps in the nexine no more than

32 !

10 lxm in length (about the distance from 'En" to the left margin in Plate XIII,34); that is similar to the endoaperture indentations in several drawings of Eucommia in the 1948 paper by Erdtman. The actual pore in the tectum will probably seldom be seen with the light microscope. We have described tongues and bridges of foot layer and globular structures that cover the pore region. Erdtman, in his 1952 book, described the colpi of Eucommia as being covered with membranes of a diffuse granular material in the center of each colpus. In addition, the rectum is no more than 0.05 l~m (50 nm) in thickness at the margin of the pore. Light microscopically, Eucommia pollen appears to be 3-colporoidate as Erdtman (1952) reported but in reality the pollen is 3-colporate. We used the term "nexine" above because it may refer to both foot layer and endexine and in Eucommia pollen ora are comprised of both layers. The funnel shape of ora can be seen in Plate XIII,34. It is also evident in this figure that both the foot layer and tectum are thin at the pore margin. The width of ora near the equator is considered to be about 5 pm. This measurement is based upon equatorial sections which show both a pore margin and fingers of endexine and foot layer extending across the os (e.g., Plate XIII,35). This means that in longitudinal section through a pore there are fingers of nexine very close under and on both sides of the opening. A plane section as shown in Plate XIII,34 is rarely encountered; most sections through the pore look like Plate VII,33 or Plate XIII,35. Occlusion of the pore and os by fingers of nexin¢ may be the reason for the difficulty in seeing and interpreting the compound aperture in this taxon using the light microscope. Eucommia pollen is highly anomalous as an inclusion in Hamamelididae. The colpus membrane of members of Hamamelididae is often granulate and the ¢xine reticulate, while Eucommia exine has widely spaced spinules and there are no granules on the colpus membrane (tectum) observable with the light microscope (e.g. Plate XV,52). Eucommia has been linked with Cercidiphyllum and Euptelea because apertures in many grains of these taxa have been reported to be of unequal length. We have no proof to the contrary, but in our SEM examinations we have seen no convincing

322

example of unequal apertures. We recognize, however, that with SEM it is not usually possible to see equally well both ends of all three apertures. Basing his conclusion upon leaf architecture and xylem anatomy Wolfe (1989) reported that Eucommia appears to have a close relationship with Stachyuraceae. He suggested that the reproductive structures of Eucommia and Stachyurus have greatly diverged while retaining a high degree of similarity in both polar foliar architecture and xylem anatomy. The syndrome of foliar characters displayed by Eucommia is like that of members of Theales and Violales and unlike that of Hamamelididae or Urticales (Wolfe, 1989). Wolfe (1989) specifically noted that the foliar and xylem features of Stewartia pentagyna (Theaceae) were like those of Eucommia. Erdtman (1952) described Stewartia pollen, like that of Eucommia, as 3-colporoidate. From his drawings it can be seen that Stewartia has narrow longitudinal ora ("oroids"), a thick foot layer and a very fine exine pattern. The development and final form of Eucommia pollen are both exceptional. While we consider that the matter of unequal length of apertures requires further observation there is no doubt that the apertures are unusual. They are involved in the same general lack of developmental concurrences as the rest of the exine. In mature pollen the shape of the furrows is extremely variable with polar ends, as seen with SEM, either tapered or rounded. Formation of components is asynchronous during development of Eucommia pollen between members of a tetrad and even on individual microspores.

Acknowledgements Financial support was provided by the Swedish Natural Science Research Council (JRR and Bj6rn Walles) and National Science Foundation of USA (JJS). We have the pleasure to acknowledge the skilled technical assistance of Elisabeth Grafstr6m, William F. Chissoe and Dr. Masamichi Takahashi. We take this opportunity to express warm thanks to Professor Bj6rn Walles for electron microscopical facilities at Stockholm University. Completion

J.R. ROWLEYETAL.

of this work was made possible through use of equipment and space provided by the Samuel Roberts Noble Electron Microscope Facility, The University of Oklahoma.

References Chissoe, W.F., Vezey, E.L. and Skvarla, J.J., 1990. Drying of pollen with Peldri II (proprietary fluorocarbon) for scanning electron microscopy. Rev. Palaeobot. Palynol., 63: 29-34. Couper, RA., 1958. British Mesozoic microspores and pollen grains. A systematic and stratigraphic study. Palaeontographica, 103B: 75-179. Cronquist, A., 1968. The evolution and classification of flowering plants. Houghton Mifflin Company, Boston, Mass., 396 pp. Cronquist, A., 1981. An Integrated System of Classification of Flowering Plants. Columbia University Press, New York, 1262 pp. Eckardt, Th., 1963. Some observations on the morphology and embryology of Eucommia ulmoides Oliv. J. Indian Bot. Soc., 42A: 27-34. Engler, A. and Diels, L., 1936. Syllabus der Pflanzenfamilien. I I th. ed. Aufl., Berlin, 916 pp. Erdtman, G., 1948. Did dicotyledonous plants exist in early Jurassic times? Geol. F6rhandl., 70: 265-271. Erdtman, G., 1952. Pollen Morphology and Plant Taxonomy. Angiosperms. Almquist & Wiksell, Stockholm, 539 pp. Erdtman, G., 1960. The acetolysis method. A revised description. Sven. Bot. Tidskr., 39: 561-564. Erdtman, G., 1969. Handbook of palynology. MorphologyTaxonomy-Ecology. An Introduction to the Study of Pollen Grains and Spores. Munksgaard, Copenhagen, 486 pp. Feuei, S. aiid Kuijt, J., 1978. Fine strm:turc of mistletoe pollen. I. Eremolepidaceae, Lepidoceras, and Tulipa. Can. J. Bot., 56: 2853-2864. Harms, H., 1930. Eucommiaceae. In: Engler and Prantl (Editors), Die naturlichen Pflanzenfamilien, (2nd ed.), 18a: 348-351. Verlag yon Wilhelm Engelmann, Leipzig. Hutchinson, J., 1926. The Families of Flowering Plants. I. Macmillan, London, 5 ! 0 pp. Hutchinson, J., 1969. Evolution and Phylogeny of Flowering Plants. Academic Press, London, 717 pp. Hutchinson, J., 1973. The Families of Flowering Plants. Oxford University Press, London, 968 pp. Ikuse, M., 1955. General survey lis' of pollen grains in Japan. J. Jap. l~:~t., 30: 225-232. Ikuse, M., 1956. Pollen Grains of Japan. Hirokawa Publishing Co., Tokyo, 304 pp. Kuprianova, L.A., I965. The Palynology of the Amentiferae. Kamarov Bot. Inst. Acad. Sci. URSS., l: 1-214. Lemesle, R., 1046. Contribution a retude morphologique et phylogenetique des Eupteleacees, Cercidiphyllacees, Eucommiacees (Ex-Trochodendracees). Ann. Sci. Nat., I l: 41-5 I. Mollenhauer, H.H., 1964. Plastic embedding mixtures for use in electron microscopy. Stain Technol., 39: I 1 l- l 15. Nemejc, F., 1956. On the problem of the origin and phyloge-

POLLEN WALL DEVELOPMENT IN EUCOMMIA ULMOIDES

netic development of the Angiosperms. Sb. Nay. Mus. Praze, Sect. B, 12B (2-3): 59-143. Oliver, D., 1891. Eucommia ulmoides Oliv. Hooker's Icon. PI, 10 (2), t. 1950. Patel, V., Skvarla, J.J. and Raven, P.H., 1983. Half pseudocolpi, a unique feature of Olinia Oliniaceae pollen. Am. J. Bot., 70: 469-473. Pedersen, K.R., Crane, P.R. and Friis, E.M., 1989. Pollen ergaans and seeds with Eucommidites pollen. Grana, 28: 279-294. Pokrovskaja, I.M., 1950. Analyse pollinique (Traduction par E. Boltenhagen). Annales du Service d'Information geologique du B.R.G.M., 24 (1958): 1-435. Rodriguez-Garcia, M T and Fernandez, M.C., 1988. A review of the terminology applied to apertural thickenings of the pollen grain: Zwischenkorper or oncus. Rev. Palaeobot. Palynol., 54:159-163. S k v a r l a , J.J., Rowley, J.R. and Chissoe, W.F., 1988. Adaptability of scanning electron microscopy to studies of pollen morphology. Aliso, 12: ! 19-175. Solereder, H., 1899. Zur Morphologie und Systematik der Gattung Cercidiphyllum Sieb. et Zucc. mit Berucksichtigung der Gattung Eucommia Oliv. Bet. Dtsch. Bot. Ges., 17. Swamy, B.G.L. and Bailey, I.W., 1949. The morphology and relationships of Cercidiphyllum. J. Arnold Arbor., 30: 187-210. Takhtajan, A., 1969. Flowering Plants: Origin and Dispersal. Transl. by C. Jeffrey, R. Bot. Gard., Kew. Smithsonian Institution Press, Washington, D.C., 310 pp Takhtajan, A.L., 1980. Outline of the classification of flowering plants (Magnoliophyta). Bot. Rev., 46: 225-359.

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Thorne, R.F., 1989. Hamamelididae: a commentary. In: P.R. Crane and S. Blackmore (Editors), Evolution, Systematics, and Fossil History of Hamamelidae. Clarendon Press, Oxford, vol. 1, pp.9-16. Tippo, O., 1940. The comparative anatomy of the secondary xylem and the phylogeny of the Eucommiaceae. Am. J. Bot., 22: 832-838. Trevisan, L., 1980. Ultrastructural notes and considerations on Ephedripites, Eucommiidites and Monosulcites pollen grains from Lower Cretaceous sediments of Southern Tuscany (Italy). Pollen Spores, 22: 85-132. Van Konijnenburg-Van Cittert, J.H.A., 1971. In situ Gymnosperm pollen from the Middle Jurassic of Yorkshire. Acta Bot. Neerl. 20: !-96. Varossieau, W., 1942. On the taxonomic position of Eucommia ulmoides Oliv. (Eucommiaceae). Blumea, 5: 81-92. Walker, J. and Doyle, J.A., 1975. The bases of Angiosperm phylogeny. Ann. M. Bot. Gard., 62: 664-723. Wettstein, R., 1935. Handbuch der Systematischen Botanik. 4th ed. Franz Deuticke, Leipzig and Vienna, 1152 pp. Wolfe, J.A., 1989. Leaf architectural analysis of the Hamamelididae. In: P.R. Crane and S. Blackmore (Editors), Evolution, Systematics and Fossil History of the Hamamelidae. Clarendon Press, Oxford, vol. I, pp.75-104. Zavada, M.S. and Dilcher, D.L., 1986. Comparative pollen morphology and its relationship to phylogeny of pollen in the Hamamelidae. Ann. M. Bot. Gard., 73: 348-381. Zhang, Yu-Long, Wang, Fu-Hsiung and Chieng, Nan-Feng, 1988. A study on pollen morphology of Eucommia ulmoides Oliver. Acta Phytotaxon., 26: 367-370.