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Comparative ultrastructure of gyrtmosperm and implications regarding the origin of angiosperms

Osborn, Jeffrey Mark, Ph.D.

The Ohio State University, 1991

U’M'I 300 N. Zeeb Rd. Ann Arbor, MI 48106

COMPARATIVE ULTRASTRUCTURE OF FOSSIL POLLEN AND IMPLICATIONS REGARDING THE ORIGIN OF ANGIOSPERMS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

Jeffrey Mark Osborn, B. S., M. S.

*****

The Ohio State University 1991

Dissertation Committee: Approved by Thomas N. Taylor Valayamghat Raghavan Fred D. Sack \ Adviser Edith L. Taylor Department of Biology To Dr. Alfred S. Osborn, Sr. ACKNOWLEDGEMENTS

This research was financially supported by several institutions and agencies. These include: the National Science Foundation (Dissertation Improvement Award; BSR- 9016397), the American Association of Stratigraphie Palynologists (Student Scholarship), Sigma-Xi, The Scientinc Research Society (Grant-in-aid of Research), the Graduate School, The Ohio State University (Graduate Student Alumni Research Award and Presidential Fellowship), and the Department of Plant Biology, The Ohio State University (Graduate Teaching and Research Associateships). The outcome of this investigation was significantly aided by their assistance and it is gratefully acknowledged. Numerous individuals have also contributed in variety of ways to both this investigation as well as my other scholarly endeavors. I would like to thank the group (Dr. L. J. Hickey, Dr. D. W. Taylor, L. Klise, and A. Muller) at the Peabody Museum of Natural History, Yale University for providing much assistance and a generous loan of Cycadeoidea material. Several members of both the Paleobotany and Departments (Drs. F. M. Hueber, S. H. Mamay, W. A. DiMichele, S. L. Wing, and R. A. Faden), National Museum of Natural History, Smithsonian Institution were also especially helpful during a my visit there. I am also grateful to Dr. T. Rich and the staff of the National Museum of Victoria for loaning the specimen of Sahnia laxiphora, and Dr. M. R. de Lima for providing sediments fi*om the Santana Formation. I am especially appreciative of the following individuals, who gave of their time in order to write supporting letters of my various grant, fellowship, and employment proposals/applications: Drs. T. N. Taylor, T. Delevoryas, E. L. Schneider, J. R. Rowley,

ni G. Piayford, D. L. Diicher, R. A. Stockey, W. L. Crepet, E. L. Taylor, V. Raghavan, D. J. Crawford, and R. E. J. Boemer. î would also like to acknowledge the assistance of Dr. W. A. Taylor, who was helpful in the early stages of this research in sharing his technical expertise on electron microscopy. Drs. E. L. Taylor, F. D. Sack, and V. Raghavan deserve much recognition and thanks. They graciously donated significant amounts of their time and effort by serving on my Advisory, General Examination, and Dissertation Reading Committees, and further contributed by providing helpful comments on this manuscript. Î am especially grateful to Dr. T. N. Taylor for the role he has played as my adviser. He has provided unrelenting guidance and encouragement at both professional and personal levels. As a student I have learned a great deal from him, and I am quite certain that as a colleague, he will remain to be a source of much knowledge and inspiration for me. Finally, I am deeply indebted to my wife, Yolanda, as well as my family for their loving support, without which this investigation would not have been possible. Yolanda has been especially understanding during the final months of preparation of this document, and I love her dearly for it

IV VTTA

29 November 1963 ...... Bora - Houston, Texas 1982-1985 ...... Undergraduate Laboratory Instructor, Undergraduate Research Assistant, Southwest Texas State University, San Marcos, Texas 1985 ...... B. S. in Marine Biology, Southwest Texas State University, graduated cum laude 1985-1986 ...... Graduate Laboratoiy Instructor, Graduate Research Assistant, Southwest Texas State University 1986-1987 ...... Assistant Instructor, Southwest Texas State University 1987 ...... M. S. in Botany, Southwest Texas State University 1987-1990 ...... Graduate Teaching Associate, Graduate Research Associate, The Ohio State University, Columbus, Ohio 1990-1991 ...... Presidential Fellow, The Ohio State University 1991- ...... Assistant Professor, Northeast Missouri State University, Kirksville, Missouri 1992 ...... International Fellow, Department of Botany, University of Alberta, Edmonton, Alberta, Canada PUBLICATIONS (Refereed Papers) Osborn, J. M. and E. L. Schneider. 1988. Morphological studies of the Nympheaeceae sensu iato. XVI. The floral biology of Brasenia schreberi. Annals of the Missouri Botanical Garden 75:778-794. Osborn, J. M., T. N. Taylor, and J. F. White, Jr. 1989. Falaeofibulus, gen. nov., a clamp-bearing fungus from the of Antarcdca. Mycologia 81: 622-626. Osborn, J. M. and T. N. Taylor. 1989. Structurally preserved sphenophytes from the Triassic of Antarctica: Vegetative remains of Spaciinodum, gen. nov. American Journal of Botany 16:1594-1601. Osborn, J. M. and T. N. Taylor. 1990. Morphological and ultrastructural studies of plant cuticular membranes. I. Sun and shade leaves of Quercus velutina (Fagaceae). Botanical Gazette 151:465-476. Taylor, T. N. and J. M. Osborn. 1991. The role of wood in understanding saprophytism in the fossil record. CFS-Courier Forschungsinstitut Senckenberg. (in press) Osborn, J. M., T. N. Taylor, and E. L. Schneider. 1991. Pollen morphology and ultrastructure of the Cabombaceae: Correlations with pollination biology. American Journal of Botany 78(9). (in press) Osborn, J. M., T. N. Taylor, and P. R. Crane. 1991. The ultrastructure of Sahnia pollen (). American Journal of Botany, (in press)

(Abstracts) Osbom, J. M. and E. L. Schneider. 1987. The reproductive biology of Brasenia schreberi J. F. Gmelin (Nymphaeaceae). Texas Academy of Science, March Program Abstr. 90: 49. Osbom, J. M. and E. L. Schneider. 1987. Wind pollination in Brasenia schreberi (Nymohaeaceae). Southwestern Association of Naturalists, April Program Abstr. 1987:72 Osbom, J. M. and E. L. Schneider. 1987. Morphological studies of the Nymphaeaceae. XVI. The floral biology of Brasenia schreberi J. F. Gmelin. IBC-87, XIV Intemational Botanic^ Congress, Berlin (West), Germany. Abstracts, 4-20-7: 224. Osbom, J. M. and E. L, Schneider. 1987. Anemophily in Brasenia (Nymphaeaceae). American Journal of Botany 74 (5): 622-623. Schneider, E. L. and J. M. Osbom. 1987. Morphological studies of the Nymphaeaceae. American Journal of Botany 74 (5): 721.

VI Osbom, J. M. and T. N. Tayior. 1988. sphenophytes from Antarctica. American Journal of Botany 75 (6, supplement): 113. Osbom, J. M. and T. N. Taylor. 1989. Cuticle ultrastmcture and micromorphology from sun and shade leaves of Quercus velutina Lam. ^lack Oak). Ohio Journal of Science (April Program Abst.) 1989: 7. Osbom, J. M., T. N. Taylor, and J. F. White, Jr. 1989. A fossil fungus possessing clamp connections from the Triassic of Antarctica. Ohio Journal of Science (April Program Abst.) 1989: 7. Osbom, J. M. and T. N. Taylor. 1989. Morphological and ultrastructural studies of plant cuticle: Sun and shade leaves of Quercus velutina Lam. American Journal of Botany 76 (6, supplement): 50. Osbom, J. M. and T. N. Taylor. 1990. Mesozoic fems from Antarctica: Morphology and ülîrasîTüCuireof in situ corystospennpollen. Ohio Jourruil of Science (April Program Abst.) 1990: 7. Osbom, J. M., T. N. Taylor, and E. L. Schneider. 1990. Exine ultrastmcture of the Cabombaceae: Correlations with pollination biology. American Journal of Botany 77 (6, supplement): 20. Osbom, J. M. and T. N. Taylor. 1990. Coiystosperms from the Triassic of Antarctica: Morphological and fine stmctural studies of in situ pollen. American Journal of Botany 77 (6, supplement): 91. Osbom, J. M., T. N. Taylor, M. R. de Lima, and P. R. Crane. 1991. Comparative pollen wall ultrastmcture of selected fossil anthophytes. American Journal of Botany 78 (6, supplement): 121.

FIELDS OF STUDY Major Field: Plant Biology Studies in Paleobotany and Palynology

vu TABLE OF CONTENTS

ACKNOWLEDGEMENTS...... iü VITA...... V LIST OF TABLES...... x LIST OF FIGURES...... xi CHAPTER I. GENERAL INTRODUCTION...... 1 Seed ; A Distinction Between and Angiosperms 1 Seed Plant Phylogeny and Angiosperm Origins ...... 5 Seed Plant Microgame tophy tes : Pollen ...... 7 Introduction ...... 7 Pollen Wall Ultrastructure ...... 9 Gymnosperm Pollen ...... 12 Angiosperm Pollen ...... 20 Dissertation Objectives ...... 22 II. MATERIALS AND METHODS...... 24 Fossil Material and Localities ...... 24 ...... 24 Corystospermales ...... 25 ...... 27 ...... 28 Gnetales ...... 29 Pentoxylales ...... 30 Techniques ...... 31 Macrophotography ...... 31 Light Microscopy ...... 32 Scanning Electron Microscopy ...... 32 Transmission Electron Microscopy ...... 33 Phylogenetic Analyses ...... 35 III. GLOSSOPTERIDALES...... 37 Introduction ...... 37 Description ...... 41 Discussion ...... 42

VUÎ IV. CORYSTOSPERMALES...... 46 Introduction...... 46 Description ...... 49 Discussion ...... 52 V. CAYTONIALES...... 60 Introduction ...... 60 Description ...... 62 Discussion ...... 66 VI. BENNETTITALES...... 74 Introduction ...... 74 Description ...... 77 Discussion ...... 80 Vn. GNETALES ...... 84 Introduction ...... 84 Description ...... 88 Discussion ...... 90 Vm. PENTOXYLALES...... 98 Introduction ...... 98 Description ...... 100 Discussion ...... 102 IX. PHYLOGENETIC CONSIDERATIONS...... 107 X. GENERAL DISCUSSION...... I l l Structural Features...... I l l Phylogenetic Interpretations of Pollen Morphology and Ultrastructure 115 LITERATURE CITED...... 119 APPENDIX A...... 148 TABLES...... 150 FIGURES...... 155 UST OF TABLES

TABLE 1. Comparison of Sahnia laxiphora pollen with other nonsaccate grains from selected extant and fossil taxa...... 150 2. List of taxa evaluated in all cladistic analyses ...... 152 3. Character matrix for all cladistic analyses ...... 153 4. Comparison of several palynological features from the major gymnosperm groups investigated ...... 154 LIST OF FIGURES

FIGURES 1 -4. Dispersed taeniate pollen (Protohaploxypinus) from the of Antarctica with possible glossopteridalean affinities ...... 155 5-10. Dispersed taeniate pollen (Protohaploxypinus) from the Permian of ^oxtarc tic a with possible glossopteridalean affinities...... 157 11-13. Corystospermalean pollen sacs from the Triassic of Antarctica ...... 159 14-19. Corystospermalean pollen sacs from the Triassic of Antarctica ...... 161 20-25. Corystospermalean pollen ...... 163 26-33. Corystospermalean pollen ...... 165 34-39. Corystospermalean pollen ...... 167 40-45. Corystospermalean pollen...... 169 46-49. arberi...... 171 50-57. Caytonanthus arberi...... 173 58-64. Caytonanthus arberi...... 175 65-70. Caytonanthus arberi...... 177 71-79. Caytonanthus arberi - Main Rachis ...... 179 80-81. Caytonanthus arberi...... 181 82-86. Cycadeoidea dacotensis...... 183 87-91. Cycadeoidea dacotensis...... 185 92-97. Cycadeoidea dacotensis...... 187 98-102. Cycadeoidea dacotensis.-...... 189 103-106. Cycadeoidea dacotensis - Frass...... 191

XI 107. Moi^hological diversity of poiyplicate, gnetalean palynomorphs recovered from Lower Cretaceous sediments of the Santana Formation, northeastern Brazil ...... 193 108-113. Gnetalean pollen (Equisetosporites spp.)...... 195 114-119. Gnetalean pollen {Equisetosporites spp.)...... 197 120-124. Gnetalean pollen {Equisetosporites spp.)...... 199 125-130. Sahnia laxiphora...... 201 131-136. Sahnia laxiphora...... 203 137. Outline drawings of exine stratification from the anthophyte taxa included in phylogenetic analyses ...... 205 138. Strict consensus cladogr^ for 4,934 , all with 34 steps, based on the data matrix in Table 3 ...... 207 139. Fifty percent majority-ruie consensus cladogram for 4,934 trees, all with 34 steps, based on the data matrix in Table 3 ...... 209 140. Strict consensus cladogr^ for 19 trees, all with 68 steps, based on the data matrix in Table 3 ...... 211

xu CHAPTER! GENERAL INTRODUCTION

Seed Plants: A Distinction Between Gymnosperms and Angiosperms The plant kingdom includes a wide array of photosynthesizing organisms that are generally classified into several broad, informally designated major groups. These include algae, , pteridophytes, and seed plants. Seed plants are no doubt the most familiar to the majority of people in that they occupy almost every habitat on earth and may attain great sizes (i.e., arborescence), in part because of their ability to produce secondary tissues. Two subgroups of seed-producing plants are recognized, and include gymnosperms and angiosperms. Four gymnosperm groups occur in present-day habitats, including Coniferales, Cycadales, , and Gnetales. These gymnosperms are characteristically restricted in their geographical ranges and exhibit a relative paucity of diversity. The fossil record, however, illustrates that gymnospermous plants were much more diverse throughout geologic time (e.g., Taylor and Taylor, 1991) in terms of the number of major groups, number of species, abundance of individuals, and geographical ranges. The earliest gymnosperms are Late (Famennian) in age, and are principally known from dispersed and -bearing cupulate organs (e.g., Long, 1960; Pettitt and Beck, 1968); however, cupulate ovules showing organic attachment to vegetative organs have recently also been recovered fi’om Famennian sediments (Fairon-Demaret and Scheckler, 1987; Rothwell, Scheckler, and Gillespie, 1989). Gymnosperm remains are 1 much more abundant in younger Paleozoic rocks (i.e., , , and Permian), represented by several orders of seed- (Pteridospermophyta; Calamopityales, Buteoxylonales, Lyginopteridales, , , and Glossopteridales), the Coidaitales and Voltziales, two groups closely related to , and two groups of enigmatic gymnosperms, Vojnovskyales and Gigantopteridales. Glossopterids extend into the Mesozoic, where three additional groups of pteridospermous gymnosperms flourished. These include: Caytoniales, Corystospermales, and . The Mesozoic was also characterized by an extinct group of cycadophytes (Bennettitales) and two additional gymnosperm groups with relatively obscure taxonomic affinities (Czekanowskiales and Pentoxylales). Angiosperms represent the geologically youngest group of plants, and have successfully dominated the majority of terrestrial habitats, as well as most freshwater sites, since their origins. Angiosperms are a ubiquitous assemblage of plants with respect to both diversity and number of taxa. They presently include approximately 215,000 described, extant species (Çronquist, 1988) and are represented by morphological forms ranging from minute floating plants to trees, shrubs, vines, epiphytes, and achlorophyllous parasites. The importance of flowering plants to both botanists and nonbotanists alike is unsurpassed by any other group of extant organisms. The economic influence of angiosperms, especially human dependence on the group as a source of nutrition, shelter, and medical drugs illuminates their position in this important category. Moreover, angiosperms have received additional attention in recent years as they represent the primary component of the earth's tropical rainforests. Angiosperms have a number of vegetative and reproductive features that distinguish them from gymnospermous plants. For example, angiosperms bear broad, petiolate leaves with complex venation patterns (i.e., reticulate and parallel) and produce chemically characteristic alkaloids. Angiospeims also have specialized conducting cells in the xylem (i.e., vessel elements) and specialized sieve tube members in the phloem which are linked both cytoplasmically and ontogenetically with companion cells; sieve-tube members also contain characteristic plastids. The ovules/ of flowering plants are generally bitegmic, and are borne and mature enclosed within a carpel/bruit wall. The majority of flowering plants exhibit relatively complex pollination mechanisms that often incorporate an polien-vector. Angiosperm gametophytes are highly reduced in size. The megagametophyte typically contains seven ceils while the microgarnetophyte (pollen grain) is three-celled, two of which are gametes. Both male gametes are functional units; two fusion events occur ("double fertilization") resulting in the formation of a diploid zygote and a triploid nutritive tissue known as endosperm. In addition to the reduced cellular complement of angiosperm pollen, it generally has a complex, nonlamellate wall (see below). However, most or these characteristics are not uniquely restricted to flowering plants; in fact several gymnosperm groups exhibit a number of angiosperm' features (e.g., Friis, Chaloner, and Crane, 1987). For example, vessel elements can be found in the wood of gnetalean gymnosperms (e.g., Kubitzki, 1990). , Ginkgo, , and all have relatively broad, petiolate leaves with intricate venation patterns, and the megagametophytes of Welwitsckia and Gnetum are more reduced than in other gymnosperms (e.g., Gifford and Foster, 1987). Moreover, pollination has been documented in Ephedra (e.g., Bino, Dafni, and Meeuse, 1984) and Zamia (e.g., Norstog, 1990; Norstog and Fawcett, 1989). To date, examinations of all characters that delimit angiosperms from gymnosperms suggest that 'double fertilization', subsequent formation of a polyploid endosperm, and sieve tubes are unique to flowering plants and represent the best criteria for determination of angiospermy (Friis et al., 1987; Taylor, 1988a). However, Friedman (1990a, b) has recently substantiated claims (e.g., Moussel, 1978) of 'double fertilization' in the Gnetales by clearly demonstrating two fusion events in Ephedra at the ultrastructural level. Significantly, this sheds new light on the suggestion to consider separate explanations for the evolution of 'double fertilization' and endosperm in terms of the subsequent adaptive significance of the latter nutritive tissue (Donoghue, 1989). Although identification of double fertilization and endosperm in the fossil record is unlikely (Taylor, 1988a), and well-preserved fossil phloem is rare (Taylor, 1990), numerous fossil angiosperms have been described. These specimens predominantly include leaves, , and that have been recovered from relatively young strata and have unequivocal affinities with extant angiosperm groups. Until recently, the oldest known angiosperms were those of floral and friiit specimens with magnolialean affinities from Albian and Cenomanian rocks. These had large, elongate receptacles with spirally attached carpels and fruits (Crane and Diicher, 1984; Diicher and Crane, 1984). However, small and simple flowers with chloranthacean and platanacean relationships have been shown to precede and occur concomitantly with the larger magnolialean types in the Albian (Friis and Crepet, 1987; Friis, Crane, and Pedersen, 1988). More recently, the oldest unequivocal angiosperm floral remains have been described from Aptian rocks of Australia (Taylor and Hickey, 1990). This fossil, like the Albian chloranthacean specimens noted above, also shows a diminutive habit and taxonomic affinities within the Magnoliidae; however, it bears features of several different families (Taylor and Hickey, 1990). Furthermore, a number of distinctive but dispersed angiosperm leaves occur in older rocks, from the Late Barremian and Aptian (e.g., Doyle and Hickey, 1976; Romero and Archangelsky, 1986). The fossil record of definitive angiosperm pollen, however, precedes that of floral and foliage specimens (see below). Moreover, several reproductive structures with "angiospermous affinities " and other enigmatic fossils from Valanginian, Hauterivian; and Barremian strata have been suggested as angiosperms (e.g., Friis and Crepet, 1987). Others have suggested that still older megafossil specimens (Triassic) exhibit some characteristics and may also represent early angiosperms (e.g.. Comet, 1989a).

Seed Plant Phylogeny and Angiosperm Origins For many years, a majority of the research that has been conducted on problems of seed plant evolution has focused on the heretofore unanswered query of angiosperm origins. Historically, there have been two general hypotheses regarding the nature of primitive extant angiosperms and their progenitors. The Anthostrobilus, or Euanthial, Theory (Arber and Parkin, 1907) and the Pseudanthial Theory (Wettstein, 1907) are contrasting postulates based primarily upon floral features characteristic of entomophüous and anemophilous pollination syndromes, respectively (e.g., Doyle and Donoghue, 1987; Friis et al., 1987). According to the Euanthial Theory primitive angiosperm flowers are bisexual and polypetalous. They also have many-parted perianths composed of free tepals and numerous spirally-arranged carpels and stamens. Such flowers are insect-pollinated and belong to the ranalean dicotyledons. These primitive angiosperms are suggested to be phylogenetically linked with the Bennettitales. The alternative hypothesis, Pseudanthial Theory, suggests that the primitive angiosperm is unisexual, apetalous, and wind- pollinated (i.e., catkins). The most primitive angiosperms according to this theory are members of the Amentiferae (Casuarinales, Fagales, Juglandales, Myricales) and are proposed to have had their evolutionary origin from gnetalean gymnosperms. However, the degree to which pollination syndromes fit into this equation, and the emphasis that should be placed on them, is somewhat questionable in light of recent discoveries. For example, several extant angiospeims show both entomophilous and anemophilous syndromes, or some intermediate combination of the two, and a number of extinct Lower to Middle Cretaceous flowering plants have been shown to have had the reproductive morphology to accommodate pollination by both wind and (e.g., Diicher, 1979; Crane and Diicher, 1984; Dücher and Kovach, 1986). Other researchers have suggested that angiosperms may have evolved from a number of pteridosperm groups. Those seed orders which have predominantly been advocated as angiosperm ancestors include the Caytoniales, Corystospennales, and Glossopteridales (e.g., Thomas, 1925; Melville, 1969, Doyle, 1978; Retallack and Diicher, 1981; see also Friis and Endress, 1990). These groups have been suggested as possible progenitors of flowering plants principally because of their reticulate-veined leaves and the morphology of their cupules, which have been variously homologized with the angiosperm carpel. Moreover, other authors have also championed the idea that the group is polyphyledc, and that certain angiosperms may have had their origins in the Czekanowskiales (e.g., Krassilov, 1977a). A large body of the information regarding angiosperm origins is derived from comparative investigations of extant flowering plants, including intense scrutiny of putatively primitive angiosperms. Examinations of such features as xylem anatomy (e.g., Bailey, 1944; Carlquist, 1975; Young, 1981), floral anatomy and morphology (e.g., Swamy, 1949; Bailey and Swamy, 1951; Canright, 1952; Endress, 1986, 1987), pollen structure and morphology (e.g.. Walker, 1974a, b, 1976; Osbom, Taylor, and Schneider, 1991), pollination biology (e.g., Gottsberger, 1988; Osborn and Schneider, 1988), and more recently, molecular characteristics have been carried out. However, even contemporary molecular techniques fail to adequately answer the perplexing question of angiosperm origins. For example, Martin and Dowd (1988) suggest either a Permian or origin based on analyses of N-îerminal amino acid sequences fromribulose-1, 5- biphosphate carboxylase (Rubisco), while Martin, Gierl, and Saedler (1989) postulate, based on DNA sequences of nuclear encoded glyceraldehyde-3-phosphate dehydrogenase (GADPH), that the origin of angiosperms was a event. However, the recent successful isolation and amplification of fossil angiosperm DNA (chloroplast encoded rfecL) from leaves (Golenberg et al., 1990), may help clarify some of the inconsistencies encountered with this endeavor. Moreover, the phylogenetic relationships between extinct and extant seed plants as well as the questions of angiosperm origins have been addressed in several cladistic investigations (Crane, 1985a, b; Doyle and Donoghue, 1986,1987). These classifications place angiosperms along with three gymnospermous groups (Bennettitales, Gnetales, and Pentoxylales) into an informally designated clade of relatively derived seed plants, referred to as the anthophyte clade. Crane (1985a, b) examined 38 characters and suggested that the anthophytes are most closely related to Corystospermales and Caytoniales, while Doyle and Donoghue (1986, 1987) linked anthophytes with Caytoniales and Glossopteridales after the evaluation of 62 characters.

Seed Plant Microgametophytes: Pollen Introduction — The term pollen has been variously defined and incorporated into usage generally from either a structural or functional perspective. Most authors observe a functional definition for extant pollen that takes into account the paramount biological role played by pollen in the life history of seed plants. Historically, this is also the case; in fact, the original Latin meaning of pollen is "fine meal or dust" and was recognized by Linneaus (in Hyde, 1944) as "the dust which is shed from the male organs of flowers and which brings about fertilization" (see Kremp, 1965, pp. 118-119). In the present investigation. Traverse's (1988) definition of pollen will be observed: "The several-celled microgarnetophyte of seed plants, enclosed in the microspore wall." Fossil pollen, however, typically does not contain microgametophytic cells nor the cellulosic component of the microspore wall (i.e., intine; see below). For this reason, workers have addressed fossil pollen fix>m a more structural viewpoint, specifically with regard to the nature of the fossilization-resistant microspore wall component (i.e., exine; see below). However, it is important to bear in mind that these fossil reproductive propagules did at one time contain both hapioid gametophytes and a cellulose wall, and functioned in a similar way to that of extant pollen. The use of pollen, both extant and fossil, as a systematic tool in evaluating phylogenetic relationships between seed plants was initially realized by Wodehouse (1928, 1936). Since that time, palynological characters have become increasingly important for phylogenetic analyses, and investigations of pollen have broadened in both number and type. The majority of studies on fossil pollen has involved the analysis of grains by light microscopy (LM). Numerous stratigraphie contributions have examined sporae dispersae grains (dispersed in the sediment) that have been recovered by maceration techniques (Barass and Williams, 1973; Doher, 1980), and addressed the nature of such palynomorphs as seen in transmitted light. Stratigraphie palynology has been useful in illustrating palynomorph distribution, diversity, and geologic occurrence. However, more detailed investigations concerning the structural aspects of both fossil and extant pollen have not been as thoroughly researched. Micromorphological and ultrastructural investigations utilizing scanning and transmission electron microscopy (SEM and TEM) reveal significant information regarding pollen structure otherwise not available by LM. Detailed surface features of the pollen grain wall (intine & exine, =sporoderm) from different plants (both inter- and intra-taxon) exhibit a wide variety of sculptiire patterns when viewed by SEM. Analyses of the sporoderm with TEM illustrate greater diversity with respect to its fine structural organization, as it is composed of multiple layers. The application of TEM in palynological studies on modem plants was initiated by Feraândez-Morân and Dahl (1952), while use of SEM did not come about until 1965 (Thornhill, Matta, and Wood, 1965). Since the inception and subsequent incorporation of electron microscopic techniques, SEM has progressively increased in the proportion of its utilization in studies of extant pollen, being used in approximately forty percent of all investigations (Skvarla, Rowley, and Chissoe, 1988). Transmission electron microscopy, however, has been employed in palynological studies at a considerably slower rate. Analyses using TEM have only been consistently incorporated in approximately twenty percent of all studies on extant pollen (Skvarla et al., 1988). Not too long after the above landmark dates in palynology, electron microscopic techniques were applied to the study of fossil spores and pollen. Ehrlich and Hall (1959) were first to employ TEM in an investigation of Eocene angiosperm pollen, while Taylor (1968) initiated paleopalynological research with SEM on several Carboniferous pteridophyte spores. Although the data have not been calculated, the percentages of both SEM and TEM use in examinations of fossil pollen are most certainly significantly smaller than the figures noted above for contemporary taxa.

Fulien Wall Ultrastructure - A vast terminology has been developed with respect to pollen wall ultrastmcture based primarily upon the stmctural aspects of the sporoderm. The range of terms created by multiple authors creates a problem when addressing homologous wall layers (e.g., similar names for stmcturally different layers may incorrectly infer homology). Of the many systems of pollen wall nomenclature proposed. 10 those of Faegri and îversen (1975) and Erdtman (1969) are most widely recognized (Zavada, 1984). The sporoderm as interpreted Faegri and Iversen (1975) consists of three major layers; an outer ektexine and middle endexine (exine collectively), both composed of resistant sporopollenin, and an inner intine composed of cellulose. The sporopoUeninous ektexine and endexine exhibit differential stainabUity in both LM and TEM preparations. The outer ektexine is considered to be tri-iayered, consisting of an outermost sculptured tectum, a middle infrastructural layer which may be "alveolar, endoreticulate, columehate, or consist of irregularly shaped granules or anastomosing rods", and an innermost unsculptured footlayer that may be lamellated or amorphous (Zavada, 1984). Erdtman (1969) also recognizes a three-layered sporoderm. The pollen wall as defined by Erdtman is composed of an outer sporopoUeninous sexine and nexine (exine coUectively) and an inner ceUulosic intine. Erdtman's terminology is based more on structural features; the sexine represents the sculptured layer of the sporopoUeninous wall component, while the nexine is unsculptured and differentiated into two layers, the outer nexine 1 and the inner nexine 2. The ceUulosic intine is not acetolysis- nor lithification-resistant and typically is not present in acetolyzed extant pollen nor fossil pollen, and is therefore generally excluded from taxonomic and phylogenetic discussions (e.g., Zavada, 1984). In addition to sporoderm structure, the presence or absence of one to several germinal pores (apertures) has been suggested to be of phylogenetic interest. Dicotyledonous angiosperms typically have three or more apertures located equatoriaUy on the pollen grain, while the poUen of monocotyledonous flowering plants has a single, distal furrow (sulcus) and is referred to as monosulcate. The monosulcate type of aperture also occurs within some dicot families and in most gymnosperms. Walker (1974a, b) and most other palynologists consider monosulcate pollen within angiosperm taxa as the primitive state. 11

The occurrence of monosulcate apertures in both primitive angiosperms and gymnosperms makes this character alone somewhat equivocal in the delimitation angiosperm pollen. However, correlative data on exine fine structure are considered to be useful criteria for distinguishing angiosperm monosulcate grains from gymnosperm monosulcate pollen (Zavada, 1984). Exine architecture is generally categorized into several general types, including alveolar and/or endoreticulate, tectate-granular, intectate-granular, tectate- columeilaie, and intectate-columeilate (e.g.. Van Campo, 1971; Doyle, Van Campo, and Lugardon, 1975; Kurmann, 1986; Vasanthy, Venkatachala, and Pocock, 1990). Zavada (1984) suggests that the alveolar type is found only in gymnosperms and the tectate- columellate organization primarily occurs in angiosperms, while the tectate-granular type is known from both gymnospermous and angiospermous pollen. Furthermore, and perhaps more perplexing is the fact that most primitive angiosperms have the tectate-granular wall organization (see below). Consequently, the distinction between pollen of these two groups has primarily been based upon the structure of the endexine (nexine 2; Blackmore and Bames, 1987). The endexine of dispersed fossil gymnosperm pollen is lamellate at maturity while the endexine of fossil angiosperm pollen lacks lamellae except in locations associated with the aperture (Van Campo, 1971; Doyle et al., 1975). This character alone, however, may not be a good criterion for distinguishing between gymnospermous and angiospermous pollen. Blackmore and Bames (1987) and Blackmore and Crane (1988) have suggested that differences in endexine structure of dispersed pollen may be indicative of ontogenetic stages rather than true structural dissimilarity. The timing of deposition of endexine materials differs between gymnosperms and angiosperms. During the tetrad stage in gymnosperm pollen, the majority of endexine is deposited; whereas in angiosperms, deposition of this wall layer is generally not initiated until the free-spore phase of development (e.g., Blackmore and 12

Crane, 1988). Moreover, the ektexine in gymnosperms and angiosperms typically differs in both timing and pattern of development. In most gymnosperms, more ektexinous material is also deposited while grains are united into tetrads, and in most conifers and this materia! is laid down in a dispersed fashion, as opposed to the relatively organized deposition of radially-aligned procolumellae or probaculae in angiosperms (e.g., Zavada, 1984). Although the structure of the sporopoUeninous portion of the pollen wall (exine) is generally delimited into two layers (e.g., ektexine and endexine sensu Faegri and Iversen, 1975, or sexine and nexine sensu Erdtman, 1969), its organization has recently been determined to be significantly more complex in several taxa (Rowley, 1981; Rowley et al., 1981; Rowley and Srivastava, 1986; Southworth, 1986a, b). The resistant outer layer consists of filamentous substnictural components, or subunits, composed of glycocalyx. Each subunit contains an axial tubule enclosed by a copious array of lateral branches (Rowley et al. 1981). The majority of this glycocalyx substructural framework is embedded within the sporopoUeninous matrix and thus protected from degradation. Consequently, it is not readily accessible to TEM stains and is thus not detected using standard TEM protocols. The organization of tubular subunits is only evident after the selective removal of sporopollenin through a series of intricate techniques which then leaves the glycocalyx substructure intact The potential use of these techniques has not been realized and it is conceivable that the glycocalyx substructure is taxon-specific, and could thus be used in a phylogenetic and taxonomic capacity.

Gymnosperm Pollen — Numerous descriptive-type studies have been carried out on both extant and fossil gymnosperm pollen using LM, and to some extent SEM (e.g.. 13

Erdtman, 1957,1965). Fewer studies, however, have addressed the fine structural nature of gymnosperm pollen using TEM. For extant groups, those works that have been undertaken have principally focused on either describing exine stratification in mature pollen or examining the various stages of microsporogenesis and sporoderm ontogeny. Moreover, only a small number of studies, as well as taxa, has been conducted on gymnosperm pollen ultrastructure relative to those of angiosperm pollen. In fact, two important and frequently cited symposium volumes on pollen fine structure lack chapters on extant gymnosperm pollen (Ferguson and Miller, 1976; Blackmore and Ferguson, 1986). More recently, however, two chapters in Blackmore and Knox (1990) dealing with the ontogeny and evolution of microspores focused on the pollen of extant gymnosperms. Modem gymnosperms are circumscribed within four orders: Coniferales, C^cadales, Ginkgoales, and Gnetales. Coniferales - The Coniferales is the most diverse of all the extant gymnosperm groups in terms of number of families, genera, and species, as well as pollen morphology and ultrastructure. Two general morphological types of pollen are produced by conifers, including bisaccate grains and nonsaccate grains. Bisaccate grains are found in all genera of the , except Larix and Pseudotsuga, and the . Moreover, some congeneric species of the pinaceous genus Tsuga produce both saccate and nonsaccate grains (see Kurmann, 1986). The exine of both saccate and nonsaccate grains is composed of an inner nexine, which is lamellated throughout, and overlaid by a sculptured sexine. The sexine of saccate grains consists of a well-defined tectum separated from the nexine by an infratectal layer of vertical partitions spaced at relatively regular intervals. This type of infrastructural pattern is generally referred to as alveolate; however, Doyle et al. (1975) have described this specific type of organization as "honeycomb-like" alveolate, as opposed to the "spongy" alveolate condition which is known firom the pollen of several 14

Pennsylvanian seed fems (i.e., Monoletss] Meduilosales). The sexine of nonsaccate pollen (i.e., Taxodiaceae, , , , ) is typically described as atectate, with a granular layer overlaying the lamellate nexine (e.g., Kurmann, 1986). Some diversity exists with regard to relative size and density (i.e., degree of packing) of granules. However, some nonsaccate conifer grains appear to have a thin, weakly-defined tectum resulting from the fusion of the outermost granules (e.g., Doyle et ai., 1975). The data noted above have come from investigations that have focused on the various ultrastructural aspects of both pollen ontogeny and mature exine stratification in the Coniferales. Those studies that have employed TEM and published transmission electron micrographs include the following: Pinaceae (Miihlethaler, 1955; Afzelius, 1956; Ueno, 1958; Praglowski, 1962; Erdtman, 1965; Ting and Tseng, 1965; Gullvâg, 1966a; Lepousé, 1966, 1969a, b; Pettitt, 1966, 1985; Ekberg, Eriksson, and Sulikova, 1968; Eriksson, 1968; Dickinson, 1970, 1971, 1976; Dickinson and Bell, 1970a, b, 1972, 1976a, b; Willemse, 1971a, b, c; Hess et al., 1973; Dickinson and Potter, 1975; Litvintseva, 1979; Moitra and Bhatnagar, 1982; Walles and Rowley, 1982; Singh, Owens, and Dietrich, 1983; Van Campo and Vernier, 1984; Kedves, 1985a, b; Rowley and Walles, 1985a, b, c, 1987, 1988; Kurmann, 1986, 1989a, b, c; Kedves, 1988a, b, c, 1990); Podocarpaceae (Gullvâg, 1966a, b; Aldrich and Vasil, 1970; Vasil and Aldrich, 1970, 1971; Pocknall, 1981a, b; Moitra and Bhatnagar, 1982; Médus, Gajardo, and Woltz, 1989); Taxodiaceae (Gullvâg, 1966a; Roscher, 1975; Kedves, 1985a; Sohma, 1985; Kurmann, 1986, 1990a); Cupressaceae (Ueno, 1959; Duhoux, 1972, 1973, 1975 Van Campo and Lugardon, 1973; Roscher, 1975; Lugardon, 1978; Pocknall, 1981c Southworth, 1986a, b); Araucariaceae (Ueno, 1959; Van Campo and Lugardon, 1973 Pocknall, 1981c); Cephalotaxaceae (Afzelius, 1956; Ueno, 1959; Gullvâg, 1966a 15

Roscher, 1975); and Taxaceae (Ueno, 1959; Gullvâg, 1966a; Pettitt, 1966; Van Campe, 1971; Roscher, 1975; Rohr, 1977; Kedves, 1988a); for review also see Ueno (1960a, b) and Kurmann (1986,1990b). Cy.cadales sM Ginkgoales - Pollen wall ultrastructuie in the Cycadales (Audran, 1964, 1965, 1970, 1978a, b, 1979a, b, 1980, 1981, 1987; Larson, 1964; Gullvâg, 1966a; Pettitt, 1966, 1977, 1982; Audran and Masure, 1976, 1977, 1978; Zavada, 1982, 1983; Kedves, 1985b; Dehgan and Dehgan, 1988; Wang, 1990; Kedves, 1990) and Ginkgoales (Wolniak, 1976; Rohr, 1977; Audran, 1987; Wang, 1990) is very similar, as it is to that of saccate conifers. The exine is composed of distinct sexine and nexine components. The nexine is lamellate throughout, and the sculptured sexine has a well-defined tectum and a honeycomb-like alveolar infrastructure. However, sexine infrastructure of these groups differs from saccate pollen of conifers in that the alveolar partitions here are much more regular in their spacing. In fact, the exine of these grains superficially appears columellate in transverse section. Gnetales - The mature pollen wall in the Gnetales is similarly two layered, consisting of a distinct nexine and sculptured sexine. Nexine organization here, as in all other extant gymnosperm pollen, is lamellate throughout. The sexine is two-zoned, composed of a thick tectum overlaying a granular infrastructural layer. Original transmission electron micrographs of gnetalean pollen have been published in the following studies: (Afzelius, 1956; Ueno, 1960a; Gullvâg, 1966a; Van Campo and Lugardon, 1973; Hesse, 1980, 1984; Zavada, 1982,1984; Kedves, 1987). Gnetalean pollen will be discussed in greater detail below (see Chapter VII), specifically as it compares in gross morphology and ultrastructure with dispersed fossil pollen that has been described as gnetalean. Fewer studies have addressed the fine structure of fossil gymnosperm pollen using TEM. Although sixteen orders of extinct gymnosperms are recognized, and fossil 16 members of the four extant ordere are also known, data on pollen ultrastructure from some groups are either entirely lacking or come from evaluation of a single taxon. An up-to-date list of these groups follows below, along with all ultrastructurally known dispersed palynomorphs; a brief description of the general exine architecture for each is also given. However, the reader is also directed to two recent, comprehensive reviews on fossil pollen morphology and ultrastructure (Taylor and Taylor, 1987b; Taylor, 1988b) from which detailed information, available prior to those publications, can be found. Calamopirvales - Nothing is known about pollen ultrastructure from this group. Buteoxvlonales - Nothing is known about pollen ultrastructure from this group. Lvginopteridales - In situ pollen has been described from Crossotheca (Millay, Eggert, and Dennis, 1978; Taylor and Taylor, 1987b), Potoniea (Stidd, 1978; Taylor, 1982), and Schopfiangium (=Cyclogranisporites, Verrucosisporites', Stidd, Rischbieter, and Phillips, 1985). Two types of exine stratification are known in this group; Crossotheca has a homogeneous organization, while Schopfiangium has an alveolar (spongy-type) exine. Meduilosales - In situ pollen of the Monoletes-typc has been described from Hallitheca (Taylor, 1971; Millay and Taylor, 1976), Bernaultia (Millay and Taylor, 1976; Taylor, 1978; Taylor and Rothwell, 1982), Rhetinotheca (Taylor 1978, 1982), Aulacotheca (Taylor, 1976a, b, 1978), Boulaytheca (Kurmann and Taylor, 1984), Schopfitheca (Taylor, 1982), Sullitheca (Taylor, 1982), Codonotheca (Taylor, 1976a, b, 1978). Sporae dispersae grains of the Monoletes-xypQ have also been described {=Schopfipollenites\ Pettitt, 1966; Abadie et al., 1978). In situ pollen is also known from Parasporotheca (=Parasporites; Millay et al., 1978, Taylor, 1982). All medullosalean pollen has the same basic type of sporoderm stratification, composed of a spongy-type alveolate sexine. Callistophvtales - In situ pollen from Idanothekion (=Vesicaspora) has been described by Millay and Eggert (1974) and Millay and Taylor (1974,1976). Although a fair amount 17 of data has been published on the nature of the callistophytalean sacci and microgametophytes, most grains sectioned simply show a homogeneous exine fine structure. Cordaitales - In situ pollen of the Florinites- and Sullisaccites-type are known from Cordaianthus (Millay and Taylor, 1974,1976), while in situ Felixipollenites-ty^ pollen is known from Gothania specimens (Millay and Taylor, 1974, 1976; Taylor and Daghiian, 1980). Dispersed Florinites palynomorphs have also been described at the ultrastructural level (Pettitt, 1966). The pollen wall of Florinites and SulHsaccites appears homogeneous in published micrographs, while that of Felixipollenites is honeycomb-type alveolate in organization. Glossopteridales - Zavada (1990a, 1991) has examined in situ glossopterid pollen from dispersed pollen sacs of the Arberiella-type (see also chapter III for additional discussion of glossopterid pollen). These grains have been shown to have a honeycomb- type alveolar organization in the region of the sacci, which may also extend into the cappus region. Voltziales - Voltziaceae: To date, in situ pollen of Williostrobus and Sertostrobus has only been described in a preliminary way in the context of two larger reviews on fossil gymnosperm pollen (Taylor and Taylor, 1987b; Taylor, 1988b). The sporoderm of these genera appears to be alveolate of the honeycomb-type. : In situ pollen of the Classopollis-typ& has been described from Hirmeriella {=Cheirolepidium; Pettitt and Chaloner, 1964) and Classostrobus (Taylor and Alvin, 1984). Three genera of sporae dispersae circumpolloid grains have been described ultrastructurally as well. These include: Classopollis (Médus, 1977; Kedves, 1985b; Rowley and Srivastava, 1986), Circulina (Médus, 1977), and Classoidites (Kedves and Pârdutz, 1973). Classopollis-type pollen has a very distinctive exine stratification. The sporoderm consists of a thick nexine 18 with well-defined lamellae, and highly complex, four-layered sexine. The sexine is composed of an innermost layer of anastomosing plate-like units which is overlaid by a thin homogeneous layer, a layer of robust rods, and a tectum with distinct supratectal spines. Coniferales - Podocarpaceae: In situ pollen from Millerostrobus (Taylor, Delevoryas, and Hope, 1987) and Trisacocladus (=Trisaccites; Baidoni and Taylor, 1982) has been described, moreover, dispersed grains assigned to Rugubivesiculates have also been suggested to have podocarpacean affinities (Zavada and Dilcher, 1988). All of the podocarpacean grains sectioned have a distinct to irregular honeycomb-type alveolar organization. Taxodiaceae: In situ pollen from permineralized Metasequoia cones has been studied by Rothwell and Basinger (1979). These Metasequoia grains show an irregularly granular to homogeneous sexine fine structure. Vojnovskvales - Nothing is known about pollen ultrastructure from this group. Gigantopteridales - Nothing is known about pollen ultrastructure from this group. Cavtoniales - Exine fine structure of in situ pollen from Caytonanthus has been described and figured by Pedersen and Friis (1986) and Zavada and Crepet (1986), and is generally reported as alveolate of the honeycomb-type. Also see chapter V for additional description and discussion of Caytonanthus pollen. Corvstospermales - Exine fine structure has been described for in situ pollen of (Taylor, Cichan, and Baidoni, 1984; Zavada and Crepet, 1985). The pollen wall is considered alveolate (honeycomb-type) by these authors. Also see chapter IV for additional description and discussion of corystosperm pollen. Peltaspermales - Nothing is known about pollen ultrastructure from this group. 19

Cvcadales - Exine Ene structure of fossil cycads is only known from one taxon, in situ pollen of Androstrobus (Hill, 1990). The exine of this genus is almost identical to that of extant cycads, composed of regularly spaced alveolar units. Bennettitales - In situ pollen has been described for Cycadeoidea (Taylor, 1973; also see chapter VI for additional description and discussion) and Leguminanthus (Ward, Doyle, and Hotton, 1989). Exine stratification of Cycadeoidea pollen is not completely known; howeveTLeguminanthus has a granular type organization. Gnetales - Nothing is known about in situ fossil poUen ultrastructuie from tliis group. Ginkgoales - Nothing is known about in situ fossil pollen ultrastructure from this group. Czekanowskiales - Nothing is known about pollen ultrastructure from this group. Pentoxvlales - Exine ultrastructuie of in situ Sahnia pollen has also been included in the two most recent reviews of fossil gymnosperm pollen (Taylor and Taylor, 1987b; Taylor, 1988b), although only described in a preliminary fashion as granular. However, Sahnia pollen has more recently been described in a more comprehensive manner (Osbom, Taylor, and Crane, 1991; see also Chapter YID). Incertae sedis (In situ) - Pollen wall fine structure is also known from the following taxa; Melissotheca (Mississippian; Meyer-Berthaud, 1989); Lasiostrobus (Pennsylvanian; Taylor, 1970; Taylor and Millay, 1977); Erdmanispermum ({Eucommiidites-Vyp€\ Lower Cretaceous; Pedersen, Crane, and Friis, 1989); and Erdmanitheca i^Eucommiidites-lypt\ Upper Cretaceous; Pedersen et al., 1989). Incertae sedis (Sporae dispersae) Exine- ultrastructure is known from the following genera: Nanoxanthiopollenites (Pennsylvanian; Taylor, 1980, 1982); Precolpatites and Marsupipollenites (Permian; Foster and Price, 1981); Cannanoropollis,Plicatipollenites, Platysaccus, and Striatopodocarpites (Permian; Foster, 1979); Protohaploxypinus 20

(Permian; Foster, 1979; see also chapter ÎIÎ); Lueckisvorites. Lunadsporites, and Ovalipollis (Liassic; Scheuring, 1974); Triradispora (Triassic; Scheuring, 1976); Monosulcites (Upper Paleozoic-Tertiary; Trevisan, 1980; Zavada and Dilcher, 1988); Eucommiidites (Triassic-Cretaceous; Doyle et al., 1975; Trevisan, 1980; Zavada, 1982, 1984); Bharadwajipollenites (Triassic; Zavada, 1990b); Granamonocolpites (Triassic; Zavada and Dilcher, 1988; Zavada, 1990b); Ephedripites (Triassic-Recent; Trevisan, 1980); Equiseiosporites (Triassic-Recent; Zavada, 1982, 1984, 1990b; Pocock and Vasanthy, 1988; see also chapter VII); Granabivesiculîîes (Triassic-Cretaceous; Zavada, 1982, 1984; Zavada and Dilcher, 1988); Punctamultivesiculites (Triassic-Cretaceous; Zavada, 1982,1984); Inaperturopollenites (Jurassic; Kedves and Pdrdutz, 1974; Kedves, 1985b); Araucariacites (Jurassic; Kedves and Pârdutz, 1973, 1974; Kedves, 1985b); Spheripollenites (Jurassic; Kedves and Pârdutz, 1973); Cycadopitys (Jurassic; Kedves, 1985b); Granamultivesiculates and Clavabisvesiculates (Cretaceous; Zavada and Dilcher, 1988); and Cyclusphaera (Cretaceous; Taylor, Zavada, and Archangelsky, 1987; Zavada, 1987).

Angiosperm Pollen — Studies on the pollen of extant angiosperm families are numerous and beyond the scope of discussion here. The fossil record of angiosperm pollen is also quite diverse with a large number of extant families recognized in relatively young strata (i.e.. Tertiary) from both dispersed grains in palynological surveys as well as in association with megafossil reproductive organs. Moreover, a growing number of dispersed palynomorphs recovered from sediments are now realized to have angiosperm affinities. The earliest unequivocal pollen is Hauterivian in age (Brenner and Crepet, 1987; Hughes and McDougall, 1987), although an abundance of grains is now known from 21

Bairemian and Aptian sediments. The majority of grains are, in general, similar in morphology and ultrastructure; they are monosulcate monads with reticulate ornamentation, tectate to semitectate with a columellate infratectum, and have a nonlamellate nexine. The most commonly occuring genera are Retimonocolpites, Stellatopollis, Clavatipollenites, Afropollis, and Liliacidites (e.g., Doyle et al., 1975, 1982; Walker and Walker, 1984; Chionova and Surova, 1988; Penny, 1989; Hughes and McDougall, 1990). These grain types distinctly resemble a variety of modem monocots and magnoliid dicots; in fact Chionova and Surova (1988) have convincingly demonstrated a number of distinct similarities between Clavatipollenites and the extant genus (). More recently, Doyle, Hotton, and Ward (1990a, b) have described fossil tetrads from late Barremian-early Aptian sediments having definitive affinities with extant Winteraceae. Several palynologists now believe that these types of fossil monosulcate, reticulate-columellate grains, with chloranthaceous and winteraceous affinities, are somewhat derived along with other extant Magnoliidae that produce this pollen morpho-type (e.g.. Walker and Walker, 1984; see Ward et al., 1989 and references therein; Doyle et al., 1990b). More primitive angiosperm pollen is suggested to have also been monosulcate, but with a relatively smooth tectum and a granular infratec tal layer (e.g., Muller, 1970; Walker, 1976; Walker and Walker, 1984). These grains are suggested to have affinities with members of several families within the Magnoliales (e.g., Magnoliaceae, Annonaceae, Degeneriaceae; see Ward et al., 1989 and Doyle et al., 1990b). However, fossil grains showing this type of morphology and ultrastructure are not known to precede the aforementioned reticulate-columellate types. In fact, one of oldest unequivocal granular angiosperm grains has recently been described from early Aptian sediments {Lethomasites; Ward et al., 1989). It has been suggested that such smooth-walled, granular 'angiosperm' 22 palynomorphs have not been widely recognized and reported because they are morphologically similar to the pollen of a number of Mesozoic gymnosperms (e.g., Muller, 1970; Zavada, 1984).

Dissertation Objectives The intent of the present investigation is to perform a detailed, systematic evaluation of pollen micromorphoiogy and ultrastructure, using SEM and TEM, from those groups of gymnosperms implicated by previous cladistic analyses as closely related to and/or possible progenitors of the angiosperms. These groups include Glossopteridales, Corystospermales, Caytoniales, Bennettitales, Gnetales, and Pentoxylales. As can be noted from the above review on exine ultrastructure of fossil gymnosperm pollen, only a paucity of data has been published on the pollen of these groups. Objectives of the study are several fold. First, I wish to ascertain as much information as possible on exine architecture from these Late Paleozoic and Mesozoic gymnosperms, so that relevant structural, functional, developmental, and systematic comparisons can be addressed. This information is not only important in the context of the present study, but also with regard to the growing database on the fine structure of fossil gymnosperm pollen. A second objective is to address the importance of in situ pollen in evaluating the reproductive biology of fossil seed plants, as well as to analyze all relevant comparisons with sporae dispersae grains. This is possible because pollen of four of the groups investigated (i.e., Corystospermales, Caytoniales, Bennettitales, and Pentoxylales) is preserved in situ. The final objective encompasses the previous two, and is directed at performing cladistic analyses of the aforementioned groups based on palynological features. Previous cladistic studies by Crane (1985a, b) and Doyle and Donoghue (1986, 1987) only included a few pollen characters, four and six, respectively. Because of the 23 nature of the present investigation, more phyiogenetically important characters are elucidated, and will also be available for inclusion in subsequent all-encompassing cladistic treatments (i.e., those evaluating numerous reproductive and vegetative characters). CHAPTER n MATERIALS AND METHODS

Fossil rvlaceriai and Localises Giossopteridaies — Glossopteridalean fossils were found within silicified peat boulders collected near Ml Augusta in the central Transantarctic Mountains, Antarctica. The permineralized peat occurs at the southeastern end of the Skaar Ridge site (Schopf, 1970; Smoot and Taylor, 1986) in the Beardmore Glacier area (84° 47' S, 163° 15' E, Buckley Island Quadrangle; Barrett and Elliot, 1973) and was collected by Dr. T. N. Taylor, Dr. E. L. Taylor , and Dr. N. R. Cuneo and their field parties during the austral summers of 1985-1986 and 1990-1991. Palynostratigraphic investigations of the Upper Buckley Formation, in which the Skaar Ridge peat occurs, indicate that the peat is Late Permian in age (Farabee, Taylor, and Taylor, 1990). Glossopterids are the most abundant elements in the Skaar Ridge peat, represented mainly by two anatomically distinct leaf types (Glossopteris schopfii and G. skaarensis; Pigg, 1988, 1990a), more recently found attached to woody stems (Pigg, 1991), and

Vertebraria rooting structures(Taylor and Taylor, 1990a). Ovulate reproductive organs are also relatively frequent components and are represented by two types of platyspermic ovules, Plectilospermum elliotii (Taylor and Taylor, 1987a) and a yet undescribed form, both of which have recently been discovered attached to megasporophylls with G/owopfôrâ-like anatomy (Taylor, 1987; Taylor and Taylor, 1990a). Glossopterid pollen organs from the Skaar Ridge peat are not yet known; however, dispersed striate bisaccate 24 25

Dollen grains (=taeniate grains') occur throughout the sUicified neat and are moroholosicallv similar to other described glossopterid pollen grains. Moreover, palynological examinations of Buckley Formation rocks (e.g., Farabee et al., 1990,1991) indicate that striate bisaccate palynomorphs are significant components; ten species assigned to three genera (Lunatisporites, Protohaploxypinus, and Striatopodocarpites) have been described. To isolate sporae dispersae grains, small pieces of Skaar Ridge peat were mechanically macerated in a mortar and pestle and subsequently processed following standard palynological protocols (e.g., Doher, 1980). Palynomorphs were also isolated from several of Farabee et al.'s (1991) Buckley residues in order to make additional observations and comparisons. Samples containing the best preserved palynomorphs were those recovered from macerations of coals, shales, and coaly shales from several Mount Achemar sites. Of those macerations evaluated for the present study, sample no. 70-6-39, collected by the late James M. Schopf, showed "good" recovery and some of the best preserved palynomorphs (see Farabee et al., 1991 for specific locality details). All residues are deposited in the Paleobotanical Collections of The Ohio State University.

Corystospermales — Corystosperm remains were recovered within a silicified peat collected north of Fremouw Peak in the Beardmore Glacier area (84° 18' S, 164° 20' E, Buckley Island Quadrange; Smoot, Taylor, and Delevoryas, 1985; Taylor, Taylor, and Collinson, 1989), central Transantarctic Mountains, Antarctica. This material was also collected during in the 1985-1986 and 1990-1991 field seasons. The peat occurs in the Upper Fremouw Formation and is interpreted as Early-Middle Triassic in age based on its palynostratigraphic record (Farabee et al., 1989, 1990) and the occurrence of the stratigraphically diagnostic vcxtébtdXQS Lystrosaurus and Cynognathus (Hammer, 1990). The permineralized megaflora from the Fremouw peat is still very much in the early stages 26 of its description; however, a wide array c-f well preserved plarts,assignable to a variety of major plant groups has already been described. Some of these include fems (Schopf, 1978; Millay and Taylor, 1990), sphenophytes (Osbom and Taylor, 1989), cycads (Smoot et al., 1985), and conifers (Meyer-Berthaud and Taylor, 1991; Yao, unpublished); the reader is directed to Taylor and Taylor (1990a and references therein) for the most current review. Moreover, the Fremouw peat is also becoming well known for the occurrence and exquisite preservation of a diverse mycoflora (e.g., Stubblerield, Taylor, and Seymour, 1987; Stubblefield, Taylor, and Trappe, 1987; Osbom, Taylor, and 'A^iite, 1989; White and Taylor, 1989, 1991). Corystosperms are also major components of the silicified flora from the Femouw Formation. They are represented by foliage (Pigg, 1988, 1990b), Pteruchus- like pollen organs (Devore and Taylor, 1988) with in situ pollen (Osbom and Taylor, 1990), as well as Rhexoxylon-type wood (Taylor and Taylor, 1990b; E. Taylor, 1991) with probable coiystospermalean affinities. To ascertain anatomical information, surfaces of sectioned slabs of peat bearing the Pteruchus-)ike pollen organs were etched in 48% hydroflouric acid for 1-5 min. Etched slabs were then serially peeled with cellulose acetate using the modified techniques of Joy, Willis, and Lacey (1956) for silicified plant material (Basinger and Rothwell, 1977). Additionally, elevated wax wells were built up over a number of pollen sacs, containing specific pollen grains of interest (i.e„ orientation, ontogenetic stage, preservation, etc.), by carefully melting dental baseplate wax over the appropriate site. Pollen was then macerated from the wells with 48% hydroflouric acid, washed several times, and stored in 50% ethanol prior to microscopic examinations. In most cases gains were macerated out individually, although some clumps were obtained. 27

A large numl^er of sectioned slabs contained nicely preserved pollen organs (Yao, unpublished); however, slabs/faces 10,717 Btop, 10,178 Btop & Cbot, and 10,925 Btop contained copious amounts of pollen that was especially well preserved. All slabs and peels are housed in The Ohio State University Paleobotanical Collections. However, the microscope slides prepared for anatomical evaluations were dissociated for electron microscopy, as described below, and are consequently no longer available.

Caytoniales — The Caytoniales is known from several Laurasian localities; however, two sites on the coast of Yorkshire, United Kingdom (Cayton Bay and Gristhoipe Bay), are the sources of the best preserved, most studied, and original type material (e.g., Thomas, 1925; Harris, 1964). The fossil bearing beds in Cayton and Gristhoipe Bays are in age (e.g., Phillips, 1875; Harris, 1953) and contain three principal, disarticulated caytonialean genera (Sagenopteris leaves, Caytonia ovulate organs, and Caytonanthus pollen organs), as well as an extremely diverse assemblage of plant remains (e.g., Seward, 1900; Harris, 1961, 1964, 1969, 1979; Harris and Miller, 1974; Harris and Millington, 1974). In the present investigation, entire Caytonanthus pollen organs (i.e., microsporophylls and attached synangia with in situ pollen) and dispersed synangia were isolated from shales collected at the Cayton Bay locality. Specimens were delicately removed from the shales with a scalpel and dissecting probes. The compressed organs were then demineralized in concentrated hydroflouric acid, followed by several aqueous washings, and hydrochloric acid, and again washed. Several demineralized specimens were also cleared (i.e., bleached) in concentrated nitric acid. Other synangia were mechanically macerated in a test tube, in order to isolate individual in situ palynomorphs. 28

Several specimens bear Caytonantkus organs (especially dispersed synangia), some with acquisition nos. and others without; however, specimen nos. J-3, 86; J-3, 91; J-3, 96; and J-3, 102 contain relatively well-preserved and easily detectable organs. All specimens are housed in the Paleobotanical Collections of The Ohio State University.

Bennettitales — In recent years, it has become increasingly clear that the Bennettitales is taxonomically much more diverse than had traditionally been thought (e.g.. Crane, 1986; Taylor and Taylor, 1991). However, the majority of information known about these plants comes from studies of permineralized North American specimens of Cycadeoidea spp. (e.g., Wieland, 1906, 1916; Delevoryas, 1968a; Crepet, 1974). These fossils are principally represented by silicified trunks with in situ reproductive organs. The specimens range in age from Upper Triassic to Lower Cretaceous and have been collected from a variety of North American localities; although several Lower Cretaceous sites in the Black Hills of South Dakota and Wyoming have yielded the majority of trunks (see Wieland, 1906 and Crepet, 1974 for historical synopses and locality details). The most substantial collections of silicified Cycadeoidea trunks are housed in the National Museum of Natural History, Smithsonian Institution (Washington, D C.) and the Peabody Museum of Natural History, Yale University (New Haven, CT). Each of these collections was visited in order to evaluate individual specimens for suitability of pollen analyses. Cycadeoid fossils in the National Museum’s collections were found to be of little use for the present investigation, because the majority of those specimens either lacked reproductive organs altogether or were primarily quartz crystalline replacements. On the other hand, the collections of the Peabody Museum contained a large number of exquisitely preserved trunks, of which several had previously been sectioned. Additionally, numerous thin sections had also been prepared from a variety of the 29 sectioned trunk portions (e.g., Wieland, 1906, 1916; Delevoryas, 1968a; Crepet, 1974). However, thin sections were only cataloged by trunk number, not according to either sectioned piece number nor face of that piece; so it was not possible to extrapolate from a thin section containing a weU preserved cone with in situ pollen to the collection of sectioned trunks. Consequently, large numbers of sectioned trunk pieces were examined with a stereomicroscope in order to identify appropriate specimens for palynological investigations (i.e., witlî pollen, pollen of a particular ontogenetic stage, etc.). Of the numerous trunk pieces examined, relatively few were found with cones bearing microsporangiate organs (i.e., in a bisporangiate [mature] developmental stage). Several pieces were identified with microsporangiate organs and taken on loan from the Peabody Museum. These included specimens of Cycadeoidea wielandi (Trunk 77), C. dacotensis (Trunks 213, 214), C. heliochorea (Trunk 722), and C. spp. (Trunks CF 78 and 00). In situ pollen was macerated from specific sites within cones by building wax wells over those sites as described above for Corystospermales. Additionally, several of G. R. Wieland's (GW) original thin sections were borrowed. The thin sections bear both GW nos. and Yale Peabody Museum nos. (YPM) and are housed, along with the sectioned trunk pieces, in the Paleobotanical Collections at the Peabody Museum.

Gnetales — A diverse gnetalean flora is known from the Lower Cretaceous (Aptian- Albian) of northeastern Brazil. Gnetalean fossils occur in the Santana Formation, one of four sedimentary units of the Araripe Group (e.g., Lima, 1978a, b). The Santana Formation is characterized by its exceptionally well preserved fauna, including fish, reptiles, and invertebrates, especially insects (e.g., Lima, 1978b; Grimaldi, 1990), and its microflora (Lima, 1976, 1978a, 1978b, 1979, 1980, 1989). The record of megafloral elements is less diverse, mainly due to the fact that this aspect of paleobotanical inquiry has 30 received the least attention in the Santana: nevertheless, those megafossils that have been examined are also well preserved (Lima, 1978b; de Oliveira-Babinski, personal communication). Gnetophytes are principally represented by a rich palynological assemblage of polyplicate pollen grains including 52 species belonging to six genera {Equisetosporites, Singhia, Welwitschiaptes, Steevesipollenites, Regalipollenites, and Gnetaceaepollenites; Lima, 1978b, 1980). Gnetalean leaves have also been recovered from the same sediments, but have only been studied and communicated in a preliminary way (Pons, Berthou, and Airneida-Campos, 1990; de Oliviera-Bablinski, personal communication). Pollen grains evaluated and described in the present investigation were isolated from Santana sediments collected by Dr. M. R. de Lima and generously donated to The Ohio State University Paleobotanical Collections. Palynomorphs were recovered from sediments according to standard techniques, as described above for glossopterid material; however, it was also necessary to separate the organic fractions of residues after macerations with hydroflouric and hydrochloric acids and all washings. Palynomorph separation was accomplished by floating grains out in a bromoform-acetone mixture, which had been adjusted to a specific gravity of 2.0 (modified Zinc Bromide flotation technique of Doher, 1980).

Pentoxylales — Several pentoxylalean specimens including leaves {Taeniopteris daintreei), ovulate organs {Carnoconites cranwellif) and pollen organs (Sahnia laxiphord) are known from Lower Cretaceous sediments (Valanginian-Aptian) of southeastern Victoria, Australia (Drinnan and Chambers, 1985, 1986). Pollen grains were isolated from a single specimen of S. laxiphora collected from the Strzelecki Group exposed at the Whitelaw road cutting (see Douglas, 1969; Drinnan and Chambers, 1985 for locality 31 details); however, several other specimens are known from stratigraphically similar rocks in the Koonwarra fish beds (Drinnan and Chambers, 1985, 1986). The specimen examined in the present investigation was initially described and illustrated as "fertile organ bearing microsporangia" (Douglas, 1969, pi. 45, figs. 1, 2, pi. 46, figs. 1, 6, pi. 48, fig. 1, pi. 51, fig. 1, text-figs. 5.2-5.Ô), but it was subsequently redescribed, reillustrated, and taken as the hoiotype of Sahnia laxiphora Drinnan and Chambers (1985), a new species. Part and counterpart of the specimen were originally assigned Geological Sur/ey of Victoria numbers (compressions - CSV 60003, 60004; light microscope slides - GSV 60934-60937, 61618-61624, and 61710; Douglas, 1969), and are now housed in the paleobotanical collections of the National Museum of Victoria (NMV P167524). Entire compressed pollen sacs containing in situ grains were isolated from the specimen as described above for caytonialean compression material. Several pollen sacs were also macerated, as for caytonialean material, in order to yield individual palynomorphs. Microscope slide preparations are deposited in The Ohio State University Paleobotanical Collection.

Techniques Macrophotography — Compression specimens of caytonialean and pentoxylalean pollen organs were photographed using a Polaroid MP-3 camera. In addition to flood lamp lighting, specimens were photographed utilizing ancillary, low-angle lighting directed from a fiber optic light source. This was undertaken in order to enhance contrast of the specimens relative to the background shales, because the surface features of these specimens, like most compressions, were quite subtle. Corystospermalean pollen sacs and masses of pollen, exposed by precisely etching within the built-up wax wells, were photographed with a Zeiss Stereomicroscope. In 32 order to illuminate the specimens, especially regions within the wax wells, fiber optic lighting was utilized.

Light Microscopy — Individual palynomorphs recovered from macerations of sediment (i.e., glossopterids, gnetopsids), compression organs (i.e., Caytonanthus, Sahnia), and permineralized organs (i.e., corystosperms, cycadeoids) were mounted on glass microscope slides in either glycerine jelly or Coverbond mounting medium, and examined using bright-fieid and Nomarski (Differential Interference Contrast) illumination. Compressed specimens of Caytonanthus and Sahnia, and cellulose acetate peels of permineralized corystosperm pollen sacs were mounted on glass slides in Coverbond and examined along with thin sections of bennettitalean cones in transmitted light. All individual palynomorphs, compressions, and histological preparations were observed and photographed with a Zeiss Ultraphot light microscope.

Scanning Electron Microscopy — Individual palynomorphs from all taxa were either pipetted directly onto aluminum stubs or through cellulose filters, under suction, which were then mounted on stubs. Cleared specimens of Caytonanthus, both synangia and rachises, were adhered to stubs with double stick adhesive tape. Once the compressed synangia had been initially mounted flat, they were teased apart using dissecting probes in order to expose the internal surfaces of synangia walls as well as their contents. Aluminum stubs were sputter coated with gold-palladium and images were recorded on either an Hitachi S-500 or a JEOL JSM-820 scanning electron microscope at accelerating voltages of 15 - 20 kV. For corystosperm material, in addition to observations of the individual palynomorphs and clumps of pollen grains, which had been macerated directly from corystosperm pollen 33 organs, several attempts were made to employ the techniques of Daghiian and Taylor (1979). This protocol facilitates the isolation of pollen as well as contiguous fossil material from cellulose acetate peels for electron microscopy (both SEM and TEM). Previously prepared microscope slides of peels containing corystosperm. pollen sacs and in situ pollen were dissociated in xylene; the peels were subsequently washed several times in xylene. Smaller portions of the peels (i.e., containing individual pollen sacs of interest, etc.) were carefully cut out and placed on Nuciepore filters. Large quantities of reagent grade acetone were flooded over the peels under suction. In previous studies (i.e., Daghiian and Taylor, 1979), the cellulose acetate peels easily dissolved in acetone leaving the sections of permineralized plant material formerly embedded within the peels fiee-standing on the filter surfaces. However, this ideal outcome was not the case in the present investigation. Here, the cellulose acetate peels never became adequately solubilized, they only partially dissolved and formed a gelatinous mass around the fossil material. This was more than likely due to either proportional differences in the chemical constituents of the specific type, brand, and/or age of cellulose acetate used in the present study, a hypothesis concurred with by Rothwell (personal communication).

Transmission Electron Microscopy — Macerates (i.e., both individual palynomorphs and clumps of pollen) were pipetted onto cellulose filters under suction and the filters were then coated on both sides with 2% agar. For compression specimens, if they were fragile and breaking apart they were also pipetted onto filters under suction, or if they had maintained their integrity following demineralization and clearing, they were simply placed onto filters with tweezers; the latter was undertaken to ensure flat embeddment and control subsequent sectioning orientation. Filters containing compressions were also embedded in 2% agar. 34

Agar embedded filters were then dehydrated in a graded ethanol series and transferred to 100% acetone, with at least four acetone changes to ensure the complete solubilization of filters. The embedded filters were then infiltrated with Spurr low viscosity epoxy resin, prepared according to the "firm" protocol, in a gradually-increasing dilution series of acetone:Spurr resin (3:1; 1:1; 1:3; 0:4; 0:4). To ensure optimal infiltration, filters were left in each dilution increment for a minimum of eight hours and under constant rotation using a Roto-Torque vial rotator. Following complete infiltration, filters were cut into four quarters, placed in shallow aluminum pans, immersed in enough 100% Spurr resin to completely cover the filters, and embedded by facilitating resin polymerization in a 70 °C oven for at least eight hours. Disks of polymerized resin were then evaluated in transmitted light (both stereo and compound microscopes) to locate and identify individual palynomorphs. Several pollen types (i.e., pentoxylalean, caytonialean, gnetalean, and bennettitalean) were relatively light tan to gold in color, indicating minimal thermal maturation of those grains (e.g.. Traverse, 1988), resulting in difficulty with grain identification because of the similar gold-colored Spurr resin. In additional and separate samples, several attempts were made to stain those pollen types with Safranin prior to TEM preparation; however, none of the grain types readily stained. Consequently, greater care was taken to identify the naturally gold-colored palynomorphs; in fact, this technique was fine-tuned enough so that the level of difficulty significantly decreased. Depending on the specific orientation of individual, embedded pollen grains as well as the orientation of sections desired (i.e., transverse or glancing sections, sections through either the polar or equatorial planes, etc.) block' outlines were stenciUed with a razor blade on the surfaces of the resin disks at the appropriate positions over grains. By comparison, stencilling block outlines over compression specimens was not as difficult and or as 35 critical, because of their larger sizes. Blocks were then cut out of resin disks with a jeweler's saw, rough trimmed with razor blades, and finely trimmed on an American Optical Specimen Trimmer using a diamond mill bit. Fine trimming was especially important for preparation of blocks containing single pollen grains. Ultrathin sections were cut from all blocks on an American Optical Ultracut uitramicrotome with a diamond knife, collected on uncoated 1x2 mm slot grids, and dried onto 0.45% formvar support films following the techniques of Rowley and Moran (1975). Although serial sections per se were not collected, serial grids were collected at intervals of every 15 to 25 sections depending of section size. Grids were either double or triple stained in varying time intervals with 1% potassium permanganate (0-30 nun), 1% uranyl acetate (0-30 min), and lead citrate (0-20 min; Venable and Coggeshall, 1965). All observations were carried out and images were recorded on a Zeiss EM-10 transmission electron microscope at accelerating voltages of 60-80 kV. When evaluating a number of grids, especially at high magnifications, specimen drift was a significant problem. In order to stabilize the support films on these, the grids were carbon coated in an Hitachi HUS-5 High Vacuum Evaporator and reexamind.

Phylogenetic Analyses — After evaluation of the comparative data ascertained through the micromorphological and ultrastructural examinations of all pollen types, a list of 16 characters for cladistic analyses was coit^iled. Although initially the list of characters was more lengthy, the present investigation is relatively conservative in its assessment of phylogenetically important characters and did not include several that have been used in other studies. Moreover, two of the six groups studied here (i.e., Glossopteridales and Corystospermales) were not included in the cladistic analyses; however, data regarding the pollen of several other seed plants was obtained from the literature. The justification for 36 the inclusion and/or rejection of all characters as well as taxa in the present phylogenetic analyses is discussed in detail in Chapter DC. In the final analyses, 15 terminal taxa were evaluated based on the total 16 characters, ten of which were multistate. Phylogenetic analyses were based on synapomorphy and parsimony, and utilized the computer program PAUP, Version 3.On (Swofford, 1990) on a Macintosh LC microcomputer. The data matrix was searched according to both Heuristic and Branch and Bound algorithms in order to elucidate the shortest . Variable options for treating the data as either ordered or unordered, with particular weights, or assigning polarities (i.e., defining the ancestral states) were also used. In instances when the program determined the presence of more than one most parsimonious tree, consensus trees (strict, semi-strict, and 50% Majority-rule) were generated. Further discussion of the various algorithms and options employed for specific analyses are also presented in Chapter IX. CHAPTER m GLOSSOPTERIDALES

Introduction The Glossopteridales represents the most dominant component of the southern hemisphere () Permian floras. Several specimens are also known from Triassic sediments; floristically speaking, however, they are appreciably less important in these younger floras. Glossopterid remains have been described from all major Gondwana continents/countries (Australia, Antarctica, southern Africa, South America, and India), and have historically played a fundamental biostradgraphic role in confirming the continental integrity of Gondwana in a geological capacity. Interestingly, there have also been isolated reports of glossopterids in two northern hemisphere localities (e.g., Delevoryas, 1969; Delevoryas and Person, 1975; Ash, 1979-1980) The group is primarily known from disarticulated leaves belonging to the genus Glossopteris Brongniart (1828). Since Brongniart's original description, a great number of specimens have been described and assigned to well over 100 species (e.g., Pigg, 1988, 1990a). In addition to Glossopteris, several other form genera of foliage are known. Gangamopteris is the second most abundant leaf type identified, followed by a number of other genera which occur much less frequently {Palaeovittaria, Rubidgea, Euryphyllum, Rhabdotaenia, Beiemnopteris, and Pteronilssonia; e.g., Pigg, 1988 and references therein). The majority of foliar specimens that have been described, as well as

37 38 other glossopterid fossils, principally occur as compressions and impressions. However, two localities have yielded well-preserved permineralized remains in recent years (the Bowen Basin, Queensland, Australia; Gould and Delevoryas, 1977; and Skaar Ridge, central Transantarctic Mountains, Antarctica; Pigg, 1988,1990a). Leaves often occur in association with relatively large axes of pycnoxylic wood (e.g., Araucarioxylon, Dadoxylon, Antarctioxylon), smaller, woody axes of Vertebraria, an anatomically anomalous rooting structure, and numerous reproductive structures. The majority of glossopterid reproductive structures ("fertiligers" sensu Schopf, 1976 and Pant, 1987) typically consist of a dorsiventrally-flattened unit (), bearing either ovules or pollen sacs, attached via a short stalk to a foliar unit with Glossopteris morphology. Plumstead (1952) was first to demonstrate the attachment of fertile organs to Glossopteris leaves; thus convincingly documenting the nature of glossopterid reproductive structures, information that had remained equivocal since the establishment of the group. To date, more than 1500 glossopterid fertile organs, circumscribed within 25- 30 form genera, have been described firom Gondwana sites since Plumstead's pioneering work (e.g., Plumstead, 1952, 1956a, b, 1958; Schopf, 1976; Pant, 1987; McLoughlin, 1990). Glossopterid ovulate organs exhibit a wide array of morphologies; forms range from being uniovulate to muitiovulate and having the typical dorsiventral symmetry to a more radially symmetrical organization. Nevertheless, they are generally classified into two groups based on the morphology of the fertile unit's subtending leaf. The first includes ovulate organs attached to an unmodified leaf of Glossopteris or GangamopterisÇc.g., Ottokaria, Senotheca, Rusangea, Austroglossa, and perhaps Scutum, Plumsteadia, Dictyopteridium, and Isodictyopteridium), while the second morpho-type encompasses fertile units subtended by modified (i.e., -like) Glossopteris or Gangamopteris leaf 39

{e.g., Liugeuorda, Denkaràa, Parîka, Hinjridia, snd Handappia; for review see Pant, 1987 and references therein). Ovulate organs are almost entirely known from compression- impression fossils with the exception of permineralized specimens from Australia (Gould and Delevoryas, 1977) and Antarctica (Schopf, 1976, Taylor, 1987). Although the permineralized ovules/seeds were found attached to a laminar unit in these investigations, none were able to demonstrate organic attachment of this unit to a subtending Glossopteris leaf or bract. Significantly, however, Taylor (1987) has substantiated that the Antarctic ovule-bearing laminar unit is anatomically similar to structurally preserved Glossopteris leaves (Pigg, 1988,1990a) that occur in the same matrices. Relative to the number of taxa and morphological diversity of glossopterid ovulate organs, fewer pollen-bearing structures have been described. However, they also exhibit some morphological variation. For example, Glossotheca and Eretmonia are typically flattened dorsiventrally, whereas Kendrostrobus has a cone-like morphology (e.g., Du Toit, 1932; Surange and Maheshwari, 1970; Lacey, van Dijk, and Gordon-Gray, 1975; Surange and Chandra, 1972a, b, 1975; Pant, 1987; Pigg, 1988). Moreover, several dispersed pollen sacs (i.e., unattached to a leaf or bract) have also been described (e.g., Nesowalesia: Pant, 1958; Arberiella, Lithangia, and Polytheca: Pant and Nautiyal, 1960). These are believed to have been produced by glossopterids because of morphological similarities with intact pollen-bearing organs, such as similar surface features, the occasional presence of an attached stalk, and the presence of morphologically similar in situ pollen grains. To date, the only permineralized pollen-bearing organs known are dispersed pollen sacs of the Arberiella-type from the Bowen Basin of Australia (Gould and Delevoryas, 1977); none have been recovered from the Antarctic silicified Permian peats yet. 40

Only limited data have been published on the pollen of glossopterids. Most information comes from examinations of in situ grains in transmitted light (e.g., Plumstead, 1956a, b; Surange and Chandra, 1972b, 1975; Pant and Nautiyal, 1960; Gould and Delevoryas, 1977). In general, grains are described as bisaccate with endoreticulations within the sacci, and having a striate proximal ornament composed of longitudinally-oriented ridges (e.g., Arberiella: Pant and Nautiyal, 1960; Gould and Delevoryas, 1977). However, some diversity in pollen morphology has been reported. For example, grains of Polytheca are described as nonsaccate, with psilate surface sculpturing and a trilete suture (Pant and Nautiyal, 1960), while grains macerated from pollen sacs of Lithangium (Pant and Nautiyal, 1960) and Kendrostrobus (Surange and Chandra, 1972b, 1975) are also nonsaccate, but bear what are referred to as monolete marks on their surfaces. Only recently have in situ glossopterid pollen grains been investigated at the ultrastructural and micromorpological levels. Zavada (1990a, 1991) has described grains from dispersed Arberiella-type pollen sacs from Hammanskraal, South Africa. The exine of these grains was found to be alveolar in the saccus region, and composed of partitions or "irregular shaped anastomosing rods" in the cappus region. Moreover, several sporae dispersae palynomorphs are morphologically very similar to the typical in situ "striate bisaccate" grains from glossopterid pollen-bearing organs (e.g., Gould and Delevoryas, 1977; Pigg, 1988; Farabee et al., 1989, 1991). One of these, Protokaploxypinus, has been studied with SEM and TEM, and the exine appears in the published micrographs to also consist of relatively robust, honeycomb-like alveolar units (Foster, 1979). In the present study, several dispersed, "striate bisaccate" palynomorphs from Antarctic rocks (Farabee et al., 1990,1991), and from the same Permian formation that has yielded the structurally preserved glossopterid foliar and ovulate organs (Taylor, 1987; Pigg, 1988,1990a) were examined at ultrastructural and micromorphological levels. 41

Description The dispersed taeniate grains examined in the present investigation are generally poorly preserved and, as a result of diagenesis, have lost relevant sporoderm structure. However, important data concerning a number of characteristics of pollen size, gross morphology, and gross ultrastructure are discernable and will be presented here. The palynomorphs evaluated in this study most closely resemble grains assigned to Protokaploxypinus by Farabee et al. (1990, 1991). Grains average 61 pm in length (saccus to saccus) and 35 pm in width (Figs. 1, 2,5). Corpi average 37 pm in length and 34 pm in width, while the lateral portions of the sacci (i.e., extending beyond the corpus in a proximal polar view) average 15 pm length and 38 pm in width (Figs. 1, 2, 5). The majority of grains examined are compressed in their polar plane, and thus do not provide information on grain heights. The cappus is distinctly taeniate, with taeniae and striae oriented parallel to the long axis of the grain. Proximal taeniae, as well as distal surfaces, and sacci are all psilate to finely scabrate in ornamentation (Figs. 1-3). Grains do show some surface pitting; however, this is interpreted as preservational in nature (Fig. 3). Taeniae range from 5 to 8 in number and average 4.2 pm in width. Both, taeniae and striae are easily detectable at the ultrastructural level as thick and thin regions of the exine. Taeniae clearly represent distinct cappus components, rather than being supratectal in origin (Figs. 4,8,10). On average, the exine measures 0.85 pm in thickness in cappus regions with taeniae and only 0.08 pm in thickness in the furrows between taeniae (i.e., striae; Figs. 8-9). Unfortunately, the actual infrastructural organization of these proximal wall components is not available from the grains that were sectioned because they show significant amounts of degradation, more than likely due to thermal/diagenetic alteration (Figs. 8-10). Some sections, however, do 42 illustrate a slight degree of differential staining in the taeniae (Fig. 8), although it is impossible to ascertain whether the thin, contiguous striae are composed of only a nexine layer, or both nexine and a thin layer of sexine as well. Grains are not truly bisaccate, but rather exhibit a transitional type of saccus organization. In particular, the saccus is attached both proximally and laterally on either side of the corpus (i.e., girdling the corpus) and continuous over the distal surface (Figs. 1, 4, 6). Distinct endoreticulations are present within the saccus (Figs. 4, 6, 7). Endoreticulations may be continuous between the outer saccus walls and the corpus (Figs. 4, 10) or discontinuous (i.e., separated from the corpus wall; Fig. 6) depending upon the plane of section. In lateral regions of the pollen grain, where the bladder is attached to the corpus, endoreticulations appear continuous (Fig. 4), whereas they are discontinuous in more medial regions (Fig. 6). Moreover, reticulations appear continuous between all walls of the bladder when it is sectioned near its margin (Fig. 7). The continuity of the endoreticulations in these grain regions is due in part to both the nature of the ultrathin sections, as well as preservational compression of grains.

Discussion Upon a cursory examination of the taeniate grains sectioned here (Protokaploxypinus), the most notable feature is the effect of preservational phenomena on exine ultrastructure. Although various patterns of exine degradation are also known to result from the effects of standard palynomorph macerations and preparation protocols for electron microscopy (e.g., Kedves, 1985b; Rowley, Rowley, and Skvarla, 1990), the exine modification seen in these Antarctic grains is clearly the result of thermal degradation. Some effects of high temperature on exine structure have been documented (e.g., Sengupta, 1977; Kedves, 1985b; Kedves and Kincsek, 1989). More importantly, however, is the fact that rocks in 43 the Beacon Supergroup of the centrai Transantarctic Mountains, from where the presently investigated palynomorphs have come, are known to have undergone extensive thermal metamoiphism (e.g., Kyle and Schopf, 1977), Permian rocks in particular (Farabee et al., 1991). Unfortunately, exine damage of the Antarctic Protokaploxypinus grains is at a level that precludes relevant systematic-type comparisons with the alveolate ultrastructure seen in other dispersed Protokaploxypinus palynomoiphs (Foster, 1979) and in in situ Arberiella- type grains (Zavada, 1990a, 1991). Despite the high degree of grain alteration, several important fine structural features have been elucidated. Most importantly, the proximal taeniae of these Antarctic Protokaploxypinus grains have been documented to represent actual thickened regions of the cappus, as opposed to being supra-exinal in origin. This is significant in that it unequivocally distinguishes these grains from palynomorphs assigned to Lunatisporites, which have also been recovered as microfloral components from the Permian coals and shales at Mt. Achemar, Antarctica (Farabee et al., 1990, 1991). Although Farabee et al. (1991) distinguish L. acutus from Protokaploxypinus simply by the presence of broader taeniae in the former, in another species of Lunatisporites, L. noviaulensis-mollis Scheming (1970), the proximal 'taeniae* have been shown to in fact be relatively small, elongated sacci completely infilled with an anastomosing system of exinous endoreticulations ("protosacci"; Scheuring, 1974). Continued examinations of other taeniate palynomorphs from the Permian of Antarctica may indicate a similar fine structural organization in those grains (i.e., Lunatisporites spp.). The taeniate grains investigated here have also provided important information regarding the structural nature of sacci. Serial sections clearly indicate that the sacci are of the eusaccate-type, with distinct separation of endoreticulations between the saccus and corpus walls in certain regions (i.e., medial) of the grain. Moreover, although these grains 44 are ftequentiy referred to as "striate bisaccates", because two prominent, latemiiy-attached bladders are easily distinguishable in a proximal polar view, the grains sectioned have clarified and confirmed that Protokaploxypinus has a saccate condition which is transitional between the mono- and bis^cate-type (Traverse, 1988). In these grains, sacci are attached both proximally and laterally to the corpus as they would be on a truly bisaccate grain. However, rather than two separate distal attachments to the lateral walls of the corpus, the sacci are fused distaliy and form a reiativeiy thin cavity which is continuous with the larger, lateral bladders. The transitional saccate condition of these dispersed Protokaploxypinus grains has significant implications if indeed they were produced by glossopterids. Most importantly is the fact that the corpus lacks a distal aperture and is completely covered by a continuous bladder on this surface. This situation would preclude germination of a pollen tube fix>m a distal sulcus, and suggests that siphonogamy was facilitated by proximal germination through one of the thin furrows (striae) in the cappus. If this was the case, it further suggests that glossopterids are more similar to several other Paleozoic gymnosperms as opposed to a number of Mesozoic groups, at least with regard to pollen structure and reproductive biology. A number of these Paleozoic gymnosperms produced either saccate (e.g., Sullisaccites) or nonsaccate (Monoletes) pollen with a distinct proximal aperture through which germination was presumed to have occurred (i.e., "prepollen"; Millay et al., 1978). Most gymnosperm groups in the Mesozoic, on the other hand, produced both saccate and nonsaccate pollen that was characterized by a prominent distal sulcus. It is interesting to note also, that the 'anomalous' glossopterid grains described from pollen sacs of Kendrostrobus, Polytkeca znd Lithangium (i.e., nonsaccate grains with a trilete- or monolete-type aperture) would presumably have also germinated proximally. 45

If proximal pollen germination was characteristic in glossopterids, then this has significant implications regarding suggestions of angiosperm origins from the Glossopteridales (e.g., Retallack and Dilcher, 1981), in that both pollen structure and biology are distinctly different. Angiosperm pollen is nonsaccate, with the exception of Lactoris (Lactoridaceae; Zavada and Taylor, 1986), and typically germinates through either a distal or an equatorial aperture, but not proximally. It should be emphasized, however, that this postulate is based only on observations of dispersed palynomorphs, and needs to be substantiated by investigations of in situ glossopterid pollen. CHAPTER IV CORYSTOSPERMALES

Introduction The Corystospermales was established in 1933 in order to classify a relatively small group of seed ferns from Triassic sediments of the Upper Umkomaas Valley, Natal, South Africa (=; Thomas, 1933). Stratigraphically, corystosperms range from Permian to Middle Jurassic and are predominantly known from Gondwana localities (e.g., Townrow, 1965; Retallack, 1976; Petriella, 1979, 1980, 1983; Anderson and Anderson, 1985; Pigg, 1988, 1990b); however, some specimens with possible corystosperm affinity have been recovered from Laurasian sites (e.g., Pteroma; Harris, 1964). Although corystospermous plants are not known from intact specimens (i.e., organic attachment), several disarticulated form genera have been found consistently within the same sediments and bear morphologically similar features, such as cuticular structure. The most frequently encountered and described genera belong to Dicroidium (leaves), Rhexoxylon (wood), Fteruchus (pollen organs), Alisporites (dispersed pollen), Pilophorosperma (megasporophylls/cupules), and (ovules/seeds). Retallack and Dilcher (1988), however, have recently proposed a collective and somewhat detailed hypothesized reconstruction for a corystospermalean pteridosperm, referring to it as Umkomasia granulata.

46 47

The cor>’Stospemi pollen organ Ptsruckus was initially described by Thomas (1933) and subsequently emended by Townrow (1962a). Pteruchus is radially symmetrical with microsporophylls attached either helically or alternately. Individual microsporophylls terminate in a flattened head and bear numerous, pendulous pollen sacs. Pollen sacs are uniloculate, each with a single, longitudinally-oriented dehiscence suture, and contain bisaccate pollen grains. Historically, information about this pollen organ has been gained only from compression-impression specimens (e.g., Thomas, 1933; Townrow, i962a; Pant and Basu, 1973,1979; Srivastava, 1974; Anderson and Anderson, 1985). However, several permineralized pollen organs similar in gross morphology to Pteruchus and containing Alisporites-type bisaccate pollen have recently been discovered in Triassic peat deposits from Antarctica (Devore and Taylor, 1988; Osborn and Taylor, 1990; Yao, in preparation). The corystospermous affinities of this pollen organ and its in situ pollen are corroborated by the concomitant occurrence of structurally preserved Dicroidium foliage (Pigg, 1988; 1990b), as well as Rhexoxylon-typQ wood (Taylor and Taylor, 1990b; E. Taylor, 1991) with probable corystosperm affinities, as principal elements in the same Antarctic peat In addition to Pteruchus and the Antarctic Pteruchus-like. organs, several other compressed microsporophyll taxa have been suggested to have affinities with the Corystospermales (see Crane, 1988), including Nidiostrobus harrisiana (Bose and Srivastava, 1973), Kachchhia navicula (Bose and Baneijee, 1984), and Pteroma thomasi (Harris, 1964). There are few detailed studies on corystosperm pollen as well. In situ pollen grains macerated from the type Pteruchus compression material were initially evaluated in transmitted light by Thomas (1933) and Townrow (1962a, b). Grains were described as being variable in size with two sacci and a single germination furrow on the distal surface; 48 ornamentation Avas reported as granular to reticulate on sacci and variable on corpi (Thomas, 1933; Townrow, 1962a, b). Numerous stratigraphie studies have also provided information on sporae dispersae grains that distinctly resemble Pteruchus poUen. These palynomorphs are most often ascribed to Alisporites (Daugherty) Jansonius (1971) and Pteruchipollenites Couper (1958). Moreover, recent palynostratigraphic studies of Triassic sediments from Antarctica have revealed a relatively diverse microflora, including Alisporites and Pteruchipolienites as primary components (Farabee, Taylor, and Taylor, 1989, 1990). Fewer grains, however, have been studied at the micromorphological and ultrastructural levels using electron microscopy. Only two investigations have focused on corystosperm pollen using SEM and TEM, and both sets of observations come from compression material (Taylor, Cichan, and Baldoni, 1984; Zavada and Crepet, 1985). Taylor et al. (1984) examined in situ pollen of Pteruchus dubius from Middle Triassic sediments from Dinmore, Ipswich, Australia, and Zavada and Crepet (1985) evaluated in situ grains from the type material of P. africanus, P. dubius, and P. papillatus from the Triassic Molteno beds in the Upper Umkomass Valley, South Africa. Both studies report exine ornamentation as psilate with artifactual pitting, and variously shaped sporopolleninous units separated by irregular lacunae within the sacci. Taylor et al. (1984) found the cappus region to gradually thin near the proximal pole and to be only slightly thicker than the distal region of the corpus, but they did not address the infrastructural nature of the cappus. Zavada and Crepet (1985), however, found the proximal cappus region to be ultrastructurally homogeneous with the occasional occurrence of small lacunae. The fine structure of Pteruchus pollen has also been addressed in two recent reviews of fossil gymnosperm pollen (Taylor and Taylor, 1987b; Taylor, 1988b). 49

lîî the present investigation, pollen grains from the Antarctic permineralized Pteruchus- like pollen organ are examined with SEM and TEM and compared with other previously described corystosperm pollen.

Description A complete and diagnostic description of the entire pollen organ will not be presented here, but rather will be addressed by Yao (in preparation) as part of a larger study on several anatomically preserved gymnosperm reproductive organs from the Triassic of Antarctica. However, several features of the poUen sacs per se, specifically as they relate to the present investigation, are noted. Permineralized pollen sacs are elliptic, unilocular, and relatively small in size, ranging from 0.7-1.1 mm in length and 0.44-0.48 mm in width (Figs. 11-13). Each sac has a single epidermal layer composed of elongate sinous cells and larger, intercalary secretory cells that occur at irregular intervals (Figs. 11-13). Three types of pollen sacs have been discovered within the peat and are believed to represent different developmental stages. In one, the individual sacs are completely devoid of pollen and have split open at a site of dehiscence (Fig. 11). The second type contains copious quantities of pollen that are completely dissociated from one another (Figs. 12-13). A third type of pollen sac, containing large amounts of pollen grains that are densely packed together, also occurs in the same matrix (Figs. 14-17). The tight packing of pollen grains here is underscored by the fact that grains macerate out of the permineralized sacs in distinct clumps (Figs. 17-18). Individual grains within clumps may have small, spherical orbicules, or orbicule-like bodies, associated with their surfaces (Fig. 19). In addition to the single-celled wall, these pollen sacs have a thin membraneous layer which occurs just inside the wall (Fig. 15) Membranes have been identified in several pollen sacs and are variable in their form. 50

Pollen grains are monosulcaîe and bisaccate with large, crescent-shaped sacci (Figs. 20- 23, 34-38). Sacci are generally found laterally attached (Figs. 20,22, 23); however, a few specimens have sacci that are distaliy inclined (Fig. 21). Pollen grains typically average 66

Jim in length (saccus to saccus) and 49 jim in width as seen in polar view. In equatorial

view, grains average 30 jim in height (Fig. 26). The corpus, as seen in equatorial view,

averages 33 jim in length and 22 jim in height (Fig. 26), while corpus width averages 49

Jim (polar view; Fig. 26). Sacci average 30 jim in length, 20 jim in height, and 36 pm in width, and have distinct endoreticulations (Figs. 25-26). The proximal wall is characterized by psilate ornamentation; although numerous pits occur on the exine surface; these are interpreted as preservational artifacts (Figs. 22, 24). In the cappus region, the exine is homogeneous and averages 1.0 pm in thickness (Fig. 28). However, some ultrastructural variability is detectable in this region. For example, some grains may show a slight degree of differential staining and/or faint lamellations here (Fig. 29). It is more than likely that this variable structure is also the result of preservational phenomena. Although grains typically have homogeneous exines in the cappus region and do not show differentially-stained sexine and nexine layers, the presence of two distinct layers is especially detectable in lateral (i.e., peripheral) corpus positions, where the layers separate to form sacci. The region of saccus attachment is characterized by a wedge-like unit, formed by the initial separation of cappus wall layers, which gradually dialates to form the saccus proper (Figs. 26, 30-32). Morphologically, endoreticulations originate at the bases of the wedge­ like unit (i.e., medially near the cappus), where they extend across the entire unit and make contact with both exine layers. In the more dialated regions, the endoreticulations separate (i.e., break apart) with the short and discontinuous reticulations attached only to the outer exine layer, which forms the outer saccus wall (Figs. 30-32). The size and ultrastmctural 51 nature of wedge-like units may vary depending on both the angle of saccus inclination and the equatorial positions from which ultrathin sections were cut (i.e., median or lateral equatorial sections; Figs. 30-31). The plane of section through grains is also paramount with regard to interpreting exine fine structure in cappus regions. In particular, in lateral equatorial sections the wedge-like units of both sacci are elongated and make contact Here the reticulations are continuous, extending between both exine layers across the entire cappus wall (Figs. 40-41). Exine fine structure in this region of the cappus is markedly different in comparison with the homogeneous wall organization seen in medial sections (cf. Figs. 28-29). Sacci are large, psilate in external ornament, and have relatively thin outer walls (Figs. 34,38-39). Endoreticulations are principally attached to the outer walls with spaces of 2-3 pm between each member of the reticulum (Figs. 26,35). Moreover, endoreticulations are rather robust, averaging 0.30 pm in width and 1.92 pm in length (Figs. 26, 45), and can easily be detected from an external view (i.e., LM or SEM; Figs. 34, 38-39). The inner saccus wall (i.e., lateral corpus wall) is relatively thin, averaging 0.38 pm in thickness, and is devoid of reticulations in medial grain positions (Figs. 26-27, 45). However, lateral equatorial sections indicate that endoreticulations are continuous between the outer saccus and lateral corpus walls (Fig. 44). A relatively broad sulcus, averaging 9.2 pm in width and extending the entire width of each grain, characterizes the distal surface (Figs. 36-38). Aperture membranes are typically not well preserved (Fig. 38); however, when they can be identified they are thin, averaging 0.22 pm in thickness, and also have a psilate surface (Figs. 36-37, 43). Although most grains lack an aperture membrane, one diagnostic feature of all grains is the presence of elevated lips that longitudinally flank the sulcus in the positions of saccus attachment (Figs. 36-38). Sacci are also attached to the corpus in distal regions by wedge­ 52 like units (Fig. 42). These distal units are similar in fine structure to those on the proximal wall with regard to both the extension and separation of endoreticulations; however, distal units are less variable ultrastructurally in equatorial positions (i.e., they do not gradually taper and fuse), because the elongate sulcus extends the entire width of each grain and prevents overlap of sacci.

Discussion Structural and Developmental Features of the Exine — One of the most notable aspects of pollen wall ultrastructure of these mature corystosperm grains is the absence of two differentially staining exine layers. Two interpretations for this condition can be suggested. The first suggests that the traditionally recognized nexine and sexine wall layers do not have varying affinities for heavy metal stains in these grains, and that they grade into one another resulting in an ultrastructurally homogeneous exine throughout (i.e., as seen in medial sections). This postulate is extremely unlikely for several reasons. For example, differentially stained nexine and sexine layers occur commonly in the majority of ultrastructurally-described fossil and extant pollen grains that have been either double or triple stained following standard procedures. Secondly, a light staining sexine and dark staining nexine have also been observed in pollen of Pteruchus africanus, P. dubius, and P. papillatus (Zavada and Crepet, 1985). A more plausible explanation for the homogeneous exine is that the grains have undergone some type of structural modification, and the absence of two distinct layers is artifactual. Exine ultrastructure of both fossil and extant pollen is known to become variously altered as a result of preservational phenomena (i.e., diagenesis), temperature effects, chemical maceration effects, and electron microscopy (EM) preparations (e.g., Sengupta, 1977; Niklas, 1980; Kedves, 1985b; Kedves and Kincsek, 1989; Rowley, 53

Rov.'iey, and Sicv'arîa, 1990). The patterns observed in exine organization of the corystosperm grains examined here most likely represents fine structural degradation due to combined diagenetic and temperature effects. Other causes of exine alteration have been ruled out because the same pollen maceration and EM preparation techniques were employed for analyses of the other gymnosperm pollen types in this study, and they showed exquisite exine preservation (e.g., Cycadeoidea, Chapter VI; Equisetosporites, Chapter VII). Moreover, rocks in the Beardmore Formation, in which corystosperm remains are preserved, are known to have undergone a fair amount of thermal maturation (Farabee et al., 1989,1990; Taylor, Taylor, and Collinson, 1989 and references therein). Although the exine of these corystosperm grains stains homogeneously throughout, it is clear that two distinct layers compose the pollen wall. This is especially evident in both proximal and distal regions of the corpus where the exine separates to form sacci. The external exine layer (sexine) splits off to compose the outer saccus wall, with discontinuous endoreticulations attached, while the internal exine layer (nexine) lacks sporopolleninous endoreticulations and composes the lateral corpus wall. Ultrastructurally, similar sexine and nexine separations characterize the origin of sacci in saccate pollen of both fossil (e.g., Millay and Taylor, 1974, 1976) and extant gymnosperms (e.g., Dickinson and Bell, 1970a; Kurmann, 1989b, c). Perhaps more interesting is the nature of exine ultrastructure observed in different positions of the proximal cappus region. In the grains examined here, the cappus appears homogeneous in medial sections, and alveolate (of the honey comb type sensu Doyle et al., 1975) in lateral sections. The prudent question to be posed is whether the exine is actually homogeneous in medial regions, or is also structurally alveolate here but has lost its sexine alveolar infrastracture as a result of preservational influences. In saccate pollen of several extant gymnosperms (e.g., Coniferales; Abies, Pinus) an alveolar infrastructure 54 characterizes the proximal wall in all regions. However, in the saccate pollen of several Paleozoic seed plants (e.g., Cordaianthus, Sullisaccites, Felixipollenites, and Vesicaspord) the alveolate structure does not extend over the corpus in the proximal wall, but is rather restricted to lateral positions where sacci overlap in attachment (Millay and Taylor, 1974). It should be noted, however, that sexine organization is known to gradually lose resolution with regard to its infrastructure in palynomorphs that have undergone increasingly more thermal maturation (as detected by relative darkness of grains; Zavada, personal communication). The corystosperm grains evaluated here were in fact quite dark in color, and possibly have lost some fine structural information. However, it is the author’s opinion that these permineralized grains have not been diagenetically altered to the degree of completely losing a possible alveolar infrastructure, but rather originally lacked this type of organization in the medial cappus region. Interestingly, the proximal cappus region of compressed in situ Pteruchus pollen also shows a homogeneous organization, with a few small lacunae (Zavada and Crepet, 1985). In addition to providing detailed data about the corpus wall, the permineralized pollen grains investigated here are especially important with regard to new information gained about corystosperm sacci. Saccus organization of Pteruchus pollen has received a significant amount of attention in previous descriptions. The majority of this attention has been focused on the nature of the endoreticulations (meshes sensu Townrow, 1933; see Taylor et al., 1984). Based on observations of grains in transmitted light, ornamentation of the saccus has been variously described as completely psilate ranging to highly reticulate (Thomas, 1933; Towmow, 1962a, b; Pant and Basu, 1973, 1979). Moreover, the early literature is relatively ambiguous as to whether saccus reticulations represent external or internal ornament Taylor et al. (1984) clarified the internal presence of endoreticulations in Australian material of P. dubius; this was subsequently confirmed in the three type 55

Sentît t^fncuîi spccics ef by Z&v&cîa aitci CTrcpct ^î9o5}. Botîi cf tlicsc stuuics found sacci to be densely filled with endoreticulations (sporopoUenin units sensu Taylor et al., 1984; infrastructural processes sensu Zavada and Crepet, 1985). Although Zavada and Crepet suggested that the endoreticulations "can be free or fused to a thin basal layer", which in the present author's opinion represents the saccus floor (i.e., lateral corpus wall), neither investigation was able to unequivocally document whether Pteruchus pollen is protosaccate or eusaccate (Scheuring, 1974) because all grains examined were extremely compressed. The permineralized grains sectioned in the present study clearly indicate that they are eusaccate, a determination afforded because grains are permineralized intact, and not compressed in their height dimension. Grains sectioned equatorially and though a medial plane illustrate the presence of well-defined, discontinuous endoreticulations. Interestingly, however, when grains are sectioned though a lateral equatorial plane, sacci superficially appear as though they are protosaccate. In these sections reticulations are continuous between the outer sacci and lateral corpus walls, because this is where sacci are tapering to their margins and beginning to slightly overlap in their attachment to the corpus. Endoreticulations in the sacci of modem Pinus strobus show a similar organization in both medial and lateral regions when respective grain positions are sectioned (Taylor et al., 1987). The fact that permineralized pollen sacs exhibiting different ontogenetic stages have been isolated is particularly interesting, and represents the first documented example of this in the Corystospermales. Pollen sacs containing completely-dissociated pollen grains are interpreted as mature, and the features of mature grains macerated from these sacs have been discussed above. Pollen sacs containing tightly-aggregated grains are interpreted as immature, but relatively close to maturity. Based on the ultrastructure and orientation of individual component grains within the larger aggregates, as well as the presence of 56 resistant membranes within pollen sacs, it is suggested that these pollen sacs were fossilized in the final stages of the 'ftee-sporing period' of development. Evidence for this hypothesis comes from the following: 1) grains do not appear to be linked into distinct tetrads, but had apparently undergone separation and were still in very close proximity to one another; 2) exine organization in the proximal cappus region is similar to that of mature grains in that it is homogeneous throughout; 3) well-defined endoreticulations are easily detectable and have apparently undergone complete ontogeny; and 4) resistant membranes just inside the pollen sac walls closely resemble peri tape tal membranes. All four of these features characterize the free-sporing period in the ontogeny of saccate pollen in modem conifers (e.g., Kurmann, 1986; 1990b). Fewer studies, however, have addressed the developmental aspects of fossil pollen, although tapetal membranes, sometimes described along with orbicules, have been reported in several Paleozoic (Taylor, 1976a, b, 1982; Taylor and Zavada, 1986) and Mesozoic (Taylor and Alvin, 1984; Zavada and Crepet, 1986; this volume, chapter VU) gymnosperms.

Systematic Comparisons — Recent phylogenetic studies of seed plants (Crane, 1985a; Doyle and Donoghue, 1986, 1987) have suggested that the Corystospermales occupy a position in the sister group of an informally designated clade referred to as 'anthophytes', and composed of Bennettitales, Pentoxylales, Gnetales, Angiospeims, and perhaps plants that produced Eucommiidites-type pollen (Pedersen et al., 1989). Crane (1985a) linked the Corystospermales to anthophytes as their direct sister taxon, while Doyle and Donoghue (1986,1987) placed the Corystospermales in a sister group to the anthophytes along with Cycadales, Peltaspermales, Caytoniales, and Glossopteridales (see Crane, 1988). Of these related groups, saccate pollen is only known from Caytoniales, 57

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1986; Zavada and Bensen, 1987). There is limited published information on the pollen produced by glossopterids (see chapter HI), with very few examples of in situ pollen known (e.g., Gould and Delevoryas, 1977; Zavada, 1990a, 1991). The majority of data comes from dispersed grains found in association with glossopterid megafossils, or from palynostratigraphic analyses. These putative glossopterid grains are relatively large, transitionally saccate {sensu Traverse, 1988), and have conspicuous longitudinally-oriented striations (taeniae) on the proximal surface of the corpus. The permineralized corystosperm grains clearly lack proximal taeniae, and only superficially resemble glossopterid grains in relative size, the bisaccate condition, and the presence of a single distal sulcus. Moreover, the taeniate grains that have been examined at the ultrastructural level show that some taeniae may actually be relatively small protosacci filled with continuous endoreticulations (Scheuring, 1974) or show a more alveolate-type organization (Foster, 1979; Zavada, 1990a; 1991). Pollen of both modem and fossil Lactoridaceae is also distinctly different from the permineralized corystosperm grains (Zavada and Taylor, 1986; Zavada and Bensen, 1987). Pollen grains are permanently united in tetrads, with individual grains having an ovoid to lenticular sulcus. Sacci are relatively reduced in size, and exine ultrastmcture is granular. By comparison to glossopterid and angiosperm pollen, the permineralized corystosperm grains more closely resemble bisaccate pollen of the Caytoniales, especially with regard to cappus ultrastmcture (see Chapter V). The infrastmctural exine of Caytonanthus pollen is composed of sporopolleninous units that can be categorized as either relatively short plate­ like alveoli, or elongated, narrow columellae. The most striking comparison between these grains and the permineralized corystosperm grains is that the infrastructural exine elements in Caytonanthus pollen also grade into a more homogeneous organization over the cappus 58 median. However, Caytonanîkiis pollen is markedly different in several features. By comparison, Caytonanthus grains are significantly smaller than the pollen of corystosperms, infrastmctural elements and endoreticulations are much more robust in size, and the sacci do not appear to attach to the corpus by a distinct wedge-like unit Based on the above comparisons, the permineralized corystosperm grains do not unequivocally compare with any of the noted major groups, but rather appear to be unique in ultrastmcture. However, comparisons with previously described corystosperm in situ compression grains and sporae aispersae grains wiin puiauve corysiosperm aniniues may be more useful. The permineralized in situ grains described here are more similar in size to pollen of Pteruchus dubius collected from Australian localities (Taylor et al., 1984) relative to the South African type pollen of Pteruchus cfricanus, P. dubius, and P. papillatus (Zavada and Crepet, 1985). The Australian grains, however, do not appear to be well preserved in either the proximal or distal regions of the corpus, thereby precluding ultrastmctural comparisons. South Pdncdm. Pteruchus pollen, however, is similar in cappus fine stmcture to the permineralized grains, in that both types exhibit a homogeneous organization. Both studies of compressed Pteruchus pollen also report exine thinning in the cappus region; this, however, was not observed in permineralized grains, and may represent an artifact of preservation. Exine thinning is known to be one first types of stmctural modifications commonly seen in corroded/degraded pollen grains (Rowley et al., 1990). Another interesting similarity between the permineralized grains and pollen from the Australian specimens is the consistent occurrence of thickened and elevated lips lining the aperture margins. The most frequently encountered dispersed grains with possible coiystosperm affinities are those assigned to the form genera Alisporites and Pteruchipollenites. Both genera are 59 morphologically similar. For example, Aiisporites is classified by such subtle differences as a slightly more rounded amb and finer endoreticulations in comparison with an ovoid shape and more robust endoreticulations in the latter (Farabee et al., 1989). In addition, Pteruchipollenites grains are reported to be slightly smaller, have a thinner sporoderm lacking a preformed sulcus, and exhibit greater diversity with respect to saccus endoreticulations. Perhaps more interesting are the sporae dispersae grains described from other Triassic sediments in Antarctica (Farabee et al., 1989). Farabee et al. (1989) noted four species of Alisporites and one species of Pteruchipollenites within their samples. Of these taxa, the structurally preserved in situ grains most closely resemble Alisporites parvus de Jersey (1962) with regard to overall size and general morphology. Moreover, the permineralized Antarctic grains are further allied with Alisporites-type palynomorphs based on their relatively large sizes, the occurrence of a distinct sulcus, and similar thickenings along the sulcus margins in some species of Alisporites (A. australis; Foster, 1979). Although the structurally preserved in situ grains described here do not unequivocally answer all of the questions concerning corystosperm pollen, they do provide new information about several important fine structural features. The importance of these permineralized specimens is underscored by the unequivocal demonstration of eusacci in this group of Mesozoic gymnosperms. Further investigations of corystosperm reproductive organs, especially fine structural studies of pollen from those microsporangiate organs suggested to be corystospermous (i.e., Nidiostrobus harrisiana, Bose and Srivastava, 1973; Kachchhia navicula, Bose and Baneijee, 1984; and Pteroma thomasi, Harris, 1964; see Crane, 1988) will no doubt provide salient information for relevant systematic and phylogenetic evaluations. CHAPTER V CAYTONIALES

Introduction The Caytoniales has received significant attention from both paleobotanists and neobotanists alike since the establishment of the order in 1925 as a new Jurassic group of "angiospermous plants" (Thomas, 1925). Since that time, numerous specimens have been identified from different strata, and the group is now known from rocks ranging in age from Late Triassic to Late Cretaceous. Geographically, the Caytoniales is relatively wide- ranging as well. Fossils are known from England (e.g., Thomas, 1925; Harris, 1964), Greenland (e.g., Harris, 1932, 1937), Poland (Reymanôwna, 1973), Sweden (Lundblad, 1948), Sardinia (Edwards, 1929), and the Soviet Union (Krassilov, 1977b); however, no caytonialean specimens have been recovered from Gondwana localities. Three disarticulated genera comprise the order, including Sagenopteris (leaves), Caytonia (ovulate organs), and Caytonanthus (pollen organs). Moreover, dispersed pollen grains assigned to Vitreisporites have also been allied with the group. No specimens have been isolated showing organic attachment, and the three definitive caytonialean taxa are held together by features of similarity, the two most prominent of which are identical cuticular morphology and the presence of Caytonanthus pollen grains in the micropyles of Caytonia ovules. In addition to Sagenopteris, Caytonia, and Caytonanthus, Harris (1964) also recognizes the dispersed seed Amphorispermum as caytonialean, but considers the putative

60 61 caytonialean microsporophyll Pramelreuthia Krausel (1949) rather as having its affinities rooted in the Bennettitales. The Caytoniales was originally suggested to share affinities with angiosperms based on the reticulate venation of Sagenopteris leaves, the carpel-like, closed morphology of Caytonia cupules, and the four-chambered pollen sacs of Caytonanthus (e.g., Thomas, 1925). However, most botanists today consider the group to be distinctly gymnospermous and more distantly related to angiosperms, a view corroborated by recent cladistic studies (Crane, 1985a; Doyle and Donoghue, 1986,1987). Thomas (1925) originally described caytonialean pollen organs as Antholithus arberi. However, at that time the generic name Antholithus had been used as a relatively noncommittal genus encompassing the reproductive organs of several different plant groups (see Harris, 1941). Both the genus and the type species were subsequently emended, and renamed Caytonanthus and C. arberi, respectively by Harris (1937), in order to definitively assign them to the Caytoniales. Caytonanthus is characterized by a pinnate morphology, with individual short pinnae arising from a main rachis in a subopposite to opposite pattern. The pinnae may fork a variable number of times and bear terminal synangia, that are typically tetrasporangiate; however, pollen sac number may also vary. In situ pollen is known from most specimens, and is relatively small, bisaccate, and has a single distal sulcus. Several studies have figured and described Caytonanthus pollen grains at the light microscopic level (e.g., Thomas, 1925; Harris, 1932, 1937, 1941, 1964; Konijnenburg-Van Cittert, 1971; Reymanôwna, 1973; Krasslilov, 1977b); however, only three investigations have examined in situ grains with electron microscopy (Krassilov, 1977b; Pedersen and Friis, 1986; Zavada and Crepet, 1986). Krassilov (1977b) evaluated grains of C. tyrmensis from the Tyrma locality, Siberia, Soviet Union with SEM, and demonstrated that both the corpus and sacci have smooth external surfaces 62 and that the sacci are filled with '’endosexinous ridges forming an irregular reticulum." Both SEM and TEM were employed in the studies of pollen from C. arberi, collected from the Cayton Bay locality, Yorkshire, England (Pedersen and Friis, 1986; Zavada and Crepet, 1986), and C. kockii, obtained from Scoresby Sound, East Greenland (Pedersen and Friis, 1986). The exine of C. arberi is described as tectate-alveolate, and is often associated with orbicules and tapetal membranes (Zavada and Crepet, 1986). Pedersen and Friis (1986) suggest that pollen grains of both C. arberi and C. kockii are similar in both external morphology and exine fine structure. Although Pedersen and Friis did not clearly illustrate exine ultrastructure in the cappus region, these authors were able to document faint nexine lamellae in some sections as well as the presence of well-defined endoreticulations within sacci. In the present study, additional Middle Jurassic material of C. arberi from Cayton Bay was examined at the micromorphological and ultrastructural levels. In addition to critically evaluating pollen grains, special attention was focused on the fine structural nature of entire synangia, as well as the main rachis of the pollen organ.

Description The Caytonanthus arberi pollen organs studied here consist of narrow, dorsiventrally flattened rachises, averaging 1.1 mm in width, and show subopposite branching. 'Branches' are short, typically 1 mm in length, and may either bi-, tri-, or quadrifurcate to support the attachment of terminal, elongate synangia (Fig. 46). Entire organs are relatively small, averaging 15 mm in width (synangium to synangium), and although the specimens investigated are not complete in length, most average 20 mm long (Fig. 46). Synangia have rounded ends, are multi-loculate, and measure 1.1 cm in length and 0.42 mm in width (Figs. 46-47). Individual locules are easily delimited at macroscopic levels 63 by the presence of distinct, longitudinal furrows (Figs. 46-47); specimens examined in the present study typically have three or four locules.

Synangia and Contents - Synangia are lined externally by a well-defined cuticle (Figs. 49-51,53, 80). This cuticular covering averages 1.32 pm in thickness and is two-parted; the inner region consists of a system of distinct, darkly-stained fibrils, or reticulations, and is overlaid by a lightly-stained amorphous region (Figs. 51, 53). This two-zoned organization is consistent in all synangia examined and is detectable in both uncleared and cleared specimens (Figs. 49, 51,53). Moreover, component pollen sacs are also lined by cuticles, which are continuous with those lining the external synangium surface (Figs. 49- 50). The occurrence of these internal' cuticles affords the opportunity for identification of individual synangium locules at the fine structural level, and suggests that at least some of the external furrows seen when evaluating gross synangium morphology (Fig. 47) are actually folds in the external cuticles (Fig. 49). Individual locules contain numerous in situ pollen grains, orbicules, and resistant tapetal membranes (Figs. 49-57). Orbicules and tapetal membranes both occur throughout entire synangia and are frequently found intimately associated with pollen surfaces (Figs. 49, 56-57,62). Membranes are thin, averaging 0.05 pm in thickness, and often lamellated (Figs. 54, 57). Membrane fine structure is distinctly different from that of the internal locule cuticles; tapetal membranes are thinner, lack reticulations, and stain more darkly (cf. Figs. 52, 54 & Figs. 51, 53). The membranes may also be slightly irregular (i.e., finely scabrate); scabrae are similar in size and ultrastructure to the scabrae on orbicule surfaces (Fig. 54). Orbicules are spherical with surface sculpturing ranging from psilate to scabrate, and are somewhat variable in size, ranging from 0.32 to 1.33 pm in diameter. 64

Orbicules are hollow, with a homogeneous wall averaging 0.40 |im in thickness (Figs. 52, 54, 57, 63). Pollen grains are relatively small, monosulcate, and bisaccate (Figs. 48, 58,60). Sacci typically show lateral attachment (Figs. 58, 64), although in several grains the sacci are distally inclined. Grains average 23 jxm in length (saccus to saccus) and 15 p.m in width (Figs. 58-60). In polar view, the corpus averages 11 pm in length and 15 pm in width (Fig. 58), while sacci range to 6 pm in length and 15 pm in width (Figs. 58,60). Reliable corpus and saccus height measurements are not available for these grains because of their compressed nature. The proximal wall is generally psilate; however, some grains have a finely ornamented surface (Figs. 58-59). The distal wall is characterized by a broad sulcus extending across the entire width of each grain and, by comparison, is significantly much more scabrate (Figs. 60-61). The sulcus averages 3.7 pm in width. Exine architecture is distinctly two- parted, composed of an inner dark staining nexine and an outer light staining sexine. Overall exine thickness averages 0.65 pm, while the sexine is typically 3.3 times thicker than the nexine, averaging 0.50 pm and 0.15 pm thick, respectively (Figs. 64-67). As for corystosperm grains, the position and plane of section is important when considering exine fine structure. For example, when transverse sections are examined from lateral equatorial positions, the sexine has an infrastructure composed of robust plate­ like units, measuring an average of 0.20 pm in width, extending between a tectum and the nexine. (Figs. 66-67). A homogeneous, basal sexine component is for the most part absent; however, a very thin layer is detectable in some grains/sections (Fig. 69-70). In more medial sections, the plate-like sexine units are significantly shorter, the tectum is thicker, and the overall sexine closely approximates a homogeneous ultrastructure (Figs. 64-65). The tectum averages 0.20 pm in thickness in lateral sections, and is continuous 65 with the outer saccus wall (Figs. 64-68). Sacci superficially appear to be completely infilled with endoreticulations; however, some discontinuity of the endoreticulum is evident in medial sections (Figs. 64-65). Endoreticulations are also robust, having similar dimensions to the plate-like units seen in lateral equatorial sections through the proximal corpus wall (i.e., cappus; Figs. 65-68). Nexine ultrastructure is characterized by the presence of well-defined lamellae (Figs. 69-70). In mature pollen grains, nexine lamellations are generally only detectable in regions of the wall that have undergone some folding (Figs. 67-69). However, several immature grains have also been isolated within synangia, and clearly illustrate lamellations throughout the entire nexine (Fig. 70). In grains of both ontogenetic stages, the lamellae are similar in thickness, averaging 0.03 |im thick. The sulcus is overlaid by a very thin exine layer, with both sexine and nexine layers present here. However, the sexine component is only present in small and patchy amounts (Fig. 68), and results in the scrabrate external morphology of the aperture (cf. Fig. 61). The thin sporoderm layer over the aperture is the result of the gradual thinning of both sexine and nexine wall layers from lateral positions (Figs. 64,68).

Main Rachis — The main rachis of Caytonanthus pollen organs also shows an intricate fine structural organization. It is dorsiventrally flattened and composed of a central region which is lined on both sides with differentially thickened cuticles (Figs. 46, 71-73). Examination of uncleared specimens reveal that the central region represents the former position of cellular tissue that is now highly coalified (Figs. 71-73). Cuticular membranes vary widely with respect to their thickness; for example, the cuticles on both rachis faces are not consistent in thickness, but rather each have thick and thin zones (Figs. 71-73). The rachis cuticle is typically lightly-stained and is three-zoned (Figs. 73, 75). In basal 66 regions, variably-sized and darkly-stained reticulations (extensions), which originate from the central, 'cellular' region, protrude into the cuticle. The relatively large extensions are surrounded by a more diffusely-stained zone, which is overlaid by an amorphous region (Figs. 73,75). Moreover, in some rachis portions, the cuticle may be multilayered (Fig. 74). Unequivocal identification of this pattern of cuticle zonation is corroborated by the concomitant occurrence and ultrastmcture of the same cuticle regions in cleared specimens (Figs. 75, 77). The main rachis is also characterized by the attachment of an ultrastructuraily complex scale-like, epidermal appendage (Figs. 72, 76-79). Each consists of a relatively thick inner, lamellated region overlaid by a thinner reticulate zone (Figs. 76, 78, 79). The lamellae are well-defined, averaging 0.05 |im in thickness, and are present in both noncleared and cleared rachises. Reticulations appear to originate from the outermost lamellae, which are slightly thicker than innermost lamellae (Fig. 79).

Discussion Structural Features of the Synangium and Main Rachis — The present investigation provides the first detailed information on the fine structure of Caytonanthus synangia. Although several early studies on this pollen organ clearly demonstrated the presence of a cuticular covering on the external synangium surface (e.g., Thomas, 1925; Karris, 1932, 1937), these works emphasized gross morphology, particularly regarding general cuticle thickness and the overall shapes of underlying epidermal cells. In all accounts, the cuticle is reported to be thin especially relative to that covering the main rachis, and the cells comprising the epidermis are described as fusiform and hexagonal in shape. Ultrastructural examinations confirm that the synangium cuticle is distinctly thinner than the rachis cuticle, and clearly indicate that the synangium epidermal 'cells' are not well- 67 preserved but rather severely coalified. This determination is based on the fact that the epidermis, as well as all other synangial cellular tissues do not show any type of structural configuration, but are simply manifested as a darkly-stained, amorphous region. One of the most interesting aspects of Caytonanthus synangia cuticles is their fine structural organization. The cuticle is principally composed of a lightly-stained, amorphous material with well-defined, darkly-stained fibrillar units, or reticulations, in its lower portion. This organization is consistent throughout the entire cuticle and in ali synangia sectioned, and provides the opportunity to stracturaliy classify this cuticle as two- zoned, with an inner fibrillar layer and an outer amorphous layer. Fibrillae are relatively irregular in size and distribution, and are basally attached to the underlying, darkly-stained region of coalified tissues. Two interpretations can be advanced to explain the structural nature of the cuticular fibrillae. The first postulates that fibrillae represent folds and/or cracks in the cuticle, resulting from various compression/compaction phenomena, that have become filled with the diageneticaUy altered cellular tissues. Evidence for this hypothesis comes primarily from the fact that fibrillae are relatively random in their distribution. A more likely explanation considers fibrillae as naturally occuring extensions of the formerly present, underlying epidermal cell walls like those observed in numerous extant plant cuticles (e.g., Holloway, 1982). This assessment is based on the similarity in overall ultrastructure and nature of attachment of fibrils with those of extant plants. Regarding the reservations noted above about the relatively irregular distribution of fibrillae in comparison with living analogs, the previous descriptions of fibrillar layers in extant cuticles have principally come from studies of mature leaves. The cuticles of extant reproductive organs have not been as thoroughly researched, and may in fact be significantly less reticulate than those on foliar organs. Moreover, Taylor, Taylor, and Archangelsky (1989) have described cuticular fibrillae at the ultrastructural level in 68

Karir.opîeris leaves, and suggested that their preserv ation is probably the result of their 'embedment' within the thick, resistant cuticle. The presence and identical fine structural appearance of cuticular fibrillae in both cleared and uncleared Caytonanthus synangia support this contention. The fine structural data on Caytonanthus synangia presented here also provide new information on the gross morphology of these structures. Thomas (1925) originally described synangia as elliptical, four-winged "anthers", and considered the short microsporophyll pinnae as androecial filaments which extended into the fused anther sacs to form a central connective. In 1937, however, Harris documented that Thomas' caytonialean "anthers" only superficially resembled the anthers of angiosperms and, principally based on their radial symmetry and nature of attachment to "filaments", suggested that the "anthers" were actually pteridospermous synangia. It is interesting to note that Harris' (1937) cross sectional reconstructions of synangia illustrated a well- defined, central connective (Text-figs. 3D, E) even though his line drawings of actual transverse sections lack such structures (Text-figs. 3A-C). However, in subsequent descriptions, discussions, and reconstructions Harris (1941,1951) clearly emphasized the absence of any type of central tissue core, including a vascular strand. The uitrathin, transverse sections of synangia prepared in the present study also confirm the lack of any type of central 'connective'. The most interesting aspect of synangium morphology about which the ultrastructural data sheds new light is the nature and degree to which individual pollen sacs are fused. In Harris' later interpretations (1951,1964), component pollen sacs of synangia are described and reconstructed as being relatively well-fused laterally, with only approximately thirty to forty percent (present author's estimate) of each pollen sac's external surface exposed. At the time of dehiscence, the individual pollen sacs are suggested to have split open along 69 their lateral margins in the central part of the synangium, while remaining fused in both the proximal and distal synangium ends (Fig. ). Based on these reconstructions it can be inferred that only the externally-exposed epidermal tissue of individual pollen sacs had a cuticular covering. This assessment is based on structural comparisons with other synangiate plant analogs, both fossil seed plants (e.g., Feraxotheca, Halletheca) and pteridophytes (e.g., Scolecopteris), as well as extant pteridophytes (e.g., marratialean ferns, Psilotum), which lack any type of 'internal* cuticle lining the margins of individual pollen sacs/sporangia beyond the point of fusion. If this was the case for Caytonanthus synangia, then the continuous (i.e., enveloping) cuticle that was externally covering each pollen sac would not be expected to be identified deeply within compressed synangia when evaluated in transverse section (Fig. 8lA, C, D). However, the synangia sectioned here reveal that a well-defined cuticle is not only present on the extanal walls of each pollen sac but rather is continuous around the majority the periphery of each pollen sac, and extends well into the central portion of synangia. This type of organization, seen in transverse sections through compressed synangia, suggests that the individual pollen sacs were either not fused at all (i.e., free; Fig. 8lB, E, F) or only slightly-fused centrally . It might be argued that the cuticular layers observed within the central part of synangia may in fact represent some type of resistant, sporopolleninous tapetal membrane, or culture sac, which lined each locule. However, this is clearly not the case; sporopolleninous tapetal membranes are also present and are strikingly different in ultrastructure from the synangium cuticles (see below). Further strengthening this position is the fact that the synangium cuticles are fine structurally identical to the unequivocal cuticle on the pollen organ's main rachis. Moreover, combined examinations of both cleared and uncleared synangia indicate that tapetal membranes are located between the external and internal 70 pollen sac cuticles and their position serves to delimit the boundaries of the pollen sac locules (Fig. 80). In addition to providing paramount, comparative data on cuticle fine structure, the present evaluation of Caytonanthus rachises also contributes important information about several other morphological aspects of this structure. It has long been emphasized that the the rachis is dorsiventrally flattened, with differentially-thickened upper and lower cuticles (e.g., Thomas, 1925; Harris, 1941, 1964). Uitrathin sections confirm the dorsiventrai nature of the rachis; however, they indicate that there is not a clear distinction in thickness between the two cuticles, but rather each has regions that vary in thickness. Moreover, early light microscopic studies document the presence of trichome bases at irregular intervals on rachises. In the present investigation, epidermal appendages on the rachis have also been identified; however, those elucidated here are more like scales than trichomes. Of particular interest is the ultrastructural complexity of these structures. They show a highly lamellated basal region with fibrils extending off of the outermost lamellae into an lightly-stained, amorphous layer that is ultrastructuraily similar to the amorphous component of the rachis and synangium cuticles. Moreover, it appears that the lamellated layer is also surrounded by a matrix of the lightly-stained cuticular layer. It is possible that the lamellated layer represents the cellulosic wall component of the epidermal appendage. An explanation for its exceptional preservation in comparison with the now severely coalified cellulosic tissue of the adjacent rachis stems fi'om the fact that it is apparently completely embedded within, rather than simply surrounded by, the amorphous (i.e., cutinous) layer which may have served as a preserving medium (see above).

Structural Features of Pollen, Orbicules, and Tapetal Membranes — The high degree of preservation exhibited by the synangium and rachis cuticles is accentuated when observing 71 structures ^within synangia. Several important features have been elucidated about pollen ultrastructure in the present study, and originate from the fact that grains are especially well-preserved, showing differentially-stained sexine and nexine layers. Significantly, the sexine inffatectal layer has been shown not to be alveolate throughout as was previously suggested (Zavada and Crepet, 1986), but rather the 'alveolae' gradually thin toward the median of the cappus resulting in a homogeneous structure in this region. In peripheral regions, the alveolae' grade into the endoreticulations within sacci. In fact, the infrastructural alveolae observed in the cappus simply represent the saccus endoreticulations along the positions where the saccus attaches to the corpus. This type of organization is also present in other Mesozoic bisaccate grains, including the pollen of glossopterids (Zavada, 1991) and corystosperms (Chapter IV). Moreover, the alveolae' of the Caytonanthus arberi grains sectioned here are strikingly more robust, especially in width, in comparison with those of grains sectioned by Zavada and Crepet (1986). However, they are more similar in overall size to the saccus endoreticulations of the grains sectioned by Pedersen and Friis (1986). The grains sectioned here also exhibit well-defined nexine lamellae, and represent the best and most comprehensive documentation of these sporoderm lamellations in the group. Although Pedersen and Friis (1986) described the nexine of C. arberi as lamellate, the individual lamellae are not convincingly distinct in their figured micrograph (Fig. 10). In fact, in most of the mature grains sectioned here lamellae are also either relatively faint or only detectable in grain regions that have undergone some folding. However, fortuitous discovery of grains preserved in early ontogenetic stages illustrates exquisite lamellae throughout the entire nexine. The reasons for the relative absence of distinct nexine lamellae in mature grains are unclear. It is unlikely, however, that lamellae disappear in the ontogeny of these grains like those in some angiosperms. This determination is based on 72 the fact that lamellae are also present in mature grains, but only detectable in folded portions of the wall. A more likely explanation for the absence of readily identifiable lamellae in mature grains is that these structures have undergone some chemical modification in development and are affected differently by preservational processes. Interestingly, based on early examinations in transmitted light Harris (1941) suggested that synangia may contain 'tapetal membranes'. He wrote: "Besides the outer cuticle, most specimens provide vestiges of an inner obscurely cellular granular membrane to which the pollen grains often adhere; perhaps this membrane is the fatty matter of the tapetum hardened in preservation." However, Zavada and Crepet (1986) were first to unequivocally demonstrate orbicules and resistant, tapetal membranes at the ultrastructural level, although they did not recover any immature grains. Documentation of immature grains within synangia, along with the concurrent presence of orbicules and tapetal membranes, also provides the opportunity to address some aspects of sporoderm development. Although numerous ontogenetic stages have not yet been isolated fi’om the material sectioned here, those immature grains examined thus far indicate that the robust alveolar units and the nexine lamellae are fairly well-developed prior to complete synthesis of the tectum. Moreover, the surface scabrae observed on mature grains are similar in size and morphology to those on orbicule surfaces, as well as to the sporopolleninous units associated with tapetal membranes. It is also interesting to note that the process of clearing specimens in nitric acid had some minor effects. Although clearing does not affect the gross ultrastructure of either pollen grains, orbicules, or tapetal membranes, as determined by comparing both cleared and uncleared specimens, it does affect the way in which these structures are stained. In cleared specimens, grains, orbicules, and membranes apparently have a greater affinity for heavy metal stains, staining darker than those in uncleared synangia. 73

Systematic Implications — The new information now available on the fine structure of Caytonanthus arberi bears significantly on current inteipretations of the group. Most importantly is the new data on gross morphology of synangia. The continuity of cuticles enveloping individual pollen sacs suggests that the sacs were not distinctly fused into a synangium, but were rather free. If this new reconstruction is accepted, then Caytonanthus pollen organs are more similar morphologically to other Mesozoic pteridosperms with free pollen sacs (i.e., Glossopteridaies, Corystospermaies, Peltaspermales). Caytonanthus could essentially represent a reduced Pteruchus-typc pollen organ, where the peltate head is reduced in size and bears fewer pollen sacs. The permineralized, Antarctic Pteruchus-likc pollen organs may help clarify this question; it will be especially interesting to leam whether or not the individual pollen sacs are fused at all basally, or are completely free. As noted above, Caytonanthus pollen ultrastructure is similar to that of the Antarctic corystosperm grains, also sectioned here, with regard to the 'alveolar' cappus infrastructure that originates from the saccus endoreticulations. The most notable difference between these grain types is the size/extent of the endoreticulations; however, this may be in part due to overall differences in grain sizes. Moreover, the fact that Caytonanthus pollen sacs are unfused undoubtedly has important implications concerning phylogenetic analyses (e.g., Doyle and Donoghue, 1986,1987). The presumed synangiate condition within the group has been characterized as apomorphic. However, the polarity assigned to this character will require modification and réévaluation, and thus may alter the topology of resultant cladograms. It will be interesting to leam how this will affect the relative terminal position of the Caytoniales in various phylogenetic trees and assessments of its relationships. CHAPTER VI BENNETTITALES

Introduction The Bennettitales (=Cycadeoidales) is an extinct group of cycadophytes which flourished during the Mesozoic (Upper Triassic-Lower Cretaceous). Although the group has been shown in recent years to be quite diverse, it is best known from silicified trunks with in situ reproductive organs (Cycadeoidea and Monanthesia) from Lower Cretaceous sediments of North America, and a variety of compression-impression foliage specimens (e.g., Pterophyllum, Ptilophyllum, Zamites), wide-ranging in both age and geographic occurrence. Although identical in gross morphology to foliage of the Cycadales, bennettitalean leaves are unequivocally distinguished from the former by their syndetocheilic stomata (e.g., Thomas and Bancroft, 1913). The group is further characterized by the presence of interseminal scales which separate individual ovules (e.g., Harris, 1969; Wieland, 1906). These two features have played a critical role in the establishment and recognition of the group as a natural unit (e.g.. Crane, 1988). More recently, additional phylogenetic emphasis has been placed on the morphology/anatomy of Cycadeoidea ovulate structures, especially regarding possible homologies with interseminal scales, and its implications on interpreting pleisiomorphic angiosperm character states (D. Taylor, 1991).

74 75

Phyîogeneîically, the Bennettitales is extremely important in that it has been allied over the years with several orders of "higher" seed plants, including Cycadales, Gnetales, and angiosperms. However, the suggested close affinities with Cycadales are generally dismissed as superficial and the two groups are now thought to be phyîogeneîically more disparate (see Crane, 1988 and references therein). Cladistic analyses, on the other hand, have supported the proposed relationship between Bennettitales, Gnetales, and angiosperms, and also linked these three groups with the Pentoxylales in a single, highly- derived clade (Crane, 1985a, b; Doyle and Donoghue, 1986, 1987). Narrower cladistic analyses focusing solely on the Bennettitales have also been carried out (Crane, 1985a, 1988), and further suggest that Triassic members of the group are the most pleisiomorphic, while Jurassic and Cretaceous specimens are relatively derived. Bennettitalean reproductive organs are relatively diverse in terms of their gross morphology as well as their general organization, in that both monosporangiate and bisporangiate forms are known. The best known pollen-bearing organs are those of Jurassic and Cretaceous specimens; monosporangiate forms include Weltrichia spp. (e.g., Harris, 1969; Sitholey and Bose, 1953,1971; Delevoryas, 1991), while the bisporangiate condition is known in Cycadeoidea spp. (e.g., Wieland, 1906, 1916; Delevoryas, 1963, 1965, 1968a, b; Crepet, 1974), Williamsoniella spp. (e.g., Thomas, 1915; Harris, 1969) anàAmarjoiia dactyloîa (Bose, 1966; Sitholey and Bose, 1971; Bose, Banerji, and Pal, 1984). These fossils consist of several dorsiventrally-flattened, often pinnate, microsporophylls that are attached in a whorl to a receptacular structure. Microsporophylls may either be fused basally into a distinct cup or attached singly around ovulate parts, and bear well-defined, adaxial synangia consisting of a variable number of pollen sacs (see Crane, 1986). 76

Triassic pollen organs are principally known from disarticulated microsporophylls, and are strikingly dissimilar in morphology to those of Jurassic and Cretaceous specimens. In the Triassic forms, the microsporophylls appear to have been arranged in a helical pattern, and the pollen sacs were apparently borne individually, rather than fused into synangia (see Crane, 1986). Several of these putative bennettitalean microsporophylls include: Leguminanthus siliquosus (Krâusel and Schaarschmidt, 1966), Haitingeria krasseri (Krausel, 1949), Leuthardtia ovalis (Krausel and Schaarschmidt, 1966), and Bennettistemon amblum , B. bursigerum, and B. ovatum (Harris, 1932). Crane (1986, 1988) suggests that these Triassic bennettitalean pollen organs are primitive within the group, based primarily on their helical phyllotaxy and nonsynangiate pollen sacs. However, the cladistic investigations which have incorporated these taxa have only been of a preliminary nature, and their definitive phylogenetic affinities remain somewhat equivocal (Crane, 1988). Relatively few studies have addressed the nature of bennettitalean pollen beyond a cursory description of grains as seen in transmitted light, and the majority of these have been illustrated in the original works on the above pollen organs. However, pollen grains are not known from several Weltrichia species, including VT. singhii, W. polyandra, and W. santalensis from the Rajmahal Hills, India (Sitholey and Bose, 1953,1971), nor from W. ayuquilana from Oaxaca, Mexico (Delevoryas, 1991). Bennettitalean pollen is typically described as relatively small, prolate in shape, and having a single, longitudinally- oriented sulcus. However, Harris (1974) described the pollen of Williamsoniella lignieri as circular in outline and monoporate. Harris’ study was of further significance because it addressed several effects of varying degrees of tension and compression on palynomorphs. Significantly fewer grains are known at the ultrastructural and micromorphological levels; only three investigations have employed electron microscopy in 77 their examinations of bennettitalean pollen. Taylor (1973) evaluated grains of Cycadeoidea dacotensis and demonstrated two differentially staining exine layers, but was unable to ascertain information about sexine/nexine fine structure. However, Taylor convincingly refuted early suggestions of cellular microgametophytes within C. dacotensis pollen by clearly documenting that these represented numerous exine folds. Interestingly, the pollen wall of Williamsoniella has also been characterized as lacking ultrastructural detail (Crepet, personal communication). More recently, however, pollen of the Triassic, dispersed microsporophyll Leguminanthus siliquosus has been shown to have a tectate-granuiar sexine and faintly lamellate nexine (Ward et al., 1989). In the present investigation, the pollen of Cycadeoidea dacotensis was reevaluated, along with grains of several other Cycadeoidea species using both SEM and TEM.

Description The microsporangiate parts of Cycadeoidea cones are extremely complex in their anatomy and morphology, and have been critically investigated by several authors (e.g., Wieland, 1906; Delevoryas, 1968a; Crepet, 1974). However, only a few salient features of the microsporangiate organs will be presented here. These principally deal with the anatomical nature of synangia, and bear upon the present study of Cycadeoidea pollen. Bisporangiate cones have elongate, pinnate microsporophylls, each bearing numerous synangia (Fig. 82). Synangia are reniform and multiloculate, typically consisting of 22 elongate locules (Figs. 82, 84). Locules may contain numerous in situ pollen grains (Figs. 86-87). The synangium wall is multilayered and relatively complex (Figs. 83-85). It is composed of an outer thick-walled, single palisade layer, three to five middle layers of thick-walled, prostrate cells, and an internal thin-walled, single cell layer which is continuous with the locule septa (Figs. 84-85). A pad of relatively thin-walled 78 parenchymatous tissue characterizes the attachment of synangia to microsporophylls (Figs. 82-83). Pollen grains are typically prolate and average 25 (xm in length and 12 |xm in width (Figs. 87-88). A few spheroidal grains have also been identified (Fig. 90). Surface sculpturing ranges from punctate to scabrate to psilate. Although the majority of grains examined were entirely punctate (Figs. 88-89), several showed punctate ornamentation with psilate 'islands' (Fig. 91) while others were more prominently psilate. The varying degree of wall smoothness may be preservational in origin. Uitrathin sections of pollen grains, as well as observations in transmitted light, indicate that grains are monosulcate

(Figs. 8 6 , 92, 101). The gross morphological nature of the aperture is more difficult to ascertain when observing pollen with SEM because grains are typically highly invaginated, with folds especially prominent in the apertural region (Figs. 8 8 , 98; see also Figs. 99- 100). The exine of Cycadeoidea poUen is two layered, with individual layers clearly delimited from one another by their differential staining properties (Figs. 92-97). The overall exine averages 0.73 pm in thickness and is composed of an outer sexine which generally stains lightly throughout, and an inner darkly staining nexine (Figs. 92-95). The sexine averages 0.50 pm in thickness and is typically 2.2 times thicker than the underlying nexine. Ultrastructuraily, the sexine has a stratified architecture, consisting of two distinct zones (Figs. 93, 95). The internal stratum is granular and measures, on average, 0.36 pm ,n thickness. This granular layer is variable in density with regard to packing of individual granules, with only a small amount of lacunal space between granules (Figs. 93, 95). Granules are constant in size, averaging 0.06 pm in diameter. In most grains, granule packing is relatively regular (Figs. 93, 95); however, some grains show less space and tighter packing of granules, which results in the sexine stratum superficially resembling a 79 homogeneous zone (Figs. 94,96). The latter case more than likely represents variation in preservation (i.e., differential compaction and folding) rather than true structural variation. The external sexine stratum is homogeneous in organization and stains slightly lighter than the granular stratum (Figs. 93-96). This homogeneous layer averages 0.14 |im in thickness and may or may not be completely continuous over the granular layer (Figs. 93- 94). In fact, the variability observed in surface sculpturing (i.e., psilate to punctate) is the result of structural variation of the homogeneous sexine layer (i.e. degree of continuity). In grains, or portions of grains, that are psilate, the outer homogeneous layer is entirely continuous over the granular layer (Fig. 94), while in scabrate and punctate grains, or exine regions, the homogeneous layer shows varying degrees of thinning (Figs. 93-94, 97). The nexine averages 0.23 |Xm in thickness and appears significantly more dense in the electron beam than the sexine (Figs. 92-97). Well-defined nexine lamellae are absent in these grains (Figs. 93-95); in fact, even structurally discernable 'faint lamellae', which often occur in nexine apertural regions (e.g., Sahnia pollen, see Chapter Vm), were absent in all Cycadeoidea grains examined. In some grains, however, several lamellated structures are seen just internal to the nexine (i.e., in the grain lumen) and superficially resemble inner nexine lamellae (Fig. 97). Albeit, these structures more than likely represent either some type of debris or poorly preserved intines, because a well-defined nexine is also contiguously present The apertural region is relatively invariable in organization. A very thin sexine layer, notably lacking in a granular zone, overlies the nexine in apertural regions (Figs. 96,102). This is the result of a gradual, lateral thinning of the overall sexine in nonapertural regions at the margins of the sulcus (Figs. 92, 96, 102). Specifically, the granular sexine component gradually thins to a point where it becomes completely absent, while the 80 homogeneous stratum is relatively constant in thickness over the thinning granular zone and then abruptly thins just at the aperture's margin (Figs. 92, 96). An extremely thin layer of the homogeneous sexine stratum overlies the aperture per se. The nexine is similar in thickness below the aperture to that in nonapertural regions and also lacks lamellae here as well (Figs. 92, 96, 101-102). Definitive orbicules and resistant membranes are not associated with any of the pollen grains examined. Several grains, however, had some residual debris closely associated with exines that superficially resemble the aforementioned structures (Figs. 101-102).

Frass — Pellets of frass are frequently found in association with synangia (Fig. 103). The frass is composed of a heterogeneous assemblage of plant material (Figs. 104-106). These remains principally include darkly-stained, fractured cell walls that exhibit varying stages of degradation (Figs. 105-106). Pollen exines are notably absent within the sectioned frass conglomerates.

Discussion As with the polypiicate, gnetalean palynomorphs sectioned in the present study (see Chapter VII), one of the most distinguishing features of the Cycadeoidea spp. pollen grains examined here is their excellent preservation. Grain surfaces show very little damage, and differentially-stained sporoderm layers are distinct and easily detectable in aU grains sectioned. The sexine is consistently composed of two well-defined strata, and stains lightly relative to the underlying nexine. Although the nexine stains exceptionally well, distinguishable lamellae in this exine layer have never been observed. This is particularly interesting, especially taking into account the overall exquisite preservation exhibited by these grains. However, it is still possible that this situation is due to 81 preservational phenomena. In particular, in the two other well-preser/ed grains investigated here, (i.e., gnetalean [Equisetosporites spp.] and caytonialean [Caytonanthus arberi]) the nexine is also darkly-stained and only exhibits faint lamellae, which are typically restricted to the nexine/sexine interface, at best in most grains sectioned. However, in both of these grain types the nexine in fact consists of distinct lamellae throughout, which were only detectable under atypical conditions. In the gnetalean grains, lamellations were identified in a grain that had undergone relatively severe preservational folding resulting in the separation of individual lamellae from one another, presumably due to varying degrees of tension and contraction. In Caytonanthus pollen on the other hand, continuous lamellations throughout the nexine were fortuitously identified in a relatively rarely encountered immature pollen grain. Consequently, it is possible that Cycadeoidea pollen also has a distinctly lamellate nexine; even though numerous grains were sectioned in the present investigation, the circumstances under which lamellae would be detectable may have not yet been encountered. Sexine organization is also interesting. The sexine is two-zoned, composed of an outer, homogeneous layer, interpreted as a tectum, and an infrastructural granular layer. The tectum is continuous, with the exception of intermittent thin areas that correspond with surface punctae; however, the tectum is not completely absent within the punctae, but rather is extremely thin in these regions. Moreover, the tectum is well-defined, and easily delimited from the underlying granular layer. In the lower portion of the infratectum, the granules are directly contiguous with the nexine. This is significant regarding comparisons with other tectate-granuiar grains in which the granular infratectum may be separated from the nexine by a homogeneous layer (foot layer sensu some authors) of variable thickness. Two other groups investigated in the present study have pollen with a tectate-granuiar exine, including the Brazilian ephedroid grains (Gnetales) and Sahnia laxiphora grains 82

^Pcîitcxylalcs^. UltiaStructurâlly) Cycadcoaicu pollen xs similsr to uotli of tîicsc ^xain types in that the granular sexine layer is in direct contact with the nexine; however, it is somewhat variable in terms of tectum fine structure. Both Cycadeoidea and the ephedroid grains have a relatively distinct tectum, while the tectum of Sahnia pollen is less well- defined, with its lower boundary gradually grading into the underlying granular infratectum. This difference in tectum architecture is interesting because the pollen of Cycadeoidea is more similar to the pollen of Sahnia rather than to the ephedroid grains in terms of gross morphology (i.e., lacking exine ribs), surface morphology, and aperture type. Perhaps a more salient comparison is one between Cycadeoidea pollen and the only other cycadeoid grain which has been examined at the ultrastructural level, Leguminanthus (Ward et al., 1989). Pollen of Leguminanthus is relatively small, averaging 23 |xm in length and 13 pm in width, monosulcate, and has a psilate surface ornament. The exine consists of lightly-stained sexine and darkly-stained, faintly lamellate nexine. The sexine is also composed of a homogeneous tectum and granular infratectum; however, the granules are significantly larger in diameter and generally more densely packed than those in Cycadeoidea. Ward et al. (1989) also suggest that a homogeneous footlayer, resulting fi'om the fusion of basal granules, overlies the darkly-stained nexine. Moreover, in comparison with Cycadeoidea the tectum of Leguminanthus pollen is much thicker and also more weakly-defined in that it grades into the underlying granular zone. Based solely on ultrastructural data, the pollen of Cycadeoidea and Leguminanthus are relatively disparate. The comparison of palynological features of these two bennettitalean taxa generally supports Crane's (1986, 1988) suggestion that the Triassic pollen organs are more primitive than fossils recovered from Jurassic and Cretaceous rocks. For example, several apomorphic characters observed in Cycadeoidea pollen include a finely-perforate 83 tectum (i.e., thin areas in punctae), a tectum with a well-defined lower boundary, an infratectum composed of relatively small and uniform granules, and a nonlamellate nexine, although this final character is somewhat questionable. However, some of these exine characters may correlate with other reproductive processes, such as pollination. It has been suggested that Cycadeoidea was self-pollinated by as these insects bored and foraged through the trunk-embedded cones (Crepet, 1972, 1974). It is interesting to note that the frass pellets sectioned here do not contain distinguishable exines. This, however, does not necessarily reflect that pollen was not eaten/transported by the beetles. For example, it is possible that the degree of sporopollenin preservation was poor after grains had passed through the insect’s gut. Numerous gaps exist m our understanding of bennettitalean pollen, with high resolution data now available for only two taxa which are relatively widely separated in terms of their stratigraphy. In order to bridge those gaps, continued ultrastructural examinations of pollen from the other poorly known Triassic and Jurassic bennettitalean pollen organs are needed. CHAPTER Vn GNETALES

Introduction Few botanists would argue that the Gnetales represents the most unique and enigmatic group of extant gymnosperms, and perhaps, of all extant plants. The order comprises three living genera. Ephedra, Gnetum, and Welwitschia, which although distinctly different in both habit and habitat, share an array of reproductive and vegetative characteristics (e.g.. Martens, 1971; Crane, 1988). Recent phylogenetic analyses (Crane, 1985a, b, 1988; Doyle and Donoghue, 1986, 1987) suggest that the Gnetales is monophyletic, and corroborate early suggestions that many of the group's unifying features are apomorphic. In fact, of all extant gymnosperms the Gnetales most closely approximates angiospermy', especially taking into account the recent fine structural documentation of double fertilization' in Ephedra (Friedman, 1990a, b). Whether or not all of the derived features observed in the group reflect true phylogeny remains somewhat speculative; for example, the petiolate, reticulate-veined leaves in Gnetum are thought to be manifestations of convergence rather than synapomorphies with angiosperms (see Crane, 1988). Nevertheless, current cladistic studies (e.g.. Crane, 1985a, b, 1988; Doyle and Donoghue, 1986,1987) group the Gnetales with the following groups in a clade of highly- derived seed plants (=anthophytes): Bennettitales, Pentoxylales, and angiosperms.

84 85

Although it is clear that all three extant genera are relatively advaneed for gymnosperms, very little is known about morphological evolution in the group over geological time and space. The megafossil record of the Gnetales is extremely sparse, with only one described fossil unequivocally assigned to the group. Drewria potomacensis is known from the Early Cretaceous (cf. Aptian) Potomac Group of Virginia (Crane and Upchurch, 1987). Another fossil, Eoantha zherikhinii KrassUov (1986) from Barremian-Aptian sediments of Mongolia, has been suggested by Crane (referred to as Eoanthus zherikhinii', 1988) to be a definitive gnetophyte; however, Krassilov (1986) originally advanced the idea that Eoantha either represents a "protoangiosperm" or some type of an intermediate morpho-type between protoangiosperms and gnetophytes. Moreover, Crane and Upchurch (1987) and Crane (1988) also suggest that several other Cretaceous (Consopermites hakeaefolius [Velenovsky and Viniklâï^ 1926], Cyperacites potomacensis [Berry, 1911], Casuarina covilli [Ward, 1895], Gurvanella dictyoptera, Cyperacites sp., and ''Potamogeton-\\k& spike" [Krassilov, 1982], Baisia hirsuta [Krassilov and Bugdaeva, 1982], and Montsechia vidali [Blanc-Louvel and Barale, 1983]) and Triassic (Hexagonocaulon minutum [Lacey and Lucas, 1981], Schilderia [Daugherty, 1941], Dechellyia gormanii and Masculostrobus clathratus [Ash, 1972]) megafossils exhibit some morphological similarities with Drewria and Eoantha and may also have gnetalean affinities. The palynological record of the Gnetales is more extensive than megafossil evidence. Dispersed polyplicate palynomoiphs that are morphologically similar to pollen of Ephedra and Welwitschia, at least at the light microscopic level, occur in sediments ranging from Lower Permian to Recent (e.g., Wilson, 1959; Scott, 1960; Bharadwaj, 1963; Pocock, 1964; Stover, 1964; Srivastava, 1968; Azéma and Boltenhagen, 1974; Elsik, 1974; Lima, 1980; Zavada, 1984, 1990b; see Crane, 1988; Pocock and Vasanthy, 1988). The most prominent similarities between these sporae dispersae, "ephedroid" grains and those of 86

Ephedra and V/ehvitsckia are their prolate shapes and ribbed, or striate, surfaces. Several genera have been described, differing from one another by such features as number of ribs, orientation of ribs (i.e, longitudinal or spiral), morphology of grain ends (i.e., more rounded or more pointed, etc.), and attachment of flap-like appendages (i.e., auriculae). The two most commonly encountered grains are those assigned to Ephedripites Bolkhovitina (1953) and Equisetosporites (Daugherty) Pocock (1964). Other polyplicate palynomorphs are recovered less frequently from palynological preparations; examples include grains referred to as Tornopollenites, Hamiapollenites, Singhia, Gnetaceaepollenites, Steevesipollenites, Welwitschiaptes, Regalipollenites, and Multimarginites. Moreover, relatively young "ephedroid" palynomorphs (i.e. Tertiary) have often been assigned to species of Ephedra (e.g., Wodehouse, 1934, 1935; Scott, 1960). It is interesting to note also that the appressed guard cells on some Pleistocene epidermal remains superficially resemble fossil Ephedra pollen (Elsik, 1975). Ephedroid grains have also been found along with three of the megafossils noted above. Although microsporangiate organs have not been identified for Drewria potomacensis, both individual and large masses of polyplicate pollen grains were recovered associated with the vegetative and ovulate organs (Crane and Upchurch, 1987). In fact, the association of these grains was one criterion for assignment of D. potomacensis to the Gnetales. The firet definitive association of Ephedripites grains with any megafossü comes from macerations of in situ grains within the pollen chambers of Eoantha zherikhinii ovules (Krassilov, 1986). In addition, in situ grains assigned to Equisetosporites chinleanus have been isolated from Masculostrobus clathratus, the pollen cones associated with Dechellyia gormanii, from the Chinle Formation (Upper Triassic) of Arizona (Ash, 1972). However, pollen wall fine structure of the Arizonan Equisetosporites grains is not characteristically gnetalean, and suggests other affinities (see below). 87

The poilerx of all three extant genera has been studied from a variet>' of perspectives. Several early investigations focused on describing the stages of microsporogenesis and nticrogametegenesis in Ephedra, GnePam, and Weh^ntschia at the gross morphological and anatomical levels. A recapitulation of these studies is beyond the scope of this work; however, the reader is directed to Martens (1971 and references therein) for the most comprehensive review. Mature pollen of Ephedra and Welwitschia is similar in its elliptic shape, overall size, and occurrence of longitudinally-oriented, psiiate exine ribs. However, pollen of Welwitschia has a distinct sulcus, whereas grains of Ephedra are generally reported to be inaperturate, but have thin areas in the exine between ribs (i.e., furrows or plicae). Several studies have addressed the nature of grains from these two genera as seen in transmitted light, especially for the purposes of morphological comparisons with fossil polyplicate grains (e.g., Wodehouse, 1935; Steeves and Barghoom, 1959; Wilson, 1959; Scott, 1960; Bharadwaj, 1963; Huynh, 1974; Kedves, 1987). Pollen wall fine structure (i.e., stratification) is also very similar in the pollen of Ephedra (Afzelius, 1956; GuUvâg, 1966a; Ueno, 1960a; Van Campo and Lugaidon, 1973; Zavada, 1982, 1984; Hesse, 1984) and Welwitschia (Gullvâg, 1966a; Ueno, 1960a; Hesse, 1984; Kedves, 1987). The exine is composed of a lamellated nexine, and a two- zoned sexine consisting of a granular infrastructural layer and a thick tectunu Both sexine components are either very thin or absent in the furrows. Gnetum pollen is strikingly different from that of Ephedra and Welwitschia, in that the former is spherical, inaperturate, and has finely spinulose surface sculpturing. Exine stratification, however, is generally similar. The exine also consists of a lamellate nexine and a two-layered sexine. Albeit, both the tectum and granular infrastructural layer are continuous and extremely thin by comparison, only thickening in the spine regions (Gullvâg, 1966a; Hesse, 1980; Zavada, 1982,1984). 88

Of the îîüiiiCrCüS gcncrâ of fossil cphsdroid. ^ & ln S , Only thicO hd.V6 been êXEinineu St the ultrastructural and micromorphological levels, and those that have been studied have come from Laurasian localities. Trevisan (1980) sectioned grains of Ephedripites sp. from the Lower Cretaceous of Italy. She found the exine of these grains to consist of an inner lamellated component (=nexine) and an outer complex, five-layered component (=sexine) which formed the "ridges and valleys" of the sporoderm. Equisetosporites chinleana grains from Arizona were initially investigated by Zavada (1982, 1984). The exine was reported to be three-layered, composed of a thin, lamellated inner layer (foot layer sensu Z^avada), an infrastmctural columeUar layer, and a thick homogeneous tectum which forms the exine ribs. More recently, however, the Arizona material of E. chinleana has been shown to be more diverse regarding both surface sculpturing of ribs and exine ultrastructure. Pocock and Vasanthy (1988) have emended E. chinleana to include the new species Cornetipollis reticulata. Cornetipollis reticulata is principally characterized by foveoreticuately ornamented ribs, larger columellae, and a nonlamellate nexine. Equisetosporites chinleana, on the other hand, is redefined as having psiiate ribs and a distinctly lamellate nexine. In the present investigation, the first ephedroid palynomorphs from Gondwana sites were examined at the micromorphological and ultrastructural levels.

Til A C * ^ B

Although six genera of polyplicate pollen grains are known from Santana sediments (Fig. 107; Lima 1978b, 1980), specimens of Equisetosporites (sensu Lima) are the most abundant in palynological preparations (Fig. 107). For this reason, grains assigned to this genus have principally been studied in the present investigation. 89

Grains are elliptic, average 41 pm in length and 13 prn in vvidtli, and have a variable number of plicae, typically ranging from 5 to 11 as seen in polar view (Figs. 108, 111). Plicae extend the majority of the entire grain lengths, but do not reach to the grain tips (Figs. 108, 110-111). Surface ornamentation of plicae ranges from psiiate to slightly scabrate (Figs. 109-110). Exine surfaces also exhibit some pitting, with pits of varying sizes (Fig. 117); however, this is more than likely represents an artifact of preservation. The overall exine averages 1.04 pm in thickness and is two-parted, with easily distinguishable sexine and nexine components, each having distinct affinities for heavy metal stains. The sexine generally stains lightly while the nexine stains darkly (Figs. 112- 113). Interestingly, however, the nexine of some grains does not stain very well at all and appears significantly less dense than the sexine of those grains in the electron beam. Moreover, the sexine of those grains stains more darkly than the sexine of grains with the typical darkly-stained nexine (Figs. 115-116). The sexine averages 0.63 pm in thickness and is generally 1.5 times thicker than the nexine. The sexine is stratified into two principal layers, including an inner granular layer and an outer homogeneous layer (Figs. 115-116, 120-121). The granular stratum averages 0.35 pm in thickness and is directly contiguous with the underlying nexine (Figs. 115-116, 120-121). Granules range in size from 0.09 to 0.26 pm in diameter, typically with smaller granules located near the nexine and larger granules near the outer, homogeneous sexine layer (Figs. 116,120). The small granules gradually grade into the larger granules which appear to fuse with the homogeneous layer (Figs. 116, 120). Some variation with regard to granule density (i.e., density of granules relative to lacunal space) also occurs between pollen grains (Figs. 114, 116,122). The homogeneous layer averages 0.24 pm in thickness. The nexine is relatively thick, averaging 0.30 pm in thickness (Figs. 115-116, 120-

1 2 1 ). In the majority of grains examined, lamellae are absent from the nexine except in 90 regions near the furrows between plicae (Figs. 121, 124) or in portions of the grain exhibiting some folding, and then usually near the interface with the sexine (Fig. 124). However, in several other grains that have undergone more severe preservational folding, individual lamellations in the nexine are significantly much more prominent (Figs. 122- 123). On average, lamellae measure 0.01 |im in thickness. The fine structure of the exine in regions associated with sporoderm furrows is especially interesting. Both sexine and nexine wall components are significantly thinner immediately below the furrows, averaging 0 . 1 0 and 0.16 pm, respectively, in these regions (Figs. 113-114, 118-119). Sexine architecture here exhibits gradual, lateral thinning of its granular stratum in marginal regions of the plicae, eventually thinning to a point where granules are completely absent over the furrow itself (Figs. 118-119). The homogeneous sexine layer does not appreciably thin over the plicae margins in concurrence with the underlying granular layer, but rather thins only slightly and abruptly just at the furrow (Fig. 119). This now slightly thinner homogeneous sexine layer overlies a now thinner nexine layer, which has also gradually thinned, at the furrow itself (Figs. 118- 119). The homogeneous sexine layer over the furrow is typically folded to a small degree, having a hinge-like' appearance (Figs. 117-119). An additional sexine layer is also present in regions of wall furrows. It occurs external to the homogeneous sexine stratum and is also homogeneous, but stains significantly more lightly (Fig. 119). Interestingly, this additional layer is thicker over marginal regions of the plicae and gradually thins as it extends toward the crests of the ribs (Fig. 119).

Discussion Structural Features of the Exine — The most striking feature of the Brazilian polyplicate grains is the fact that they are exquisitely preserved at the ultrastmctural level 91 and exhibit well-stained exine layers. A darkly-stained nexine and lightly-stained sexine generally characterize the sporoderm of these ephedroid grains. This type of differential staining is the most typically observed situation in fossil pollen, especially when grains have been stained with potassium permanganate in addition to uranyi acetate and lead citrate. Interestingly, however, some of the sections evaluated in the present study apparently did not readily stain with potassium permanganate, although contiguous sections on the same grid did. These sections show an opposite staining reaction, with the nexine staining lighter than the sexine. Although sections that are reasonably well-stained with potassium permanganate illustrate better overall contrast in the electron beam, the opposite staining sections also afford important information. In particular, the infrastructural granules are actually more easily resolved in the sections with a more darkly-stained sexine. Interestingly, however, nexine lamellae are generally not well-defined, nor easily detectable in either type of stained sections. When lamellae are detectable, they are typically faint, occur in irregular intervals, and are most pronounced in outermost regions of the nexine (i.e., at the nexine/sexine interface). The relative absence of nexine lamellae in these grains is similar to most of the other Mesozoic grain types sectioned for this study; although they are markedly different from the pollen of all three extant gnetalean genera which have very distinct lamellated nexines. However, it is clear that the nexine in the sectioned Brazilian grains is unequivocally lamellate throughout. Grains that have undergone a relatively high degree of preservational folding, but have not been altered either diagenetically or thermally, illustrate definitive lamellae that have individually separated from one another. An especially interesting structural feature of the Brazilian grains is the nature of the pollen wall in regions of the plicae. In these regions, the sexine has gradually thinned to a 92

single, homogeneous layer that forms a folded, hinge-like structure directly over the furrow. Analogous sexine structures have not been reported in any of the other fossil polyplicate grains that have been sectioned to date (i.e, Ephedripites: Trevisan, 1980; Equisetosporites: Zavada, 1982, 1984; Pocock and Vasanthy, 1988). However, similar folds are prominent in the sporoderm furrows of extant Welwitschia pollen (Kedves, 1987; Plate IV, Figs. 2, 3). Kedves suggests that these exine folds are the result of crumpling of the tectum, and are manifested further in the concomitant separation of both the tectum and infratecum from the nexine in a number of grains that he had sectioned, especially immature ones. This 'artifactual explanation' for exine folds in the furrows of extant Welwitschia pollen, however, may not be accurate. In the present author's opinion, the Welwitschia grains figured in Kedves (1987) do not appear to be fixed well, nor is a comparison of this type relevant between immature and mature pollen. Moreover, the sexine folds in the Brazilian ephedroid grains are interpreted as naturally occurring structures, rather than as artifacts of preservation or electron microscopy preparation. This inteipretation is based on the fact that almost all grains sectioned are exceptionally well- preserved throughout, and do not show gross separation of sexine and nexine sporoderm components at any positions throughout sectional view. This is notably the case in the specific grains that show prominent furrow hinges'. These furrow 'hinges' may in fact have important functional implications, specifically relating to harmomegathy. Pollen grains of extant plants are known to exhibit a variety of harmomegathic modifications in shape and volume in order to accommodate for alterations in their relative degree of hydration. This is especially important for the pollen grains of anemophilous plants, which need to minimize dehydration while being completely immersed in a desiccating medium. Blackmore and Barnes (1986) identify three principal mechanisms by which pollen grains are harmomegathically altered, including expansion 93 and contraction of the exine, exine folding, and volume modification of exine lacunae. The operative element with regard to the Brazilian ephedroid grains is exine folding. In extant grains, folds in the sporoderm typically occur in apertural regions, they may also occur in specific nonapertural regions or in nonspecific sites altogether (Blackmore and Barnes, 1986). The ephedroid grains sectioned here lack a well-defined aperture; however, the exine is appreciably thinner in the furrow regions in comparison with rib regions. This is almost identical to pollen of extant Ephedra which also lacks a distinct aperture, but is sometimes referred to as polycoipate because of the numerous thin-walled furrows. Although exine folds have not specifically been reported in extant Ephedra pollen, two species of this gnetalean genus (£. trifurca and E. nevadensis) have been shown to be well-adapted for wind pollination (Niklas and Kerchner, 1986; Niklas, Buchmann, and Kerchner, 1986; Niklas and Buchmann, 1987; Buchmann, O'Rourke, and Niklas, 1989). Moreover, insect pollination has also been documented in two species of Ephedra (Bino, Dafini, and Meeuse, 1984; Bino, Devente, and Meeuse, 1984; Meeuse et al., 1990a, b). Although poUenkitt is not present on the exine surface of Ephedra pollen (Hesse, 1984), grains of E. aphylla have an anomalous capacity to cohere to one another as well as to adhere to insects (Meeuse, 1990b). Documentation of entomophily in the Gnetales may also affect interpretations of fossil ephedroid pollen, and have implications regarding polarity assignments of important pollen grain features in phylogenetic analyses. For example, as noted above, several ephedroid palynomorphs bear flap-like exine extensions, or auriculae (e.g., Elateroplicites, Elaterosporites, Galeacornea, Steevesipollenites, Regalipollenites-, Elsik, 1974; Lima, 1980). Crane (1988) has suggested that these types of auriculate ephedroid grains are more specialized than the nonauriculate ephedroids, and further implicitly suggested that the presence of such "specialized forms" in the fossil record adds to the "evidence of insect pollination in the group." 0 4

Whether or not anricuiate ephedroid grains are more advanced than nonauriculate ones, and how they should be polarized in phylogenetic studies remains a matter of conjecture; however, ultrastructural examinations of these forms may help elucidate this problem. At least two auriculate ephedroid forms occur in the Santana Formation (i.e., Steevesipollenites, Regalipollenites; Lima, 1980); continued fine structural investigations of these palynomorphs are in progress.

Systematic Comparisons — Ephedroid palynomorphs are especially abundant in the Cretaceous, when certain groups of gymnosperms, presumably including Gnetales, flourished, and angiosperms were undergoing massive radiations (see Crane, 1988 and references therein). Even upon a cursory examination, it is evident that the diverse assemblage of fossil ephedroid grains currently known, represents a distinctly artificial group. In fact, several palynostratigraphic studies have emphasized that different, dispersed ephedroid grains may share affinities with either Gnetales, Coniferales, Monocotyledoneae, or Araceae in particular (e.g., Mchcdlishvili and Shakmoundes, 1973; Elsik, 1974). Moreover, although only two genera of dispersed ephedroid grains have previously been studied at the fine structural level (i.e., Ephedripites and Equisetosporites), exine architecture of these grains clearly demonstrates disparate taxonomic affinities. Only one species of Ephedripites pollen has been sectioned (Trevisan, 1980). These grains are monosulcate and bear longitudinally-oriented ribs. Both rib morphology and exine stratification of this ephedroid species suggests gnetalean affinities, based on its tectate-granular sexine that gradually thins toward the furrows, and nexine that is apparently lamellate throughout. The recently emended ephedroid palynomorph, Equisetosporites chinleana, and the newly separated species Cornetipollis reticulata, are 95 both strikingly different in surface morphology and exine nne structure from one another as well as from Ephedripites (Pocock and Vasanthy, 1988). Both Equisetosporites and Cornetipollis are nonaperturate and have a discontinuous tectum (=semitectate) that is underlaid by infratectal columellae. Cornetipollis has more or less longitudinally-oriented ribs with foveo-reticulate ornamentation, stouter columellae than those of Equisetosporites, and a nonlamellate nexine. Based on these features, Cornetipollis is thought to be angiospermous. In Equisetosporites, exine ribs are psiiate and more spiral in their orientation, columellae are more variable in distribution as well as narrower in diameter, and the nexine is three-layered with distinct lamellae in an intermediate position (Pocock and Vasanthy, 1988). In addition to Pocock and Vasanthy's work on these two ephedroids, the fine structure of E. chinleana grains was also initially realized to be 'nongnetalean,' principally because of its columellar vs. granular infratectum (Zavada, 1982,1984). Moreover, despite the presence of nexine lamellae in E. chinleana, Zavada (1990b) has subsequently suggested that these grains are definitively more angiospermous than gymnospermous and most similar to the pollen of Dahlgrenodendron natalensis (Lauraceae) and Spathiphyllum (Araceae). Of the three ultrastructurally known fossil polyplicate palynomorphs, the Brazilian grains sectioned in this study most closely resemble Ephedripites both in surface morphology and exine stratification. The exine of Ephedripites consists of an inner lamellated nexine (layer A) and an outer complex, five-layered sexine (layers Bj, B 2 , B3 , C, D; Trevisan, 1980). The most prominent layers of the sexine are the three innermost. These consist of a very thin, homogeneous band (Bj) appressed to the lamellated nexine, successively overlaid by a layer of "anastomosing units" (=granules; B 2 ) and a homogeneous layer (=tectum; B 3 ). Layer C is a thin and discontinuous layer just outside of the tectum and is intermixed to overlaid by layer D, that consists of variably sized 96

"giobuiets.” The comparison between the Brazilian grains and Ephedripites is based principally on a similar tectate-granular sexine organization and faintly lamellate nexine. Although Trevisan (1980) did not specifically describe the inffatectum of Ephedripites as granular, but rather referred to it as consisting of "anastomosing units", it is clear firom her transmission electron micrographs that these "units" are indeed granules. Another interesting comparison with Ephedripites is the possible occurrence of a sexine layer in the Brazilian grains that is analogous to Trevisan’s C layer. In the ephedroid grains sectioned in the present investigation a thin, homogeneous sexine layer is present external to the tectum in the plicae and absent over the crest regions of the sporoderm ribs. This additional sexine layer may also have functional significance relating to harmomegathy, as it is restricted to furrow regions, and occurs concomitantly with sexine hinges (see above). A layer analogous to the D layer observed in Ephedripites grains has not been detected in the Brazilian grains. Zavada (1984) suggested that the giobuiets making up the D layer in Ephedripites may represent tapetal remains; however, in the present author's opinion they are more likely fragments of adhering palynodebris. When the Brazilian grains are systematically identified using Pocock and Vasanthy's (1988) "key to polyplicate or ridged and furrowed pollen," they key out very close to Ephedripites; however, they also show some features of Ephedra. The key only employs micromorphological and ultrastructural data, is based in part on information acquired from the literature, and includes three dispersed taxa (Cornetipollis, Equisetosporites, Ephedripites), Ephedra, and Spathiphyllum (Araceae). In the present author's opinion, however, the key is slightly flawed concerning two characters that delimit Ephedripites, including "tectum almost disappearing in furrows" and "infratectum non-granular. " Data on this genus were gained from Trevisan's (1980) descriptions; however, it does not appear that Pocock and Vasanthy critically evaluated the published micrographs taking into 97

üuiiuuîît their îow resolution. In particular, in the present author's opinion a tectum is indeed present in the furrows; although it is thinner here, it does not "almost disappear" (see Trevisan, 1980; Plate U, Fig. Ic) as Pocock and Vasanthy (1988) suggest, and in fact is similar to the tectum in extant Ephedra pollen. Furthermore, regarding the fine structural assignment of "non-granular" to Ephedripites, it has been stated above that the infratectum of Ephedripites is interpreted as granular in the present study. In addition, when the Brazilian ephedroids are compared with the pollen of extant gnetophytes, both in surface morphology and exine fine structure, they are in fact more similar to the pollen of Ephedra than that of Welwitschia, based principally on the lack of a distinct aperture in the former. Exine stratification of the pollen of both extant genera are almost identical. Based on these comparisons with extant and fossil taxa, the Brazilian ephedroid grains sectioned in this investigation appear to have their affinities unequivocally rooted in the Gnetales. Using fine stractural features, the Brazilian grains are clearly most similar to Ephedripites in comparison to Equisetosporites, although originally assigned to the latter genus by Lima (1980). Additional fine structural studies which focus more critically on the systematics of the various Santana ephedroids will undoubtedly provide paramount data toward emendation of at least several grains assigned to Equisetosporites, and perhaps others. CHAPTER Vm PENTOXYLALES

Introduction The Pentoxylales was established in 1948 for silicified gymnospermous plant remains from Jurassic rocks at Nipania in the Rajmahal Hills, Bihar Province, northeastern India (=Pentoxyleae; Sahni, 1948). Since that time, there have been several additional studies of the Rajmahal material (Vishnu-Mittre, 1953; Rao, 1974,1976,1981; Stewart, 1976; Bose, Pal, and Harris, 1984, 1985), and numerous compression fossils from southern hemisphere localities of Mesozoic age have also been assigned to the group (e.g.. New Zealand, Harris, 1962, 1983; Australia, New South Wales, White, 1981 [see also Walkom, 1921]; Queensland, Turner, personal communication; Victoria, Drinnan and Chambers, 1985, 1986 [see also Douglas, 1969]). Four genera have been established; Pentoxylon for the characteristic woody stems, Carnoconites for ovulate organs, Sahnia for microsporangiate organs, and Nipaniophyllum for leaves. Although not all of these plant parts have been found connected, the suite of organs is often collectively referred to as the Pentoxylon plant (for review see Bose et al., 1985; Crane, 1985a, 1988). The pollen-producing organs of the Pentoxylales were initially described by Vishnu- Mittre (1953) from the Rajmahal Hills. Sahnia nipaniensis consists of numerous filiform microsporophyUs, each 1-2 cm in length, borne on a Pentoxylon-lype spur shoot. The microsporophylls (=microsporangiophores sensu Bose et al., 1985) were originally

98 99 thought to be whorled and fused at their bases to form a cup (Vishnu-Mittre, 1953). However, Rao (1974, 1981) and Bose et al. (1985) have shown that the microsporophylls are not fused proximally, but rather borne either irregularly or in a spiral on a collar-like structure surrounding the conical shoot apex (see also Drinnan and Chambers, 1985). Each microsporophyll consists of a single main branch bearing numerous short branches on ail sides. The side branches bear four to seven unilocular, pyriform sporangia borne on short stalks. The sporangium wall is a single cell thick with a possible line of dehiscence marked by cells with thickened periclinal walls (Vishnu-Mittre, 1953). A second species of Sahnia (S. laxiphora) based on compression material from the Lower Cretaceous of Australia is similar in morphology to 5. nipaniensis (Drinnan and Chambers, 1985,1986). Pollen grains from Sahnia nipaniensis were initially described as boat-shaped, monocolpate with a smooth wall, and averaging 25-26 x 10-25 pm in size by Vishnu- Mittre (1953). However, the preservation of pollen grains in the Sahnia material from the Rajmahal Hills is generally poor (Bose et al., 1985), and examination of pentoxylalean pollen beyond these original observations is especially sparse. Sukh-Dev (1980) isolated in situ pollen from the type material of S. nipaniensis and compared these grains with other dispersed pollen from Rajmahal HUls rocks using transmitted light microscopy. The pollen from S. nipaniensis was found to most closely resemble the sporae dispersae genus Cycadopites (see Sah and Jain, 1964); pollen isolated from Australian material of S. laxiphora has also been compared with this dispersed pollen genus (M. E. Dettmann [personal communication] in Douglas, 1969, p. 242). Preliminary fine structural analyses of Sahnia pollen, employing electron microscopy, were provided by Taylor and Taylor (1987b) and Taylor (1988b) as part of a comprehensive review of sporoderm ultrastructure in fossil gymnosperms. The present study expands these earlier observations, provides additional details of the ultrastructure 100 and micromoipholog>' of in situ Sahnia laxiphora pollen grains, and contributes new data with which to assess the systematic affinities of the Pentoxylales.

Description Sahnia laxiphora consists of a short length of axis bearing numerous (>30) microsporophylls. Microsporophylls are unbranched for most of their length, but in their distal half they bear numerous simple, spherical to ovoid pollen sacs, each on a short stalk (Fig. 125). Occasionally, short side branches of the microsporophyll have more than one attached pollen sac. Pollen sacs contain numerous, highly compressed in situ pollen grains (Fig. 128). The walls of the pollen sacs can be recognized but are not well preserved (Figs. 130-134). Pollen grains are small, ovoid, and monosulcate. In polar view the grains measure 26 |im in length and 23 lim in breadth (Figs. 126-127,129). The sulcus is relatively broad and the exine in this region is often strongly invaginated. Exine ornamentation is psüate (Figs. 126, 129). The pollen wall is composed of two distinct layers, with an outer sexine approximately six times the thickness of an inner nexine (Figs. 130-133). The sexine has a variable affinity for heavy metal stains but generally stains lightly throughout. The sexine is relatively thin, averaging 0.82 um in thickness, and in many specimens consists of an inner granular layer which averages 0.23 pm in thickness and grades into an outer ultrastructurally homogeneous layer. The inner layer consists of relatively large granules (ranging up to 0.12 pm in diam.) separated by irregular lacunae of variable sizes. Exine granules have approximately the same size, shape, and staining density as the granular elements composing orbicules associated with the surfaces of grains and pollen sac walls (see below). At the interface with the nexine, granules and lacunae are particularly 101 pronounced (Fig. 131), often giving the appearance that the sexine is slightly separated from the underlying nexine (Figs. 131-133). The granules also vary considerably in their distinctness, in particular outer granules, where there is no clear separation from the more homogeneous outer part of the sexine (i.e., tectum). The tectum in these grains is relatively thick, averaging 0.45 |im in thickness. The consistency of wall construction in all grains examined suggests that the features described are not a result of preservational phenomena, but ratlier reflect differences in the nature of the sporopoiieninous wall. The broad sulcus is clearly visible in ultrastructural sections. In some grains there is an extremely thin sexine layer overlying the nexine along the sulcus; this originates from a gradual, lateral thinning of the nonapertural sexine (Figs. 130, 132). In other grains, evidence of a sexine layer in this area is completely absent (Fig. 133). The nexine stains more densely than the sexine (Figs. 130-133). This darkly staining layer averages 0.13 |xm in thickness, and is characterized by the occasional presence of faint lamellae, averaging 0.04 pm thick. Lamellae may occur in both apertural and nonapertural nexine regions (Figs. 131-132) and are typically most conspicuous close to the nexine/sexine interface. Apertures are often inconspicuous in section view because of the strongly invaginated sporoderm (Fig. 130). Folding of the apertural nexine is particularly complex, perhaps due to the absence of a sexine layer in this region of the grain. In some specimens the nexine appears to be inrolled independently of the sexine (Figs. 135-136). Tapetal membranes and orbicules frequently occur within pollen sacs. Lamellated tapetal membranes average 0.09 pm in thickness, are typically appressed against the interior surface of the pollen sac walls, and are often associated with orbicules (Figs. 130, 134). Although many orbicules have variable shapes in section view, most are circular to oval and range from 0.29 to 0.71 pm in diameter. Ultrastructurally, orbicules are similar 102 to the sexine; although they have a more distinct granular structure (Figs. 130, 134). Orbicules are also found appressed against the exine surfaces of some grains (Fig. 129).

Discussion Structural Features of the Exine — The two-parted nature of the Sahnia sporoderm is not unusual for fossil pollen grains; however, sexine organization is somewhat unique. The presence of lacunal spaces in non-apeitural regions of the sexine, at the nexine interface, is especially interesting. Several interpretations of this ultrastructural organization can be postulated. It is possible that the lacunal region represents an early ontogenetic stage of alveolar wall development, which would later develop in a centrifugal fashion. This hypothesis is unlikely because of the diffuse occurrence of sexine lacunae throughout the whole grain in section view and the restriction of these spaces to an interface position. Alveolar sporoderm development has been reported for other fossil gymnosperms, in particular medullosalean pollen (e.g., Monoletes). Here, fairly uniform lacunae initially occur between the sporopoiieninous units throughout the entire sexine; the alveolate infrastructure subsequently becomes more pronounced following concomitant sexine and lacunal expansion (Taylor, 1982). The most likely explanation for the lacunal region is that it represents a system of infrastructural spaces between relatively large, irregularly-shaped granules. Although the granules are relatively non-distinct, with regard to their appearance in the electron beam, they have approximately the same sizes, shapes, and density as the granular orbicules associated with sporangial walls and pollen exines. The external region of the sexine has a markedly different fine structure, being characterized by a homogeneous organization, suggesting that the sporopoiieninous granular units have undergone some fusion. The 103 homogeneous layer is interpreted as a weakly-denned tectum, in that it does not have a well-defined lower boundary, but rather grades into the granular zone.

Comparison with. Extant and Fossil Taxa — Details now available on the structure of Sahnia poUen remove an important gap in previous knowledge of the Pentoxylales (Crane, 1985a), and contribute to the accumulating information on the micromorphology and ultrastructure of in situ fossil gymnosperm pollen (Taylor and Taylor, 1987b; Taylor, 1988b). These new data therefore provide an improved basis for comparing the pollen of Pentoxylales with that of other seed plants. Several features distinguish Sahnia pollen from the grains of other extant and fossil gymnosperms (Table 1). The absence of a saccus distinguishes Sahnia pollen from that of most conifers (including the earliest fossil representatives of the group), cordaites, and several groups of seed ferns including Caytoniales, Corystospermales, and Glossopteridales (see Taylor and Taylor, 1987b; Taylor, 1988b for review). Sahnia pollen is ovoid in shape and has a distal sulcus; this also distinguishes it from pollen of certain conifers that have spheroidal, inaperturate grains (e.g.. Araucaria), as well as meduUosan and iyginopterid seed ferns in which the pollen has a proximal trilete or monolete mark rather than a distal sulcus (Taylor and Taylor, 1987b; Taylor, 1988b). Granular sexine organization distinguishes Sahnia pollen from that of cycads and Ginkgo which are characterized by an alveolar sexine infrastructure (Audran and Masure, 1976, 1977, 1978; Audran, 1987; Dehgan and Dehgan, 1988; Hill, 1990), as well as the alveolate Iyginopterid seed fem genus Schopfiangium (Stidd et al., 1985). Excluding the Peltaspermales, for which details of pollen ultrastructure are not yet known (Taylor, 1988b), these comparisons leave four groups of extant and fossil seed plants 104

(Bennettitales, Eucommiidites-^\snXs, Gnetales, and certain angiosperms) in which at least some taxa have ovoid pollen with a distal sulcus and a granular sexine infrastructure. Eucommiidites pollen is easily distinguished firom that of Saknia by the presence of two prominent, lateral, groove-like thinnings in the exine, and the more rounded ends of these grains as seen in polar view. Pollen of extant Gnetales (except the inaperturate grains of Gnetum) is distinguished from that of Sahnia by the presence of prominent longitudinal plications. However, both Eucommiidites and gnetalean pollen, as well as pollen of Bennettitales and certain angiosperms, are similar to that of Saknia in having a distinct granular component in the outer part of the sporoderm. Both within and among these groups the details of exine stratification vary considerably, particularly with regard to granule size and position with respect to the differentially stained sporoderm layers. Eucommiidites pollen that occurs in the micropyles of seeds of Erdtmanispermum balticum (Pedersen et al., 1989), as well as pollen from the fossil bennettitalean microsporophyll Leguminanthus siliquosus (Ward et al., 1989) have the granular sporoderm layer separated from the darkly staining inner layer by a structurally homogeneous, lightly staining zone. Similarly, in certain angiosperms, for example the Early Cretaceous putative angiosperm grain Lethomasites fossulatus (Ward et al., 1989), a very thin homogeneous layer (foot layer) may be present, although the darkly staining inner layer may be entirely absent. In contrast, in Sahnia pollen the granules are in direct contact with the darkly staining inner layer. A basically similar arrangement, but with finer granules, occurs in Eucommiidites pollen (Doyle et al., 1973; Trevisan, 1980), including that isolated from Erdtmanitheca texensis (Pedersen et al., 1989), pollen of the bennettitalean Cycadeoidea dacotensis (Chapter VI) as well as pollen of extant Welwitschia and Ephedra (Gullvâg, 1966a; Van Campo and Lugardon, 1973; Kedves, 1987). Pollen of Cycadeoidea dacotensis also lacks lamellae in the darkly staining inner sporoderm layer. 105

These comparisons show that pollen of Sahnia iaxiphom is not unequivocally identical to any previously described in situ pollen, but it is clear that several characters of Sahnia pollen occur within a relatively small assemblage of seed plants including Bennettitales, Gnetales, Eucommiidites-^lmts and certain angiosperms. Because Sahnia pollen lacks the specialized features that distinguish Eucommiidites and gnetalean grains (i.e., lateral grooves, longitudinal plications), it may be suggested that the strongest similarities are with the pollen of certain Bennettitales.

Systematic Implications — Since the Pentoxylales were first described it has been recognized that they exhibit a unique combination of characters seen in several groups of seed plants (e.g., Sahni, 1948). Based on a number of vegetative and reproductive features, including the nature of pollen as seen through transmitted light, the Pentoxylales have been compared with a variety of gymnosperm taxa including Coniferales (Araucariaceae), Ginkgoales, MeduUosales, and Bennettitales (e.g., Rao, 1981; Bose et al., 1985) and even the angiosperm family Pandanaceae (Liliopsida; Meeuse, 1961). As a consequence, the relationships of the group have been regarded as enigmatic (e.g., Andrews, 1961; Stewart, 1983). More recently, cladistic analyses of seed plants (Crane, 1985a; Doyle and Donoghue, 1986) have tended to support earlier suggestions (Ehj-endorfer, 1976) that the Pentoxylales are closely related to the Bennettitales and have placed both groups with the Gnetales and angiosperms in a clade of relatively derived seed plants informally termed the anthophytes (Crane, 1985a; Doyle and Donoghue, 1986), which may also include Eucommiidites-plz.ni's (Pedersen et al., 1989). The new information now available on pollen ultrastructure in the Pentoxylales, and the comparisons presented above, broadly support this systematic hypothesis. Further resolution of relationships of the Pentoxylales will hinge on the clarification of other 106 aspects of the reproductive structure and biology of this enigmatic, but important group of Mesozoic seed plants. CHAPTER IX PHYLOGENETIC CONSIDERATIONS

One of the stated goals of the present investigation was to perform phylogenetic (i.e., cladistic) analyses of all six groups of gymnosperms examined utilizing the newly ascertained data on pollen micromorphology and ultrastructure. However, the glossopteridalean and corystospermalean pollen types both were determined to have undergone a relatively high degree of diagenesis, thus altering a large number of characters. Although several important structural features concerning these grains were documented, both were determined to have lost phylogenetically important information, especially with regard to exine infrastructure. Consequently, the phylogenetic analysis component of the investigation was redirected, focusing on the groups that showed well- preserved pollen, including Caytoniales and the gymnospermous 'anthophytes' (Bennettitales, Gnetales, and Pentoxylales). Although most botanists, especially neobotanists, generally consider the term "anthophytes" as a reference to flowering plants (i.e., members of the Anthophyta), in 1986 the term was also used as an informal designation for a clade encompassing several major groups of highly derived seed plants (Doyle and Donoghue, 1986). Since the term's initial use in this cladistic capacity, it has been increasingly incorporated into the literature, particularly in other phylogenetic analyses (e.g., Donoghue and Doyle, 1989; Crane, 1990). The "anthophyte" clade includes angiosperms as the sister group of Bennettitales,

107 108

Gnetales, and Pentoxylales. The clade, whether specifically referred to as "anthophytes" or not, is held together by a number of both vegetative and reproductive features (e.g., Doyle and Donoghue, 1986, 1987; Crane, 1985a, 1990). These characters include syndetocheilic stomata, complex, 'flower-like* reproductive structures with aggregated microsporophylls, nonsaccate pollen with granular exine structure, and bitegmic ovules. Moreover, Pedersen et al. (1989) have suggested that Eucommiidites pollen-producing plants may also have their affinities nested within the anthophyte clade, based on the highly reduced morphology of their microsporophylls and their granular pollen wall. In addition to those anthophyte grains sectioned in the present investigation (i.e., Bennettitales, Cycadeoidea dacotensis', Gnetales, Equisetosporites sp.; and Pentoxylales, Sahnia laxiphora) data on several other fossil and extant anthophyte grains have been included in the preliminary phylogenetic analyses presented below. These grains included a number of gymnospermous forms, including Eucommiidites pollen-producing plants, and several dispersed grains with putative angiospermous affinities (Table 2; Fig. 137). Moreover, Caytoniales was also included in all analyses as the outgroup (Table 2; Fig. 137) because this group has been suggested to be the sister group to anthophytes (Doyle and Donghue, 1986,1987), and good data on Caytonanthus arberi were readily available as this material was examined in the present study and found to be exquisitely preserved. AH terminal taxa were evaluated based on 16 char^ters (see Appendix A). Only preliminary analyses have been conducted thus far, and results are relatively ambiguous. When initial computer analyses were carried out using PAUP's default settings (i.e., equal weights of all characters, characters unordered, and standard ancestral settings), more than 4,000 most parsimonious trees were achieved, all with the shortest tree length of 34 steps. As would be surmised from this large number of trees, the cladograms show an array of different topologies, and the resultant strict consensus tree 109

shows a large polytomy (Fig. 138). However, a fifty percent majority-mle consensus cladogram of those same trees is more resolved, showing several branches, some of which are supported relatively regularly (Fig. 139). Interestingly, Cycadeoidea dacotensis, Leguminanthus siliquosus, and Sahnia laxiphora are grouped together relatively closely, and the Brazilian ephedroid grains {Equisetosporites sp. [Santana]) are nested together with Ephedripites sp. 1. Gnetum, however, is consistently linked with the Eucommiiditean taxa. The inclusion of Gnetum within this clade of otherwise strictly Eucommiidites plants most certainly indicates separation of these taxa based on character no. 4, pollen shape. Other tree topologies can be achieved if the default settings for character ordering and ancestral states are manipulated; however, none of these modifications result in the production of a single most parsimonious tree. Several other experiments were also carried out by manipulating character weights. Although character weighting introduces bias into the analysis from the outset, it appears that some manipulation of weights is necessary in order to compensate for particular characters that are more than likely of less phylogenetic significance. Nevertheless, such characters affect tree topology, or lack thereof, in uniting terminal taxa into clades which, based on a multitude of other morphological data, clearly have different relationships. In the analyses in which character weighting was employed, still no single most parsimonious cladograms were produced, but significantly fewer trees were determined resulting in more resolved strict consensus trees. For example, several characters believed to be more phylogenetically informative, mostly of the fine structural-type, were either weighted by two (saccate pollen [2]; exine tectate [11]; tectum ultrastructure [12]) or three (gross exine architecture [8]; exine partitioning [9]; sexine ultrastructure [10]; aperture type [14]). This weight set was also concurrently evaluated by ordering all characters and treating those states assigned zero as ancestral. In this analysis, 19 trees were generated having a 110

îTTîîiîîTiiirîi Icn^tlx Gx 6S stcps^ uic stncî consensus troc is presenter in Figure 140. AlLiiou^ii this tree is clearly more resolved than the one based on unweighted and unordered characters in terms of showing some branching patterns; the proposed relationships of the terminal taxa, however, are still somewhat equivocal. For example, in this particular case Welwitschia is segregated away from Gnetum and Ephedra, and resolved as a sister group of Lethomasites. This relationship, to a large degree, is based upon their monosulcate aperture type and their overall size, and, in the present author's opinion does not necessarily represent true phylogenetic affinity. Although the cladistic experiments carried out thus far have not clarified the relationships of the anthophyte groups evaluated, they have only been preliminary in nature. Additional analyses are currently in progress in an attempt to resolve the ambiguities observed in the cladograms produced thus far without incorporating any bias into the analyses. However, based on these preliminary results it appears that phylogenetic assessments, based solely on palynological data, of such widely disparate groups are not very illuminating. In addition. several other phylogenetically unrelated factors (e.g.. pollination) may also bias these types of analyses by potentially affecting the morphology and fine structure of fossil pollen (see Chapter X). GENERAL DISCUSSION

The information available on Mesozoic seed plants is less extensive in comparison with Paleozoic forms, and the majority of this has come from descriptive studies on gross morphology, principally of vegetative organs. The new, comparative data presented here on the micromorphology and fine structure of pollen from several Mesozoic (Corystospermales, Caytoniales, Bennettitales, Gnetales, and Pentoxylales) and Paleozoic (Glossopteridales) gymnosperms begin to fill the gap in previous knowledge of the reproductive structure and biology of these groups. The specific morphological and structural aspects of the various pollen types, as well as relevant systematic comparisons, have been presented in the discussion sections of each of the foregoing chapters. These data wül not be recapitulated here, but several are summarized in Table 4. Because of the critical and systematic nature with which all pollen types were investigated, a number of structural characteristics have been elucidated within multiple grain types and have important implications regarding several functional and phylogenetic interpretations, and may bear significantly on cladistic analyses which utilize palynological data.

Structural Features Sacci — Three of the groups examined have saccate pollen, including Glossopteridales, Corystospermales, and Caytoniales. However, the sacci from each of

111 112 these groups are luorphologicaiiy and structurally dissimlar. Both the Antarctic corystosperm grains and pollen of Caytonanthus arberi have well-defined, laterally attached sacci and are clearly bisaccate. The Antarctic taeniate grains (i.e., glossopterid), on the other hand, have a saccus that girdles the corpus and apparently does not attach distally. This type of saccus organization bears significantly on interpretations of the events associated with pollination and siphonogamy of Protohaploxypinus pollen- producing plants (Glossopteridales; see also Zavada, 1991). An especially interesting aspect of saccus ultrastructure is the nature of the internal sporopolleninous units, or endoreticulations. The most prominent features of the endoreticulations available for study by electron microscopy are their overall sizes and extent within sacci. In both the Permian taeniate grains and the Triassic corystosperm grains from Antarctica, individual endoreticulations are relatively narrow. This may be in part due to diagenesis, which has definitely affected these two grain types in a number of other ways. However, based on the regularity of endoreticulation sizes in the numerous grains sectioned, it is more than likely the case that these structures were in fact naturally narrow in size. By comparison, endoreticulations within the sacci of Caytonanthus arberi pollen are much larger. Both Antarctic grain types also show attachment of endoreticulations to the outer wall of the sacci, and thus distinctly separated from the lateral corpus walls (i.e., eusaccate). In bisaccate grains of C. arberi, the endoreticulations appear to be continuous between the outer saccus and lateral corpus walls (i.e., protosaccate). The sizes of endoreticulations may also play a role in the morphological determination of a particular grain as 'eusaccate' or 'protosaccate'. For example, the Antarctic grain types may be 'eusaccate' because of the delicate nature of their endoreticulations which would conceivably be easily ruptured, or separated, from the underlying nexine during saccus expansion; whereas the relative 113 robustness of C. arberi endoreticulations would preclude tlieir separation from tlie corpus wall. Several other factors may also play a role in the morphological appearance of fossil grains as eusaccate or protosaccate (e.g., grain ontogeny, saccus size, and sectioning events), and are addressed below.

Apertures — Another feature which is particularly interesting and was detected in all grain types, with the exception of the dispersed taeniate grains, is the fine structure of the thin portion of the wall, either the distinct aperture (i.e., sulcus) in grains from Antarctic corystosperms, Caytonanthus arberi, Cycadeoidea dacotensis, and Sahnia laxiphora or the furrows in the polyplicate gnetalean palynomorphs. In all of these grains, this region of the exine is characterized by a gradual thinning of the sexine, primarily the infratectal layer, in lateral positions to a point where the sexine component is either completely absent or only present as an extremely thin layer. Moreover, in all grain types the nexine waU component is present in the aperture-, or plicae-associated region, and in fact is typically the same thickness as in other sporoderm regions. A distal aperture is not readily detectable in the Permian taeniate grains sectioned here (i.e., Protohaploxypinus); as noted these grains have a continuous, girdling saccus that is apparently not attached to the distal surface of the corpus.

Nexine Orgardzation — Nexine lamellae were only detected in three of the pollen types investigated, including Caytoniales, Gnetales, and Pentoxylales. Of these groups, the nexine was found to be lamellate most regularly in only pentoxylalean pollen, and these lamellae were only faint in outline and primarily restricted to the nexine/sexine interface in apertural regions. Lamellae in caytonialean and gnetalean grains were only detected under rather anomalous conditions. Well-defined lamellae in the nexine of Caytonanthus arberi 114 were only observed in grains thaï had undergone some preser»ationai folding or in infrequently occurring immature grains. Although one to two faint lamellae were identified at the nexine/sexine interface in only a few of the Brazilian ephedroid grains, these palynomorphs were only characterized as having unequivocally lamellated nexines after sectioning a single grain that had also undergone relatively extensive folding. These data clearly demonstrate that lamellae may in fact be present in particular pollen grain nexines that might otherwise be characterized as nonlamellate. The fact that numerous grains were sectioned, and sectioned serially, in the present study is the basis for this premise. Although many grains of Cycadeoidea dacotensis were also sectioned, and lamellae were not detected in the nexine of any; this may simply reflect the fact that the 'right* grain has not yet been sectioned. For example, in another ultrastructurally well- known gymnosperm pollen type (Monoletes', MeduUosales), out of thousands of sections cut from numerous grains, nexine lamellae have only been detected in an extremely small number of sections (T. Taylor, personal communication). The reasons for the relative absence of distinct nexine lamellae in most mature pollen of the various gymnosperms are unclear. It is unlikely, however, that lamellae disappear in the ontogeny of these grains like those in some angiosperms, because lamellae are in fact present in grains, but only detectable in folded portions of the wall. A more likely explanation for the absence of readily identifiable lamellae in mature grains is that these structures have undergone some chemical modification in development and/or are affected differently by preservational processes. Based on this information, a more conservative phylogenetic assessment of nexine fine structure is suggested (see below). 115

Phyiogeneîic Interpretations of Poüen morpîioîogy and Uîtrastructure When evaluating the overall morphology and fine structure of fossil pollen it is important to bear in mind that not only are the observed features manifestations of actual biological phenomena, but several physical processes may have also affected grain morphology/ultrastructure. One category of physical modification of pollen structure is diagenesis. The results of diagenesis can be severe (i.e., possible loss of all exine infrastructure with increasing level of thermal maturation), and have been discussed at some length above, as well as illustrated in a few examples. Two other major types of physical alteration can affect fossil pollen, as well as extant pollen, and relate to preparation techniques for electron microscopy. First, variable effects of staining can alter the appearance of pollen in the TEM, depending on both the types of heavy metal stains used as well as the duration of time that sections are stained in each. Secondly, drastically different types of exine ultrastructural patterns can be observed in pollen grains as a result of sectioning effects. Of these three physically altering processes, the potential effects of sectioning are the most significant in terms of providing information that is phylogenetically inaccurate. Although the effects of diagenesis and extreme staining protocols can also be serious, these can often be more easily detected as artifacts. Another major problem that can result from sectioning is the misinterpretation of the plane of section. The Antarctic corystosperm grains sectioned in the present study provide an excellent example of how the sectioning plane can be critical in the characterization of two features that are generally considered phylogenetically significant. These include cappus ultrastructure and saccus type. For example, this grain type could potentially be classified as either tectate-alveolate, or tectate-columellate, in the cappus region and protosaccate if observations are based only on a few sections through a lateral plane of a single grain. Several other grain types sectioned here could possibly illustrate similar 1 1f, misleading information as well. Tne literature may include such descriptions of pollen based on sections through nonrepresentative exine regions, and, in some cases, data from these studies may have been incorporated into larger phylogenetic syntheses. In light of this, results from the present investigation are significant in that they underscore the importance of sectioning numerous grains, sectioning them serially, and knowing the plane of section from which recorded micrographs have come. Although physical phenomena which may have affected the morphology and structure of fossil pollen are extremely important, the biological factors which have 'shaped' the form of these ancient reproductive propagules are especially interesting. Pollen morphology and ultrastructure are generally considered conservative features in terms of evolutionary change, and numerous authors have interpreted these characters, especially exine infirastructure, as solely representative of an historical, or phylogenetic process (i.e., evolution from some common ancestor). However, it is now realized that pollen morphology and fine structure, both in extant and fossil grains, also reflect certain developmental events (e.g., Taylor, 1982, 1990; Taylor and Zavada, 1986), as well as other functional processes (i.e., the role of 'external' reproductive phenomena such as pollination; Taylor and Millay, 1979; Taylor, 1988b; Osbom et al., 1991). With regard to pollen ontogeny, the exine of Caytonanthus arberi exhibits distinctly different stratification patterns during various stages of development Immature grains not only show well-defined nexine lamellae, but they also lack a well-defined tectum and infratectal alveolar imits that are seen in mature grains. Consequently, if only an immature C. arberi pollen grain were sectioned in a palynological analysis, then the exine of this species would more than likely be dubiously reported as intectate and granular. Moreover, it is also possible that the ontogenetic age of pollen grains may contribute to their characterization as protosaccate. For example, in some fossil 'protosaccate' grains the 117 sacci are relatively small (e.g., Triadispora', Scheuriag, 1976), and grains are often found in tetrads and are thought to have been dispersed in this way. The possibility exists that these grains were morphologically 'protosaccate' only, because either the sacci had not yet fully expanded, or they were genetically destined to remain small in size. In extant Pinus banksiana pollen (e.g., Dickinson and Bell, 1970a), endoreticulations separate from the corpus wall while grains are in the tetrad stage; however, Finus sacci clearly expand during subsequent developmental stages following dissolution of the callose wall to attain relatively large sizes. Functionally, sacci are also believed to play several critical roles, principally during pollination and pollination-related events. It is clear that sacci efficiently increase overall grain sizes without significantly increasing grain weight, and thereby provide for greater dispersal by wind. Others have suggested that sacci have a number of other functions, which include: to harmomegathically maintain volumetric continuity; to physically ensure that the distal aperture is oriented against the nucellus to maximize siphonogamy; and to reduce competition with other pollen grains by maximally occupying the micropylar space, thereby physically preventing other grains from entering this space (e.g., Zavada and Taylor, 1986; Zavada, 1991). Based on these functional roles it is interesting to note the relative sizes of sacci and endoreticulations of the saccate grains sectioned in this study. Sacci of the glossopterid and corystosperm grains are relatively large, especially the corystosperms, and the endoreticulations within these sacci are relatively narrow. However, in Caytonanthus arberi, sacci are relatively small and filled with robust endoreticulations. Based on the relative size of these units, it is possible that grains of C. arberi were heavier than the pollen of other Mesozoic anemophilous seed plants. As a consequence, the relatively short geologic history of caytonialean plants may have in part been due to their inability to compete reproductively during a period of time when insect 118 pollination is believed to have been becoming more prominent, and wind-pollinated plants were in need of maximizing their efficiency. Although the primary emphasis here has been on developmental and functional effects on pollen form, a phylogenetic element may also be equally important. However, the present investigation underscores a conservative approach in assessing the relative emphasis that should be placed on the manifestation of phylogenetically relevant data in fossil pollen morphology and ultrastructure. Based on the above discussion, it also encourages steering away from 'homologizing' particular exine features of fossil pollen, especially when those data are taken from published micrographs in the literature. Most importantly are interpretations of the endexine and the ectexinous foodayer. Exine layers of fossil grains are variable in their stainabilities, and certainly in the presence or absence of lamellations in the wall. In fact, it is distinctly possible that the endexine per se is absent from many fossil grains. In an analogous system, in many extant pollen types the underlying intine frequently acts as support for the endexine and, consequently, both are lost following acetolysis (Blackmore and Crane, 1988), and presumably also during lithification in the case of fossil grains. Finally, based on the above considerations of the noted physical and biological factors which potentially affect pollen morphology and fine structure, the fact that a single most parsimonious tree was not achieved in any of the preliminary cladistic analyses presented in Chapter IX is more easily understandable. Continued investigations of the various aspects of the reproductive structure and biology of the Mesozoic plants studied in this investigation, as well as others, will no doubt certainly provide more information necessary to better elucidate phylogenetically important characters, and better resolve enigmatic cladograms. LITERATURE CITED

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Van Campo, M. 1971. Précisions nouvelles sur les structures comparées des pollens de gymnospermes et d’Angiospermes. Comptes Rendus Hebdomadaires des Séances de l'Académie des Sciences, Series D, Paris 272:2071-2074. and B. Lugardon. 1973. Structure grenue infratectale de l'ectexine des pollens de quelques gymnospermes et angiospermes. Pollen et Spores 15: 171-187. and D. Vernier. 1984. Les grains de pollen. Séries morphologiques et structures de l'exine. Giornale Botanico Italiano 118:255-270. Vasanthy, G., B. S. Venkatachala, and S. A. J. Pocock. 1990. The evolution of angiospermid poUen characteristics: Conjectures and queries. The Palaeobotanist 38: 131-146. Vasü, i. K. and H. C. Aldrich. 1970. A histochemicai and ultrastructural study of the ontogeny and differentiation of pollen in Podocarpus macrophyllus D. Don. Protoplasma 71:1-37. and .1971. Histochemistry and ultrastructure of pollen development Podocarpus macrophyllus (D.) Don. In J. 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APPENDIX A Characters and character states for the data matrix (Table 3) analyzed to generate the cladograms presented in Figures 138-140.

1. Pollen unit: 0, monad; 1, tetrad. 2. Saccate pollen: 0, saccate; 1, nonsaccate. 3. Grain symmetry: 0, radial; 1, bilateral. 4. Pollen shape: 0, prolate/spherical; 1, suboblate/spherical; 2, oblate. 5. Pollen size: 0, large ^ 50 |im); 1, medium; 2, small (< 20 jim). 6. Pollen striate: 0, nonstriate; 1, striate. 7. Sculpture origin: 0, lacking suprasculpture; 1, with suprasculpture. 8. Gross exine architecture: 0, ’spongy’ alveolate (Mo«o/efôj-type); 1, honeycomb’ alveolate (cycad type); 2, granular, 3, columellate. 9. Exine partitioning: 0, sexine > nexine; 1, sexine > nexine (1.5-4 x); 2, sexine » nexine (>4 x). 10. Sexine ultrastructure: 0, one-layered; 1, bi-layered; 2, tri-layered. 11. Exine tectate: 0, tectum continuous or finely perforate; 1, semitectate, reticulate, or foveolate; 2, intectate. 12. Tectum ultrastructure: 0, weakly-defined; 1, well-defined. 13. Nexine ultrastructure: 0, lamellate throughout; 1, lamellate at aperture margins; 2, nonlamellate. 14. Aperture type: 0, monosulcate; 1, bi- to tri-sulcate; 2, inaperturate.

148 149 15. Aperture membrane: 0, not conspicuously sculptured; 1, some sculpture; 2, conspicuous sculpture. 16. Nexine thickness over aperture: 0, thicker than nonapertural regions; 1, equal to nonapertural regions; 2, thinner than nonapertural regions. Table 1. Comparison ofSahnia laxiphora pollen with other nonsaccate grains from selected extant and fossil taxa

Hïÿïertaxon denus/Species Shape Aperture Exine Ôrgamzatlon o{ outer part of Ôigamzation o^ inner part of type ornamentation sporoderm sporoderm

Pentoxylales Sahnia laxiphora Ovoid Monosulcate Psilate Two layered Faintly lamellate granular/homogeneous

Inceitae Sedis®>f’ Cycadopites Ovoid M onosulc^ Psilate ? 7

Inceitae Sedis*^ Monosulcites sp. 1 Ovoid Monosulcate Psilate Single layer Faintly lamellate faintly granular to homogeneous

Inceitae Sedis® Eucommiidites sp.2 Ovmd Monosulcate Psilate Three layered Lamellate with two additional homogeneous/granular/homogeneous lateral fiurows

Inceitae Sedis*^ Eucomnùidites pollen Ovoid Monosulcate Psilate with Two layered Lamellate from Erdtmanitheca with two additional occasional large granulré/homogeneous lexensis lateral furrows granules on the proximal surface

Inceitae Sedis<* Eucommiidites pollen Ovoid Monosulcate Punctate Three layered Lamellate from Erdtmanispermum with two additional homogeneous/granular/homogeneous baldcum lateral furrows 5 Bennettitales®"^ Cycadeoidea dacotensis Ovoid Monosulcate Punctate, scabrate Two layered Nonlamellate granular/homogeneous

Bennetütalesë Leguminanthus Ovoid Monosulcate Psilate Three layered Faintly lamellate siliquosus homogeneous/granular/homogeneous

Gnetales® Ephedripites sp.I Ovoid, Monosulcate Psilate Tliree layered Faintly lamellate plicate homogeneous/granular/homogeneous

Gnetales^"'j'k Ephedra Ovoid, Inaperturate Psilate Two layered Lamellate plicate granular/homogeneous

Gnetalesh"ij"k Welwitschia Ovoid, Monosulcate Psilate Two layered Lamellate plicate granuWhomogeneous

AngiospeimaeB Lethomasites Ovmd Monosulcate Foveolate, fossulate Three layered Absent fossulatus very thin homogeneous/ granular/homogeneous

Angiospermae^ Pandamis Spheroidal, Monopoiate, Typically echinale, ? ? ovoid rarely pseudocolpate occasionally psilate, finely granulate or regulate Table 1. Continued

Cycadalest*'*t'>®"P>94 Extant and Ovoid Monosulcate Psilate, foveolate, Two layered Lamellate fossil genera fossulate, rugulate alveolate/homogeneous

GinkgoalesP'9 Ginkgo biloba O vdd Monosulcate Rugulate, vemicate Three layered Lamellate homogeneoiis/alveolate/homogeneous

Coniferales'>s>‘ Araucaria, Spheroidal, Inapeiturate Scabrate, finely Irregularly granular throughout Lamellate Agathis ovoid papillose

aSah and Jain, 19(54; '’Kedves, 1985b;

V, Table 2. List of taxa evaluated in aU cladistic analyses.

Terminal taxa Data source Bennettitales Cycadeoidea dacotensis Chapter VI Leguminanthus siliquosus Ward et al., 1989 Pentoxylales Sahnia laxiphora Chapter Vm Gnetales Equisetosporites sp. Chapter VH Ephedripites sp. 1 Trevisan, 1980 Ephedra spp. Van Campo and Lugardon, 1973j Hesse, 1984; Kedves, 1987 Welwitschia mirabilis Hesse, 1984; Kedves, 1987 Gnetum spp. GuUvâg, 1966a; Hesse, 1980; Zavada, 1984 Eucommiidites-plants Eucommiidites sp. 1 Doyle et al., 1975 Erdtmanitheca texensis Pedersen et al., 1989 Erdtmanispermum balticum Pedersen et al., 1989 Angiospermous sporae dispersae grains Equisetosporites chinleana Zavada, 1984; Pocock and Vasanthy, 1988 Cornetipollis reticulata Pocock and Vasanthy, 1988 Lethomasites fossulatus Ward et al., 1989 Caytoniales (Outgroup) Caytonaruhus arberi Chuter V

152 Table 3. Character Matrix for all cladistic analyses. (See text for explanation of characters)

OTUs Characters

1111111 1234567890123456 Cycadeoidea dacotensis 0110100211012001 Leguminanthus siliquosus 01101002210?00?1 Sahnia laxiphora 0110100221001011 Equisetosporites sp. 0110110211013212 Ephedripites sp. 1 011011020207001? Ephedra spp. 0110010201010211 y/elwitschia mirabilis 011001021101001? Gnetum spp. 010120020101027? Eucommiidites sp. I 011110020101017? Erdtmanitheca texensis 0111100211070111 Erdtmanispermum balticum 0111100222010110 Equisetosporites chinleana 0110010311110211 Cornetipollis reticulata 0110010321112211 Lethomasites fossulatus 0110000221112072 Caytonanthus arberi 0017100111010012

153 Table 4. Comparison of several palynological features from the major gymnosperm groups investigated.

ChaiatSer Glossopteridales Corystospermales Caytoniales Bcnneaitales Gnetales Pentoxylales

S grain length 61 pm 66 pm 23 pm 25 pm 41 pm 26 pm

X grain breadth 35 pm 49 pm 15 pm 12 pm 13 pm 23 pm

X corpus length 37 pm 33 pm 11 pm “- -

% corpus breadth 34 pm 49 pm 15 pm - - -

X saccuslength 15 pm 30 pm 6pm ---

X saccus breadth 38 pm 36 pm 15pm ---

Ornament Taeniate/psilate Psilate Psilate/ Punctate/ Psilate/ Psilate faintly-scahate psilate faintly-scabrate

Overall Homogeneous Homogeneous Tectate- Tectaie- Tectaie- Tectaie- ultrastructure alveolate granular granular granular (honeycomb­ like type)

X exine thickness 0.85 pm 1.0 pm 0.65 pm 0.73 pm 1.04 pm 0.82 pm (@ taeniae)

X sexine thickness — -- 0.50 pm 0.50 pm 0.63 pm 0.69 pm

X tectum thickness -- 0.20 pm 0.14 pm 0.24 pm 0.45 pm

5i-ifra*<>ç»ojri — - 0.30 pm 036 pm 0.35 pm 0.23 pm thickness

Inâatectal element — - Aveolae width Granule diam. Granule diam. Granule diam. typesize 0.20 pm 0.03-0.11 pm 0.09-0.26 pm 0.04-0.12 pm

X nexine thickness — - 0.15 pm 0.23 pm 0.41 pm 0.13 pm

Nexine lamellae Absent Absent Present Absent Aesent-faint Present-faint present & 0.03 pm 0.01 pm 0.04 pm X thickness

Orincules present Absent Absent Present Absent Absent Present and Jc diameter 0.32-1.33 pm 0.29-0.71 pm

Membranes Absent Absent Present Absent Absent Present present 0.05 pm 0.09 pm & X thickness

154 Figures 1-4. Dispersed taeniate pollen (Protohaploxypinus) from the Permian of Antarctica with probable glossopteridalean affinities. 1. Proximal view of pollen grain showing taeniate cappus and girdling saccus. Cracks are artifacts of preservation. (SEM, xl,000). 2. Proximal view of a grain wMch appears to be bisaccate and thus superficially resembles Striatopodocarpites. However, this grain is less compressed than the one illustrated in Fig. 1, and still shows a girdling saccus. (SEM, x1,000). 3. High magnification of the same grain in Fig. 2 showing psilate to slightly scabrate surface on both the saccus (upper part of figure) and the taeniae; pits are interpreted as artifacts of preservation (SEM, x3,000). 4. Transverse section through grain showing intermittent thick (taeniae) and thin (striae; arrow) regions in the cappus wall, and endoreticulations extending between the outer saccus and distal corpus walls. (TEM, x3,080).

155 156 Figures 5-10. Dispersed taeniate pollen (Protohaploxypinus) from the Permian of Antarctica with probable glossopteridalean affinities. S. Proximal view of pollen grain showing girdling saccus and taeniate cappus. (SEM, xl,000). 6. Relatively medial transverse section through a grain showing broad taeniae and less well-defined striae (arrow) in the proximal cappus wall, and endoreticulations within the saccus that are separated from the corpus wall. (TEM, x1,650). 7. Lateral transverse section through the saccus of the same grain illustrated in Fig. 6 showing continuous endoreticulations between the proximal and distal saccus walls. (TEM, x2,100). 8. Transverse section through a single taenia showing a slight degree of differential staining, although distinct sexine and nexine layers are not distinguishable. (TEM, x40,000). 9. Transverse section through a single cappus groove; note the poorly preserved flanking taeniae. (TEM, x40,000). 10. High magnification of same grain as in Fig. 4 showing taenieae and striae (arrow) in the cappus, and continuous endoreticulations between the outer saccus (S) wall and distal corpus (C) wall. (TEM, xl2,500).

157 158 Figures 11-13. Corystospermalean pollen sacs from the Triassic of Antarctica. 11. Oblique transverse section through a pollen organ showing several presumed, dehisced pollen sacs, split open along a suture and completely devoid of grains, as weU as several sacs containing copious amounts of pollen in the upper right of the figure. Note also the intercalary secretory cells in the pollen sac walls (arrow). (LM, x20). 12. Oblique, longitudinal section through several pollen sacs showing numerous in situ grains. (LM, x32). 13. Oblique, transverse section through two pollen sacs containing dissociated bisaccate grains. Note also the single-celled pollen sac wall. (LM, xllO). All Figs. from 10,717 Btop 2oo .

159 w m a ef* .q%%A

'/ y g # Æ f / j

160 Figures 14-19. Corystospermalean pollen sacs from the Triassic of Antarctica. Î4. Oblique longitudinal section through several pollen sacs containing numerous, tightly aggregated pollen grains. (Lîvî, x43). 15. High magnification of the lower part of the same pollen sac illustrated in Fig. 16 showing single-celled wall with contiguous resistant tapetal-like membrane (arrow) and tightly aggregated grains. Note also the presence of endoreticulations within pollen grain sacci. (LM, x313). 16. High magnification of pollen sac in lower right of Fig. 14. (LM, xl40). 17. Macerated clumps of aggregated grains from the same pollen sacs illustrated in Fig. 14. (x98). 18. Several aggregated grains broken off of one of the clumps in Fig. 17. (SEM, x650). 19. High magnification of the proximal wall from a single grain within a larger clump; note the small, spherical orbicule-like body (arrow) on the grain surface. (SEM, x10,000). All Figs. from 10,717 Btop 2oo.

161 ^ MJf

f

162 Figures 20-25. Corystospermalean pollen. 20. High magnification of grain showing rounded corpus and laterally attached sacci. (LM, Nomarski; proximal focal plane; xl,iOO). 21. Equatorial view of pollen grain showing distally inclined sacci. (LM, Nomarski; xl,100). 22. Proximal view of grain (SEM, x8G0). 23. Equatorial view of grain showing broad, distal sulcus. (SEM, x800). 24. High magnification of cappus showing psilate surface ornamentation. Note also that the small pits are artifacts of preservation. (SEM, x4,000). 25. Transverse section through the outer saccus wall showing endoreticulations (SEM, x3,500).

163 m ÆÊ

164 Figures 26-33. Corystospermalean pollen. 26. Relatively medial, equatorial section through a pollen grain showing overall ultrastructure. Note the homogeneous cappus wail, wcugc-Iikc units at the sites of sacci attachment, and discontinuous endoreticulations within sacci. (TEM, xl,775). 27. High magnification of the lateral corpus wall (TENl, x20,100). 28. High magnification, transverse section of the cappus wall from the same grain illustrated in Fig. 26 showing homogeneous fine structure (TEM, x31,500). 29. High magnification, slightly oblique section of the cappus from a different grain showing partial differential staining of the wall (TEM, x20,100). 30. Wedge-like attachment of a laterally attached saccus showing endoreticulations extending between 'upper' and 'lower' wall layers. Note also the poorly preserved, homogeneous cappus wall at left of figure. (TEM, x5,150). 31.Wedge-like attachment of a distally inclined saccus. (TEM, x4,150). 32. High magnification of saccus attachment showing homogeneous cappus wall, separation of wall layers, and discontinuous endoreticulations. (TEM, x20,100). 33. High magnification of a different wedge-like unit showing continuous endoreticulations; note the diagenetic modification of the wall. (TEM, x20,100).

165 fV

28

> y : '

166 Figures 34-39. Corystospermalean pollen. 34. High magnification of grain showing broad sulcus and laterally attached, highly ornamented sacci. (LM, Nomarski; distal focal plane; xl,100). 35. Internal surface of saccus showing general organization and size of the endoreticulurn (SEM, x2,000). 36. Distal view of grain showing intact apertural membrane and 'elevated lips' (arrow) at the sites of sacci attachment flanking the margins of the sulcus (SEM, x800). 37. High magnification of the apertural region in Fig. 36 showing the psilate apertural membrane surface and preservational pitting (SEM, x3,500). 38. Distal view of grain showing ruptured apertural membrane and well-defined 'lips' flanking the margins of the sulcus. (SEM, x800). 39. High magnification of the outer saccus wall showing its psilate ornament; note also the robustness of the internal endoreticulations which are discernable from this external view (SEM, x5,000).

167 m

168 Figures 40-45. Corystospermalean pollen. 40. Relatively lateral, equatorial section through the cappus wail showing elongated regions of saccus attachment; note that both wedge-like units nearly fuse centrally and that endoreticulations are continuous between outer and inner wall layers. (TEM, x2,0ô0). 41. High magnification of the central cappus region from a more lateral section of the same grain illustrated in Fig. 40. (TEM, x20,100). 42. Saccus attachment to the distal wall showing endoreticulations extending between both wall layers. (TEM, x8,050). 43. High magnification of the apertural membrane. (TEM, x20,100). 44. Lateral, equatorial section through a saccus showing continuous endoreticulations between the outer saccus and lateral corpus walls and superficially appearing "protosaccate". Note also the extensive diagenetic modification of both the proximal and distal corpus wall at left of figure. (TEM, x2,100). 45. Medial, equatorial section through a saccus showing eusaccate condition with discontinuous endoreticulations attached to the outer saccus and lateral corpus walls. (TEM, x2,600).

169 %

170 Figures 46-49. Caytonanthus arberi. 46. Morphology of pollen organ showing dorsiventral rachis and branching 'pinnae' with terminal, elongate 'synangia.' (x3.6). 47. Three dispersed (one complete) 'synangia' showing elongate morphology and longitudinally oriented grooves previously thought to represent sutures, but are considered here to be external folds in the individual pollen sacs. J-3; 102. (x8). 48. High magnification of a bleached 'synangium' showing in situ pollen grains and numerous spherical orbicules. (LM, x600). 49. Ultrathin transverse section through a single, cleared 'synangium' showing numerous darkly stained in situ pollen grains (P) and orbicules (O) seen in various planes of section. Note also the margins of each pollen sac (PS) which are delimited by both an external cuticle (C) and 'internal' cuticle (arrow heads). Individual pollen sacs are well-defined by their cuticular boundaries as well as the space (S) seen between them. Although the organic material which composed the pollen sacs has been removed, the locations of their locules is easily delimited by the restricted occurrence of pollen grains and orbicules. (TEM, x1,320).

171 h - f j i m - i - i

PS K

172 Figures 50-57. Caytonanthus arberi. 50. Ultrathin transverse section through a single, uncleared 'synangium' showing two darkly stained pollen sacs (PS). Only one locuie (L) is visible in die upper pollen sac and is continuous with a possible dehiscence zone (arrow). Tlie region of the pollen sac wall lining the locuie (box a) is illustrated in Fig. 52 and the pollen sac cuticle (box b) is shown in Fig. 51. (TEM, x640). 51. High magnification of same general area depicted in box b in Fig. 50 showing the cuticular covering lining the outer wall of each pollen sac. Note the darkly stained reticulations extending into the lighter stained cuticle (TEM, x20,100). 52. High magnification of same general area depicted in box a in Fig. 50 showing lamellated, tapetal membranes lining the internal surface of the locuie. Note also several associated hollow, spherical orbicules. (TEM, x20,100). 53. High magnification of the external cuticle from a cleared pollen sac; note that the darkly stained reticulation are still present. (TEM, x20,100). 54. High magnification of a thin locuie region from a cleared pollen sac showing tapetal membranes delimiting the boundaries of the locuie and numerous orbicules. Note also the fine scabrae on the surface of each orbicule (TEM, xl2,500). 55. Fold in a portion of the pollen sac cuticle with several orbicules, which have been liberated fi’om a locuie, associated with it (SEM, x6,000). 56. Single pollen grain within the locuie of a cleared pollen sac; note that the grain is covered by a thin tapetal membranes (arrow) and orbicules. (SEM, x2,000). 57. Ultrathin section through a grain like that illustrated in Fig. 56 showing pollen exine (E) and associated membranes and orbicules. (TEM, x20,100).

173 174

f Figures 58-64. Caytonanthus arberi. 58. Proximal view of pollen grain showing psilate surface and several orbicules; note also the saccus endoreticuiation in the right saccus. (SEM, x3,000). 59. High magnification of proximal wall. (SEM, xl0,000). 60. Distal view of pollen grain showing broad sulcus extending the entire width of the grain. (SEM, x3,000). 61. High magnification of apertural membrane showing scabrate morphology. (SEM, xl0,000). 62. Distal view of pollen grain completely covered with orbicules. (SEM, x2,000). 63. High magnification of orbicules showing finely scabrate surface sculpture. (SEM, x10,000). 64. Equatorial transverse section of pollen grain through a relatively medial plane showing overall ultrastructure and two layered exine; note the lateral sacci with robust endoreticulations and thin exine over the aperture (A). Also note that this grain has been liberated from a locuie and is appressed against the external cuticle of the pollen sac. (TEM, x7,734).

175 rjeSSBrsSSaSS^BB

?ilTi?i<

176 Figures 65-70. Caytonanthus arberi. 65. Relatively medial equatorial section through a pollen grain showing overall fine structure and lightly stained sexine and darkly stained nexine. Note tlie robust endoreticulations within the sacci which extend into the proximal cappus wall at the sites of cappus attachment; thus the cappus appears 'alveolar', except in the central region where it is nearly homogeneous. (TEM, x6,786). 66. More lateral section of the same grain illustrated in Fig. 65 showing the change in fine structure of the cappus. The wall is no longer centrally homogeneous, but is rather alveolar' throughout. (TEM, x6,786). 67. Oblique section through a grain showing the cappus (upper wall) with a well-defined tectum and infratectal alveolar/endoreticular units, and underlying, darkly stained nexine. Note also the nexine lamellae in the folded region of the lower half of the grain. (TEM, x20,100). 68. Oblique-transverse section through two distally inclined sacci showing nexine lamellae in the folded regions of the wall and thinning of both sexine and nexine wall layers over the aperture membrane (upper center of figure). (TEM, x20,100). 69. High magnification of left saccus in Fig. 68 showing nexine lamellae. (TEM, x40,000). 70. Transverse section of an immature grain showing well defined lamellae throughout the entire nexine and and partially developed sexine; note the incomplete tectum as well as a thin sexinous basal layer. (TEM, x40,0CK)).

177 178 Figures 71-79. Caytonanthus arberi - Main Rachis. 71. Transverse section through an entire uncleared rachis showing the darkly stained cellular tissue and lightly stained cuticle; note also that the cuticle on both sides is variably thickened. (TEM, x2,10G). 72. Transverse section through a different region of the rachis showing the presence of an external, scale-like appendage. (TEM, x2,650). 73. High magnification of a portion of the same rachis illustrated in Fig. 72 showing coalified cellular tissue (CT) with darkly stained reticulations (R) protruding into the cuticle. (TEM, xl0,100). 74. High magnification of the cuticle showing occasional multilayered appearance. (TEM, x20,100). 75. High magnification of a similar region illustrated in Fig. 73 fi-om a cleared portion of rachis showing the former location of cellular tissue (FCT) and the remaining reticulations (R) extending into the cuticle. (TEM, xl0,100). 76. Scale-like appendage showing darkly stained lamellate basal region and lightly stained cuticle. (TEM, x1,600). 77. High magnification of rachis showing attachment of external appendage. (TEM, x 10,100). 78. High magnification of appendage showing basal lamellae. (TEM, x25,100). 79. High magnification of appendage showing reticulations (arrow) extending off of outermost lamellae into lightly st^ e d cuticle. (TEM, x40,000).

179 180 Figures 80-81. Caytonanthus arberi. 80. Outline drawing of the same 'synangium' illustrated in Fig. 50 clarifying it's ultrastructure. Two pollen sacs (stippled) are visible in the micrograph and delimited externally by a continuous cuticular covering (dark lines). The pollen sac locules (L) are compressed, lined by tapetal membranes, and contain pollen grains, orbicules, and dissociated tapetal membranes. A possible line of dehiscence is seen in the upper pollen sac. 81 A) Generally accepted, hypothesized reconstruction of a single synangium, showing three laterally fused pollen sacs. (Redrawn from Harris, 1951). B) New, hypothesized reconstruction of fertile region showing unfused pollen sacs. (Modified from Harris, 1951). C) Transverse section of a 'synangium' like that in Fig. 81A showing cuticle (black lines), cellular tissue (stippled), and internal locules. D) Hypothesized compression of 'synangium' in Fig. 8 ID showing resultant location and extent of pollen sac cuticles. E) Transverse section of three unfused pollen sacs like that in Fig. 8 IB. F) Hypothesized compression of pollen sacs in Fig. 8IE showing resultant location of external pollen sac cuticles.

181 182 Figures 82-86. Cycadeoidea dacotensis. 82. Longitudinal section through a multiloculate synangium showing only a few pollen grains in the lower right locuie; note also the parenchymatous pad of tissue which attaches the synangium to the microsporophyll. G\V#107; YPM#5084. (LM, x36). S3. Tangential section through a synangium showing parenchymatous attachment to microsporophyll, thick outer wall of palisade cells, and two locules separated by a central space. GW#307; YPM#5105. (LM, x31). 84. Transverse section through a multiloculate synangium palisade outer wall, 13 locules, and central space. GW#103; YPM#5086. (LM, x37). 85. High magnification of synangium wall showing single, outer layer of thick-walled palisade cells, three to four middle layers of prostrate cells, and an inner amorphous lining which also forms the locuie septa (arrow); note also several pollen grains within the locules. GW#177; YPM#5081. (LM, xl76). 86. High magnification of locuie contents showing overall size and elliptic shape of several in situ pollen grains. GW#307; YPM#5105. (LM, x425).

183

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84

184 Figures 87-91. Cycadeoidea dacotensis. 87. Several in situ pollen grains from the lower right iocuie of the synangium illustrated in Fig. 82. GW#107; YPM#5084. (LM, x680). 88. Slightly folded pollen grain showing elliptic shape and punctate surface. (SEM, x2,500). 89. High magniRcation of pollen surface showing punctate ornament. (SEM, X15,000). 90. Slightly compressed pollen grain showing anomalous spherical shape. (SEM, x2,500). 91. Exine surface from a different grain showing several psilate regions amongst the punctate sculpture. (SEM, xl0,000).

185 186 Figures 92-97. Cycadeoidea dacotensis. 92. Ultrathin transverse section through a pollen grain showing overall ultrastructure, and lightly stained sexine and darkly stained

nexine; note the thinning of the sexine over the aperture (A). (TEm, x4, 150). 93. Transverse section through two grains showing thin tectum with intermittent thin areas corresponding to surface punctae, granular infratectum, and dark staining nexine. (TEM, x40,000). 94. Transverse section through a folded region of a grain showing exine stratification and thin regions in the tectum (arrow); note also the more densely packed granules in the infratectal layer. (TEM, x31,500). 95. Oblique section showing tectum and well-defined infratectal granules; note that the granules are directly contiguous with the nexine. (TEM, x31,500). 96. High magnification of the apertural region from the same grain illustrated in Fig. 92 showing lateral thinning of the sexine and a consistently thick nexine; note that infratectum thins more gradually than the tectum and that only a very thin sexinous layer covers the aperture per se. (TEM, x20,100). 97. Transverse section through two grains showing pseudo-lamellae (arrow) within the lumen of the lower grain. Note also the relatively wide thin area in the tectum of the upper grain. (TEM, x25,100).

187 188 Figures 98-102. Cycadeoidea dacotensis. 98. Folded pollen grain. (SEM, x3,000). 99. Ultrathin section through a highly folded grain. (TEM, x4,150). 100. High magnification of a folded grain showing the ruptured exine in the apertural region; note the exine has broken in region only occupied by nexine. (TEM, xl0,100). 101. Transverse section through a grain showing overall ultrastructure, thin exine in the apertural region (A), and association of tapetal-iike material. (TEM, x6,500). 102. High magnification of a different section from the same grain illustrated in Fig. 101 showing that the tapetal-like and orbicule-like (arrow) material more than likely represents maceration debris. Note also the presence of a thin layer of sexinous material over the aperture per se in the upper part of the figure. (TEM, x25,100).

189 < r

190 Figures 103-106. Cycadeoidea dacotensis-VrsiSS. 103. Transverse section tiirough the synangiate region of a bisporangiate cone showing microsporophylls with synangia sectioned in various planes and masses of ffass (arrows). GW#309; YFM#5i07. (LM, xll.5). 104. Ultrathin transverse section through a frass mass. (TEM, x2,650). 105. Contents of frass showing relatively amorphous, darkly stained plant cell walls; note that pollen exines are absent. (TEM, x20,100). 106. High magnification of frass contents showing amorphous plant cell walls. (TEM, x40,000).

191 k # - ' 'V

& \/.r.'A ■•> 'A“a'4k. i ty i

f - e x S / '

192 Figure 107. Morphological diversity of polyplicate, gnetalean palynomoiphs recovered from Lower Cretaceous sediments of the Santana Formation, northeastern Brazil. Genera include Equisetosporites (A-D), Singhia (E-F), Weiwitschiaptes (G), Sieevesipolleniîes (K-K), RegaiipoUenites (L), and Gnetaceaepoiienites (m-Q). (Redrawn from Lima, 1980).

193 MU . . I ......

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j u j '^ n y j '.i.ifiin Ü

194 Figures 108-113. Gnetalean pollen {Equisetosporites spp.). 108. Overall morphology of pollen grain showing relatively large size and broad ridges. (SEM, x2,700). 109. High magnification of exine showing psilate to slightly scabrate surface. (SEM, xl0,000). 110. High magnification of grain tip showing termination of the plicae and the psilate/scabrate grain surface (SEM, x13,000). 111. Overall morphology of a smaller grain than the one illustrated in Fig. 108 showing narrower ridges. (SEM, x2,700). 112. Ultrathin transverse section through a pollen grain showing overall ultrastructure, distinct ridges and furrows (plicae), and lightly stained sexine and darkly stained nexine. (TEM, x5,150). 113. High magnification of upper portion of grain illustrated in Fig. 112 showing overall exine stratification. (TEM, x20,100).

195 y#»

196 Figures 114-119. Gnetalean pollen {Equisetosporites spp.). 114. Transverse section through a slightly folded grain showing overall ultrastructure. (TEM, x7,179). 115 High magnification of two appressed ridge regions from the same grain illustrated in Fig. 114 showing thick homogeneous tectum, granular inffatectum, and darkly stained nexine. (TEM, x31,500). 116. Different section from the same region of the same grain as illustrated in Fig. 115 showing opposite staining pattern; note the more distinct appearance of the infratectal granules. (TEM, x31,500). 117. Exine surface of two contiguous ridges showing slight folding of the wall within the furrow (arrow). (SEM, x10,000). 118. Transverse section of two furrows showing hinge-like foldings of the exine (arrows). (TEM, x40,000). 119. Transverse section of two ridges and one furrow showing gradual, lateral thinning of the sexinous granular inffatectum (G) and tectum (T) toward the furrow. Note also the presence of an external, homogeneous sexine layer (arrow) within the cavity of the furrow and the basal, hinge-like sexine folding. (TEM, x50,500).

197 ■t-.'j.'îôiôïâïs

198 Figures 120-124. Gnetalean pollen {Equisetosporites spp.). 120. Transverse section through a slightly folded grain showing thick, homogeneous tectum and granular inffatectum; note that the granules grade into the lower portion of the tectum. (TEM, x31,500). 121. High magnification of sporodemi showing homogeneous tectum, granular inffatectum, and darkly stained nexine. (TEM, x81,000). 122. Transverse section through a highly folded grain showing portions of the nexine (N) in which individual lamellae (arrow) have separated. (TEM, x25,100). 123. High magnification of the same grain illustrated in Fig. 122 showing nexine lamellae. (TEM, x64,000). 124. Transverse section of a nonfolded grain showing faint nexine lamellations (arrow) in the region below a furrow. (TEM, x31,500).

199 ...

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200 Figures 125-130. Sahnia laxiphora. 125. Morphology of pollen organ showing several microsporophylls and simple pollen sacs (arrow). (x2). 126. Distal view of two pollen grains showing psilate exine surface; note the strongly invaginated exine over the sulcus of the lower grain. (SEM, x2,000). 127. Distal view of pollen grain showing broad sulcus. (LM, xl,659). 128. Numerous compressed in situ grains within one pollen sac; note thin, darkly stained nexines and thicker, lightly stained sexines. (TEM, x3,000). 129. Proximal surface of pollen grain with associated orbicules. (SEM, x3,100). 130. Transverse section through portion of a single pollen sac showing three in situ grains (one complete); note coalified pollen sac walls (W), numerous orbicules associated with the walls of the pollen sac (arrows), and strongly invaginated exine at the sulcus. (TEM, x8,050).

201 /Æ S ' i . ;

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202 Figures. 131-136. Sahnia laxiphora. 131. High magnification of nonapertural sporoderm showing two-zoned sexine and compressed nexine, note homogeneous outer part of sexine, large granular units separated by irregular lacunae internally, and faint lamellae at the nexine surface. (TEM, x40,u0G). 132. High magnification of apertural sporoderm showing more distinct granular units of the internal sexine; note gradual thinning of sexine in the apertural region, thin sexine layer overlaying actual aperture (arrow), and more prominent nexine lamellae. (TEM, xl9,500). 133. Portions of two compressed pollen grains, each in the apertural region, showing lacunae at sexine/nexine interface and thinning of sexine at the aperture margin; note also the orbicules associated with the exine of a third grain in upper right of figure. (TEM, x10,000). 134. Section of pollen sac wall (W) with appressed orbicules and associated lamellated tapetal membranes (arrow); note granular substructure of orbicules. (TEM, x20,000). 135. High magnification of invaginated nexine in the apertural region from same pollen grain in Fig. 130. (TEM, x25,100). 136. High magnification of sporoderm showing prominently invaginated exine in the sulcus; note coil-like appearance of nexine. (TEM, x20,100).

203 é Ê

iiiiii ""iiiiiiiiii ' _____

204 Figure 137. Outline drawings of exine stratification from the anthophyte taxa included in phylogenetic analyses. (All exines xl0,300). Drawings are based on tracings of either sections presented here or of published transmission electron micrographs, and include tire following: Bennettitales - Cycadeoidea dacotensis (Chapter VI), Leguminanthus siiiquosus (Ward et al., 1989); Pentoxylales - Sahnia laxiphora (Chapter VIII); Gnetales - Equisetosporites sp. (Santana; Chapter VII), Ephedripites sp. 1 (Trevisan, 1980), Welwitschia mirabilis (Hesse, 1984), Ephedra spp. (Van Campo and Lugardon, 1973), Gnetum spp. (Hesse, 1980); Angiospermous Sporae Dispersae Grains - Lethomasites fossulatus (Ward et al., 1989), Equisetosporites chinleana (Pocock and Vasanthy, 1988), Cornetipollis reticulata (Pocock and Vasanthy, 1988); and Caytoniales (Outgroup) - Caytonanthus arberi (Chapter V). It should also be noted that the published figin-e of Equisetosporites chinleana (Pocock and Vasanthy, 1988) was noted as x4,990; however, this was interpreted as an inaccurate magnification and treated as x14,990, based on comparisons with the noted magnifications of Cornetipollis reticulata in the same publication, as well as other published figures of Equisetosporites chinleana (i.e., Zavada, 1984).

205 BENNETTITALES PENTOXYLALESGNETALES

Cycadeoidea dacotensis Equisetosporites sp. (Santana)

Sahnia laxiphora

Leguminanthus siiiquosus EUCOMMIIDITES-PLANTS Ephedripites sp. 1

ANGIOSPERMOUS SPORAE DISPERSAE GRAINS Eucommiidites sp. 1

Welwitschia mirabilis Lethomasites fossulatus

Erdtmanitheca texensis

Equisetosporites chinleana

Ephedra spp.

Cornetipollis reticulata Erdtmanispermum balticum Gnetum spp.

CAYTONIALES (OUTGROUP)

Caytonanthus arberi

206 Figure 138. Strict consensus cladogram for 4,934 trees, all with 34 steps, based on the data matrix in Table 3. Characters were treated as unordered and with equal weights.

207 Cycadeoidea dacotensis

Leguminanthus siiiquosus

Sahnia laxiphora

Equisetosporites sp. (Santana)

Ephedripites sp. 1

Ephedra spp.

Welwitschia mirabilis o 00 Gnetum spp.

Eucommiidites sp. 1

Erdtmanitheca texensis

Erdtmanispermum balticum

Equisetosporites chinleana

Cornetipollis reticulata

Lethomasites fossulatus

Caytonanthus arberi Figure 139. Fifty percent majority-rule consensus cladogram for 4,934 trees, aU with 34 steps, based on the data matrix in Table 3. Characters were treated as unordered and with equal weights.

209 Cycadeoidea dacoteiisis

Leguminanthus siliquosus

Sahnia laxiphora

Equisetosporites sp. (Santana)

Ephedripites sp. 1

■60- Ephedra spp. ■80 Equisetosporites chinleana K) t—& O •64- •95 Cornetipollis reticulata

Weiwitschia mirabilis

Gnetum spp.

■64 Eucommiidites sp. 1

100- Erdtmanitheca texensis

Erdtmanispermum balticum

Lethomasites fossulatus

Caytonanthus arberi Figure 140. Strict consensus cladogram for 19 trees, all with 68 steps, based on the data matrix in Table 3. Characters were treated as ordered and some were weighted either by two (nos. 2,11,12) or by three (nos. 8, 9,10, 14); see text for discussion.

2 1 1 Cycadeoidea dacotensis

Leguminanthus siliquosus

Sahnia laxiphora

Equisetosporites sp. (Santanci)

Equisetosporites chinleana

Cornetipollis reticulata

Ephedra spp.

Gnetum spp.

Eucommiidites sp. 1

Erdtmanitheca texen.sis

Erdtmanispermum balticum

Ephedripites sp. 1

Weiwitschia mirabilis

Lethomasites fossulatus

Caytonanthus arberi

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