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8703576

Kurmann, Marie Helena

POLLEN WALL ULTRASTRUCTURE AND DEVELOPMENT I SELECTED GYMNOSPERMS

The Ohio State University Ph.D. 1986

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POLLEN WALL ULTRASTRUCTURE AND DEVELOPMENT

IN SELECTED GYMNOSPERMS

DISSERTATION

Presented in Partial Fulfillment of the Requirements

for the

Degree Doctor of Philosophy in the Graduate School of

The Ohio State University

by

Marie Helena Kurmann, d i p l . Natw. ETH, M.S.

*****

The Ohio State University

1986

Reading Committee:

Dr. W.A. Jensen Approved by

Dr. G.L. Floyd

Dr. F.D. Sack L) JUjua---- Adviser Department of Botany @ 1986

MARIE HELENA KURMANN

All rights reserved Meinen Eltern gewidmet ACKNOWLEDGMENTS

I thank with deepest sincerity the members of the

Graduate Studies Committee of the Department of Botany for their time and support. I also wish to thank the members of my reading committee, Drs. William A. Jensen, Gary L.

Floyd and Fred D. Sack for their helpful suggestions and comments. Dr. Thomas N. Taylor, Department of Botany, and

Dr. Robert M. Pfister, Department of Microbiology, are acknowledged for providing facilities to carry out research. Special thanks go to my fellow graduate students

Kathleen Pigg, Louise Lewis, Linda Mull-Young and Finlay

Bryan and to Dr. Sara P. Stubblefield for their thoughtful assistance and inspirations. Most importantly, I thank my husband, Dr. Chris H ill, for his support and love, which made th is a l l worthwhile. VITA

October 10, 1955...... Born - Menznau, Luzern, Switzerland

1978 - 1979...... Graduate Teaching Assistant, Swiss Federal I n s titu te of Technology, Zfirich, Switzerland

197 9...... Diploma, Swiss Federal I n s titu te of Technology, Ztirich, Switzerland

1979 - 1981...... Student Exchange Fellow, Kansas S ta te U niversity, Manhattan, Kansas

1981...... Graduate Teaching Assistant, Kansas S ta te U niversity, Manhat t an , Kansas

1981...... M.S., Kansas State U niversity, Manhat ta n , Kansas

1981 - 1983...... Graduate Teaching Assistant, The Ohio S tate U niversity, Columbus, Ohio

198 2...... Student Scholarship Award, American A ssociation of Stratigraphic Palynologists

1983 - 1985...... Graduate Research Assistant, The Ohio S tate U niversity, Columbus, Ohio

1985...... Graduate School Alumni Research Award, The Ohio S tate U niversity Columbus, Ohio

1985 - 1986...... Presidential Fellow, The Ohio S tate U niversity, Columbus, Ohio PUBLICATIONS

Kurmann, M.H. & T.N. Taylor, 1984: Comparative ultrastructure of the sphenophyte spores Elaterites and Equisetum. Grana 23: 109 - 116.

Kurmann, M.H. & T.N. Taylor, 1984: The u ltra s tru c tu re of Boulaya fertilis (Medullosales) pollen. Pollen Spores 26: 109 - 116.

Kurmann, M.H., 1985: An opal phytolith and palynomorph study of extant and fossil soils in Kansas (U.S.A.). Palaeogeogr., P alaeoclim atol., Palaeoecol. 49: 217 - 235 .

Taylor, T.N. & M.H. Kurmann, 1985: Boulavatheca, th e new name for the seed fern pollen organ Boulaya. Taxon 34: 666 - 667.

Archangelsky, S., Taylor, T.N. & M.H. Kurmann, 1986: Ultrastructural studies of fossil plant cuticles: Ticoa harrisii from the early Cretaceous of Argentina. Bot. J. Linn. Soc. 92: 101 - 116.

Kurmann, M.H. & T.N. Taylor (in p ress): Sporoderm ultrastructure of Lophosoria and Cvatheacidites: systematic and evolutionary implications. PI. Syst. Evol.

Archangelsky, S., Taylor, T.N. & M.H. Kurmann (in p re s s ): Ultrastructural studies of fossil plant cuticles. II. Tarphvderma gen. n . , a Cretaceous conifer from Argentina. Amer. J. Bot.

PUBLISHED ABSTRACTS OF PAPERS PRESENTED AT PROFESSIONAL MEETINGS

Kurmann, M.H., 1980: Oekologische Untersuchungen von Trespen-Halbtrockenrasen in der Nordschweiz, mit besonderer Berticksichtigung der Wurzelsysteme. Ber. Geobot. Inst. ETH, Stiftg. Riibel, Ztirich, Heft 47: 26.

Kurmann, M.H., 1982: An opal phytolith and palynomorph study of extant and fossil soils in Kansas. Ohio J. Sci. 82: 18.

V Kurmann, M.H., 1982: The ultrastructure of Elaterites trlferens spores. Bot. Soc. Amer. Misc. Ser. Publ. 162: 59 - 60.

Kurmann, M.H. & T.N. Taylor, 1983: Carboniferous seed fern pollen ultrastructure: On the grains of Boulaya f e r t i l i s . Ohio J. S ci. 83: 17.

Kurmann, M.H. & T.N. Taylor, 1983: Sphenophytespore ultrastructure: A comparison between Elaterjtes and Equisetum. Ohio J. Sci. 83: 17 - 18.

Kurmann, M.H., 1983: The u ltra s tru c tu re of pollen extracted from Boulaya fertilis. Amer. J. Bot. 70{5), Part 2: 72-73.

Kurmann, M.H., 1984: Some aspects of p o llen wall development in Tsuqa canadensis. Ohio J. Sci. 84: 6 - 7.

Kurmann, M.H. , 1984: Pollen wall development in selected gymnosperms. Abstr. Int. Org. Palaeob'ot. Conf. , 2nd, Edmonton, Canada.

Kurmann, M.H., 1984: Pollen wall development in Tsuqa canadensis■ Abstr. Int. Palynol. Conf., 6th, Calgary, Canada: 85.

Kurmann, M.H., 1984: Sporoderm ontogeny in selected gymnosperm pollen g ra in s. Ann. Meet. Amer. Assoc. Stratigr. Palynol., 17th, Arlington, Virginia: 12.

Kurmann, M.H. & T.N. Taylor, 1985: The comparative ultrastructure of the fossil Cvatheacidites (Cretaceous) and extant Lophosoria spores. Ohio J. Sci. 85: 18 - 19.

Archangelsky, S., Taylor, T.N. & M.H. Kurmann, 1985: Estudios ultrastructurales de cuticulas fosiles del Cretacico de Patagonia, Argentina. Palaeobot. Symp., 6th, Tucuma'n, Argentina.

Kurmann, M.H., 1985: Comparative sporoderm ultrastructure of Cretaceous Cvatheacidites and extant Lophosoria. Amer. J. Bot. 72: 923 - 924.

Archangelsky, S., Taylor, T.N. & M.H. Kurmann, 1985: Ultrastructure of fossil plant cuticle. Amer. J. Bot. 72: 889.

vi Kurmann, M.H., 1985: Some evolutionary perspectives to the study of pollen and spore fine structure. Symposium on pollen ultrastructure. Ann. Meet. Amer. Assoc. Stratigr. Palynol., 18th, El Paso, Texas: 17.

Kurmann, M.H., 1986: Pollen wall development in Abies concolor (Gord. & Glendl.) Lindl. Amer. J. Bot. 73: 632.

FIELD OF STUDY

Major Field: Palynology

v ii TABLE OF CONTENTS

p age

DEDICATION...... ii

ACKNOWLEDGMENTS...... i i i

VITA...... iv

LIST OF TABLES...... X

LIST OF FIGURES...... xi

LIST OF PLATES...... x ii

CHAPTER

I. INTRODUCTION...... 1

Exine stratification...... 7 Exine stratification in extant gymnosperms...14 Exine development in extant gymnosperms...... 21 Glycocalyx concept...... 27

I I . MATERIALS AND METHODS...... '...... 33

M a te ria ls ...... 33 Light microscopy...... 34 Transmission electron microscopy...... 34 Scanning electron microscopy...... 36 Terminology...... 37

I I I . POLLEN WALL DEVELOPMENT IN ABIES CONCOLOR...... 39

Formation of the tetrads...... 41 Tetrad period ...... 44 Free spore period...... 46 Mature pollen wall...... 50 page

IV. POLLEN WALL DEVELOPMENT IN TSUGA CANADENSIS. ...7 0

Formation of the tetrads...... 72 Tetrad period ...... 73 Free spore period...... 76 Mature pollen w all...... 78

V. POLLEN WALL DEVELOPMENT IN TAXODIUM DISTICHUM. .97

Formation of the tetrads...... 99 Tetrad period ...... 100 Free spore period...... 101 Mature pollen w all...... 103

VI. DISCUSSION...... 119

Microspore mother cell surface coat...... 126 Callose wall...... 128 Microspore surface coat...... 131 E ktexine ...... 135 Endexine ...... 141 I n ti n e ...... 145 Tapetum...... 146 Homology of po llen wall la y e r...... s 147

VII. BIBLIOGRAPHY...... 154

ix LIST OF TABLES

TABLE PaSje 1. Time of appearance of tetrads in Abies concolor, Tsuqa canadensis and Taxodium distichum ...... 38

x LIST OP FIGURES

FIGURE p a g e

1. Exine stratification...... 3

2. The three major types of ektexines...... 4

3. Terminological scheme to describe sporoderm stratification...... 11

4. Exine structural types within the extant gymnosperms...... 22

5. Model of microspore surface coat and exine d eposition...... 31

139. Summary diagram of developmental stages of sporoderm in Abies concolor...... 120

140. Summary diagram of developmental stages of sporoderm in Tsuqa canadensis...... 122

141. Summary diagram of developmental stages of exine in Taxodium distichum ...... 124 LIST OF PLATES

PLATE p a g e

I. Microspore mother cells of Abies concolor in premeiotic stages (Figs. 6 - 14)...... 53

II. Meiotic stages of Abies concolor microspore mother cells (Figs. 15 - 20)...... 55

III. Early tetrad phase in Abies concolor (Figs. 21 - 3 0 )...... 57

IV. Late tetrad phase in Abies concolor (Figs. 31, 3 2 )...... 60

V. Early free spore phase in Abies concolor (Figs. 33 - 38)...... 62

VI. Sporopollenin accretion in Abies concolor during the free spore phase (Figs. 39 - 43)...... 64

VII. Mature pollen grains of Abies concolor (Figs. 44 - 50) ...... 66

VIII. Peritapetal membrane (Fig. 51) and sporoderm of acetolyzed pollen of Abies concolor (Figs. 52 - 5 5 ) ...... 68

IX. Formation of tetrads in Tsuqa canadensis (Figs. 56 - 60) ...... 81

X. Tapatal c e lls (Figs. 61, 62) and microspores (Figs. 63 - 65) of Tsuqa canadensis in early tetrad period ...... 83

XI. Tetrad period in Tsuqa canadensis (Figs. 66 - 7 1 )...... 85

XII. Early free spore phase in Tsuqa canadensis (Figs. 72 - 76) ...... 87 PLATE page

XIII. Late free score Dhase of Tsucra canadensis microspores (Figs. 77 - 80) and p e rita p e ta l membrane (Fig. 81) ......

XIV. Mature pollen arains of Tsuga canadensis (Figs 82 - 85) ......

XV. Sporoderm of mature pollen in Tsuga canadensis (Figs. 88 - 93)...... 93

XVI. Sporoderm of acetolvzed pollen in Tsuga canadensis (Figs. 94 - 101)...... 95

XVII. Microspore mother c e lls in Taxodium distichum (Figs. 102 - 107)...... 105

XVIII. Dvads and te tra d s in Taxodium distichum (Figs. 108 - 111)......

XIX. Proximal pollen wall formation in Taxodium distichum (Figs. 112 - 117) ...... 109

XX. Late tetrad phase in Taxodium distichum (Figs. 118 - 122)......

XXI. Free spore phase in Taxodium distichum (Figs. 123 - 128)......

XXII. Sporoderm of mature pollen in Taxodium distichum (Figs. 129 - 132)...... 115

XXIII. Mature pollen (Figs. 133 - 136) and p e rita p e ta l membrane (Figs. 137, 138) of Taxodium distichum ...... 117

xi i i C h a p te r I

INTRODUCTION

The origin of the flowering plants (Angiospermae),

referred to by Darwin as 'that abominable mystery1, remains

to this day a major evolutionary problem. Obstacles to the

study of angiosperm origin include the apparent scarcity of

relevant megafossils linking early angiosperms with other

gymnospermous seed plants and the absence of r e li a b le

features with which to identify them. Since microfossils are ubiquitous in the fossil record, the problem has been approached by studying pollen grains both of extant and extinct seed plants. Pollen morphology, including the fine structure of the wall termed the exine, has long been the subject of intensive study with light and, more recently, with electron microscopy. In the evaluation of pollen characters, researchers have concentrated mostly on the various morphological components of the exine since this wall incorporates a large number of taxonomically important

features. Moreover, owing to its resistance to decay and degradation, the exine is usually well preserved during

fossilization.

1 The mature ex in e consists of several la y e rs, and various schemes have been proposed to describe this s tr a tif ic a tio n . The schemes most widely used currently were in s titu te d by Paegri (1956) and Erdtman (1952, 1966).

Faegri (1956) distinguished two main components within the exine, the outer ektexine and the inner endexine, based on their differential stainability with basic fuchsin. He further subdivided the ektexine into tectum, columellae and foot layer. Erdtman (1952) proposed a system which divides the exine into an outer, sculptured sexine and an inner, non-sculptured nexine. In 1966, he further subdivided the nexine into an outer nexine 1 and an inner nexine 2 based on staining differences (Fig. 1).

With the initiation of phylogenetically oriented studies of pollen walls, it became apparent that there is considerable variation in the structure of pollen exines.

Van Campo & Lugardon (1973) e s ta b lish e d three major types of ektexines that they termed alveolar, granular and columellar (Fig. 2). The alveolar type is characterized by the partitioning of the sporopollenin units below the tectum into a system of lacunae. This type is c h a ra c te ristic of many gymnosperms and appears to be lacking in angiosperms (e.g., Lepous^, 1969a, Van Campo&

Sivak, 1972; Dickinson, 1976; Audran & Masure, 1977, 1978;

Zavada, 1983). The granular type is characterized by a layer of aggregated granules of sporopollenin beneath the 3

ERDTMAN (1952,1966) FAEGRI (1956)

e k to sex fn e

SEXINE

•n d o se x in e Intratectum EKTEXINE

footlayer

NEXINE

ENDEXINE

Fig. 1: Terminological schemes currently most widely used to describe exine stratification. tectum

infratectum

footlayer ALVEOLAR

tectum

infratectum

footlayer GRANULAR

tectum iii i i i infratectum footlayer COLUMELLATE

. 2: The three major types of ektexine recognized by Campo & Lugardon (1973). 5

tectum. This type is found both in gymnosperm and

angiosperm pollen (e .g ., Doyle et a l . f 1975; Roscher, 1975;

Walker, 1976; Le Thomas, 1980, 1981; Zavada, 1984b).

Columellar ektexines are characterized by a layer of

radially oriented rods beneath the tectum. Within the

extant seed plants, this type is considered to be

restricted to angiosperm pollen (e.g., Heslop-Harrison,

1968b; Walker & Doyle, 1975; Nowicke & Skvarla, 1979). As

pointed out by Doyle et al. (1975), there are also

different patterns in the endexine, in that mature

gymnosperm pollen may possess a lam ellated endexine as

opposed to the non-lamellated one of most angiosperm

pollen.

In reviewing the taxonomic and phylogenetic

significance of fossil dispersed pollen, Zavada (1984b)

indicated that this clear demarcation of pollen wall

structure between angiosperms and gymnosperms breaks down

among several Mesozoic taxa. He also pointed out that the

widely used systems to describe exine stratification have

been applied so loosely as to disregard and confuse

homologies of the various pollen wall layers among the

major groups of seed plants. In this respect, the value of

developmental studies has long been recognized in assessing homologies, and ontogenetic studies of pollen walls can be utilized in elucidating homologies between the various wall

layers of gymosperm and angiosperm pollen. 6

Development of the pollen wall has been extensively studied in a number of angiosperms, and these investigations have revealed a variety of wall deposition modes (e.g., see Hideux's (1979) extensive review for pre-1979 studies; Abadie & Hideux, 1980;. El-Ghazaly, 1982;

Rowley et al., 1981a; Owens & Dickinson, 1983; Southworth,

1983; Dunbar & Rowley, 1984; P e t t i t t e t a l . , 1984; Zavada,

1984a; Hideux & Abadie, 1985; El-Ghazaly & Jensen, 1986;

Rowley & Skvarla, 1986). Only a few gymnosperm taxa, however, have been investigated with respect to their sporoderm ontogeny (Lepouse", 1971; Willemse, 1971a-e;

Dickinson, 1971; Rohr, 1977; Audran, 1981; Moitra&

Bhatnagar, 1982; Zavada, 1983; Pennell & B ell, 1986) and these studies have presented evidence that there are differences between gymnosperms and angiosperms especially in the timing of wall layer deposition. In order to describe accurately the differences and to gain better insight into homologies, additional, detailed developmental data on extant gymnosperm p o lle n w alls a re needed. This would not only allow for the establishment of valid criteria with which to specify phylogenetic relationships, but would also provide a basis to interpret the fine structure of fossil pollen grains more critically.

The main objective of the present study, therefore, was to undertake an in depth study of pollen wall development in selected gymnosperms in order to give a 7 detailed account of the deposition modes and the timing of deposition of the various wall layers. The results are utilized in elucidating homologies between the various wall la y e rs in gymnosperms and angiosperms. Abies concolor

(Gord. & Glen.) Lindl., Tsuqa canadensis (L.) Carr, and

Taxodium distichum (L.) Rich., all members of the

C o n ife ra le s,, were chosen because th is order shows the greatest variation of exine morphological types within the extant gymnosperms. Abies concolor produces bi-saccate pollen grains and the mature ektexine is of the tectate-alveolar type. Tsuga canadensis pollen grains are non-saccate but show an equatorial frill suggestive of a rudimentary saccus. The ektexine has an interesting stratification in that it consists only of a tectum and footlayer and an infratectal layer is not elaborated.

Taxodium distichum produces non-saccate pollen and the ektexine is of the atectate-granular type. All three taxa have a lamellated endexine.

Exine stratification

The literature on exine stratification reveals a bewilderingly wide range of terms and interpretations. The terminology has been used inconsistently and this poses a problem for palynologists in trying to establish systematic and phylogenetic relationships utilizing exine stratification. As mentioned earlier, the two schemes currently most widely used for describing the stratification of the exine were introduced by Erdtman (1952) and Faegri (1956) (Fig.

1). These terminological schemes are based on the organization of angiosperm pollen walls as revealed by

light microscopy. Erdtman (1943) had earlier introduced

the terms ektexine and endexine for the morphologically

two-layered exine. Faegri and Iversen (1950) and Iversen &

Troels-Smith (1950) adopted this terminology. Faegri

(1956), however, redefined the exine subdivisions based on

their stainability with basic fuchsin. The outer layer or

ektexine sensu Faegri (1956) is stained intensely by basic

fuchsin and can be subdivided into three zones based on morphological features: tectum, columellae and footlayer.

The inner layer or endexine sensu Faegri (1956), is not or

only faintly stained by basic fuchsin.

In 1952, Erdtman revised his earlier terminology. He dropped the terms ektexine and endexine sensu Erdtman

(1943) and introduced new terms which he considered

topographically more accurate. He renamed the outer

'sculptured' layer of the exine the sexine and the inner

'non-sculptured' layer the nexine. He further subdivided

the sexine into an ektosexine (tegillum) and an endosexine

(baculae), and the nexine into ektonexine and endonexine.

In 1960, he further subdivided the ektonexine into nexine 1 and nexine 2, and substituted the term endonexine with nexine 3 (Erdtman, 1960a). In 1964, he revised his interpretation yet again and equated the term ectonexine with nexine 1. In 1966, he compared his terminological scheme with the one of Faegri (1956) and pointed out that his nexine 1 is equivalent to Faegri1s footlayer and his nexine 2 to Faegri's endexine.

The study of pollen wall morphology in taxa other than angiosperms, such as the conifers, and the advent of electron microscopy, led to a further proliferation of terms and their usage. This prompted several workers to review the subdivisions of the sporoderm used by various authors. Wittman & Walker (1965), for example, stated that the diversity of terms created for various parts of the exine, and their varied use, constitute serious obstacles to communication and to understanding the phylogenetic significance of the various layers. In order to clarify the problem, they provided a diagnostic key for identifying terms and they proposed to return to a simple nomenclature which follows the principles established by Iversen &

Troels-Smith (1950). This suggestion, however, overlooks the advances made as a result of cytochemical and ultrastructural studies. Reitsma (1970) suggested that a terminology based on ■interpretation1 (e.g. stainability, development, phylogeny) should be rejected. His attempt to unify terminology, however, is not very helpful if one is interested in biologically important aspects, such as 10 pollen wall homologies and the phylogenetic significance of the various wall layers.

In 1975, a European working group on the structure and terminology of sporopollenin walls (Cerceau-Larrival et al., 1975) proposed a general terminological scheme for the exine, and they provided some recommendations in the hope that the use of terms would become more uniform and thereby enhance communication and understanding. Their resolution is based on the systems of Erdtman (1952) and Faegri (1956) and is illustrated in Fig. 3. For the set of envelopes covering the cell contents of a pollen or spore they propose to use the term sporoderm. The sporoderm, in the case of pollen grains, is subdivided into two walls, the exine and the intine. The exine, consisting of sporopollenin, is acetolysis-resistapt and is preserved during fossilization. The intine, consisting of cellulosic material, is destroyed by acetolysis and is usually not preserved during fossilization. The term layer they restrict to parts of the wall with special features. The layers, in their opinion, should be subdivided into strata rather than sublayers. They also recommended to use the terms footlayer and endexine if it is possible to distinguish two strata within the nexine. In cases where it is impossible to recognize two strata, the term nexine should be retained. They did not specify, however, on what 11

WALL LAYER STRATA LAYER

s tectu m p 0 infratectum ektexine R EXINE 0 footlayer D nexine E endexine R

M INTINE INTINE

©

Fig. 3: Terminological schemes to describe exine stratification as proposed by Cerceau-Larrival et a l. (1975). (Modified from Cerceau-Larrival et al., 1975). 12 criteria the distinction of strata should be based, i.e. stainability, ultrastructure, and/or ontogeny.

The subdivision of the exine into ektexine and endexine proposed by Faegri (1956), based on their differential stainability with basic fuchsin, has been further substantiated by various other cytochemical tests.

Early TEM investigations confirmed a subdivision of the exine into ektexine and endexine because these wall layers also exhibit differential stainability with the TEM stains uranyl acetate and lead citrate (e.g., Larson et a l.

(1962), Larson (1964) and references quoted therein).

Using ultraviolet microspectrophotometry, Southworth (1969,

1983a) showed that the absorption spectrum of the endexine in Ambrosia and Gerbera pollen differs from that of the ektexine. Willemse (1972) investigated changes in the autofluorescence of pollen walls during development and after various chemical treatments and recorded that mainly the ektexine showed autofluorescence. Using the protein stains aniline blue-black and coomassie blue, a staining difference between ektexine and endexine was demonstrated in Gerbera pollen by Southworth (1973). Various researchers tested the solubility of pollen and spore walls using ethanolamine and these studies established that the endexine is usually more resistant to hydrolysis by ethanolamine than the ektexine. Southworth (1974), testing the solubility of pollen and spore exines of 40 genera, 13

showed that the ektexine of fresh angiosperm and gymnosperm pollen is readily dissolved in ethanolamine, while the endexine does not dissolve. Pollen exines of aged gymnospermous and monocotyledonous pollen, however, are more difficult to dissolve. Combining differential hydrolysis with selective staining, Kress & Stone (1982)

established that the exine of most monocotyledons is

ektexinous in composition. Rowley and his collaborators

showed that dissection of the exine exposes a so-called

glycocalyx (e.g., Rowley, 1978, 1981; Rowley et al. 1981a,

b). They suggested that the stainability of the exine,

particularly the endexine, is dependant on the 'transfer of

nutrition, recognition, etc. molecules' in the final stages

of pollen development. In discussing exine stratification,

Kress (1986) suggested that cytochemical criteria might be

equally or more useful than ontogenetic evidence in

distinguishing strata by pointing out a conflict between

chemical and ontogenetic criteria in the genus Ipomoea.

The ontogeny of pollen walls has been used as another

criterion to characterize exine stratification.

Heslop-Harrison (1971), for example, discussed developmental sequences in the formation of pollen walls in

angiosperms. He summarized earlier investigations by

stating that the ektexine is usually formed during the

tetrad phase, while the endexine is accumulated in the free spore phase, after dissolution of the callose wall. This same observation prompted Godwin et al. (1967) to divide the exine of Ipomoea into a primary exine and a secondary exine. The primary exine (tectum and columellae) develops during the tetrad phase and is formed on a so-called primexine matrix. The secondary exine, however, forms in the free spore phase by a different process. They pointed out that the secondary exine exhibits the same electron density as the primary exine. Southworth (1983b), also using developmental timing in naming strata, referred to the exine layers in Gerbera as exine-1 and exine-2. The exine-1 is patterned during the tetrad phase and the exine-2 is synthesized in the free spore phase. Zavada

(1984b) also used ontogenetic criteria in his discussion of exine stratification. Since the development of the exine in most gymnosperms is completed during the tetrad phase, he recognized merely sexine and nexine for the gymnosperms on this ontogenetic basis. According to his interpretation, therefore, no endexine is formed in gymnosperms. He excluded the use of the term endexine for the morphologically identical layer in gymnosperms purely because of differences in developmental timing. As a result he maintains that the ektexine of angiosperms is homologous with the entire exine in gymnosperms. 15

Exine stratification in extant gymnosperms

In the following comparison of exine stratification in extant gymnosperm pollen, most of the published electron micrographs have been examined. In order to facilitate comparison, Faegri's terminology will be used in instances where two strata can be distinguished within the non-sculptured layer even if the authors used another terminological system. In the cases where no such differentiation can be made, the term nexine is retained as suggested by Cerceau-Larrival et al. (1975). The extant gymnosperms are classified into four orders (Knoll &

Rothwell, 1981): Cycadales, Ginkgoales, Coniferales and

G n e ta le s.

Cycadales - The exine ultrastructure of mature pollen in the Cycadales has been illustrated by a number of researchers (Afzelius, 1956; Ueno, 1960; Larson, 1964;

Gullv&g, 1966a, b; P ettitt, 1966; Audran, 1980; Audran &

Masure, 1976, 1977). Audran & Masure (1976, 1977) examined representatives of most genera. They post-stained their sections with potassium permanganate which did not reveal a difference in stainability between the outer and inner wall layers. Therefore they used the terminological scheme of

Erdtman (1952) and divided the exine into an outer sexine and an inner nexine. The sexine is composed of two strata, the ektosexine and the endosexine. The ektosexine in cycads is a homogeneous layer which covers the endosexine. 16

The endosexine consists of elements similar to the columellae of angiosperms, but they fork and anastomose.

The nexine is three-layered and homogeneous all around the spore body. The sexine elements rest on the outermost part of the nexine (nexine 1) which is irregular in thickness.

The middle layer (nexine 2) is electron transparent and of uniform thickness (approx. 6 nm). The innermost nexine

layer (nexine 3) is narrow and lamellated and reveals white

lines. Sometimes this layer becomes mixed with the intine.

In their discussion these authors pointed out that the

interpretation of the nexine in cycadalean pollen is

debatable, but they reject the presence of a footlayer as

interpreted by Larson (1964), because of differential

susceptibility of the sexine and nexine to degradation by

acetolysis (Audran, 1978a, 1980). However, if the

stainability criterion is applied, the nexine 1 (sensu

Audran) equals the footlayer (sensu Larson) and the nexine

3 {sensu Audran) equals the endexine.

Audran & Masure (1976) remarked that the term

'alveolar infrastructure' (Van Campo, 1971) should not be applied rigidly to the endosexine of cycads, because there are some differences from the alveolar structures of saccate gymnosperms. The lacunae and rods, in radial section, appear relatively regular, more like the columellae of angiosperms than the infrastructure in the wall of saccate conifer pollen. 17

Ginkgoales - The exine ultrastructure of mature pollen walls in the Ginkgoales has been studied by Ueno (1960) and

A udran & Masure (1978). The exine in Ginkgo biloba is composed of an ektexine and an endexine. The ektexine is further subdivided into a tectum, an infratectum and a footlayer. The endexine is thick and strongly lamellated.

A udran & Masure (1978) wrestled with the terminology and decided that Faegri's (1956) scheme is better adapted for describing exine stratification in Ginkgo. Ueno divided the exine into sexine and nexine, and he further subdivided the nexine into an ektonexine and an endonexine. From his illustration it is apparent that his ectonexine can be referred to as the footlayer and his endonexine as the en d e x in e .

Coniferales - Exine stratification in the Coniferales is more diverse than in the Cycadales and Ginkgoales. The mature pollen grains of some species of all extant families of conifers have been investigated ultrastructurally.

These families are the Pinaceae, Taxodiaceae, Cupressaceae,

Araucariaceae, Podocarpaceae, Cephalotaxaceae and Taxaceae

(F o s te r & Gifford, 1974).

Pinaceae - Electron micrographs of the pollen wall in the following genera have been published: Abies (Ueno,

1958; Lepouse, 1969a), Cedrus (Ueno, 1959; Gullv&g, 1966a),

Larix (Ueno, 1958; Gullv&g, 1966a), Picea (Ueno, 1958;

Erdtman, 1965), Pinus (MGhletaler, 1955; Ting & Tseng, 18

1965; Pettitt, 1966; Hess et al,, 1973; Litvintseva, 1979),

Phvllocladus (Pocknall, 1981b), Pseudotsuqa (Ueno, 1958;

GullvSg, 1966a) and Tsuga (Ueno, 1958; Sivak 1973), In

Abies. Cedrus. Picea, Pinus and Phvllocladus. the pollen grains are bi-saccate and the exine consists of an ektexine and an endexine. The ektexine is subdivided into tectum, alveolar infratectum, and footlayer. The endexine is lamellated and differs from the ektexine as evidenced by its different staining reaction with TEM stains. The only apparent exception is found in Pinus balfouriana where the endexine is reported to be granular (Ting & Tseng, 1965).

In Larix and Pseudotsuqa, the pollen grains are non-saccate and the exine consists of an outer, granular layer, and an inner, lamellated one. Gullv&g (1966a) did not in fact refer to these layers using either Erdtman's or Faegri's scheme, but it is reasonable to term the granular layer the ektexine and the lamellated layer the endexine. In Tsuga, one species produces saccate pollen and the others non-saccate ones. The non-saccate grains show a 'frill' around the equator, which has sometimes been referred to as a vestigial saccus (Bassett et al., 1978). The exine consists of an outer ektexine and an inner, lamellated endexine. The ektexine shows an interesting stratification in that no infratectal layer is present; it consists only of a convoluted tectum and a footlayer. 19

Taxodlaceae - Exine ultrastructure has been studied in mature grains of the following genera: Cryptomeria

(Roscher, 1975; Sohma, 1985), Cunninghamia (Roscher, 1975),

Metasequoia (Roscher. 1975; Sohma, 1985), Sciadopitvs

(Roscher, 1975), Sequoia (Gullv§g, 1966a; Roscher, 1975;

Kedves, 1985), Sequoiadendron (Roscher, 1975), Taiwania

(Roscher, 1975) and Taxodium (Roscher, 1975). The pollen wall in these taxa is divisible into an outer ektexine and an inner, lamellated, endexine. The ektexine consists of a granular layer and sometimes in addition a footlayer can be recognized. A tectum is absent. In most of the genera, orbicules adhere to the outer surface of the granular la y e r.

Cupressaceae - Mature pollen wall stratification of the following genera has been'described: C allitris

(Roscher, 1975), Cupressus (Van Campo& Lugardon, 1973;

Lugardon, 1978), Juniperus (Ueno, 1959; Van Campo&

Lugardon, 1973), Thu.ia (Roscher, 1975) and Libocedrus

(Pocknall, 1981c). The exine consists of an outer, granular ektexine and an inner, lamellated endexine as in the Taxodiaceae.

Araucariaceae - Published electron micrographs of exine stratification include the genera Aqathis (Van Campo

& Lugardon, 1973) and Araucaria (Ueno, 1959; Van Campo&

Lugardon, 1973). The pollen wall shows an outer, granular ektexine and an inner, lamellated layer. 20

Podocarpaceae - Pollen grains of extant podocarps are

saccate. The exine ultrastructure of the following genera has been investigated: Acmopyle (Gullv&g, 1966a, b),

Dacrvcarpus (Pocknall, 1981a), Dacrvdocarpus (Pocknall,

1981a) and Podocarpus (Ueno, 1960; Pocknall, 1981a). The

exine shows a similar stratification to that of saccate pollen of the Pinaceae. The ektexine consists of a tectum,

an alveolar infratectum and a footlayer. The endexine is

lamellated.

Cephalotaxaceae - The sporoderm of Cephalotaxus has

been investigated by Ueno (1959), Gullvtg (1966a), and

Roscher (1975). It consists of a granular ektexine and a

lamellated endexine. A tectal layer in the ektexine is not p r e s e n t .

Taxaceae - Exine ultrastructure has been studied in __

Taxus (Gullv&g, 1966a; P ettitt, 1966, Roscher, 1975) and

Torreva (Ueno, 1959; Roscher, 1975). Their exines consist of a granular ektexine and a lamellated endexine.

From this survey of exine stratification in the

Coniferales it can be concluded that there are two basic exine structural types within this order of gymnosperms.

The saccate pollen grains have a three-parted ektexine with an alveolar infratectum. The non-saccate pollen grains have an ektexine which is granular. The endexine in both

types is lamellated. The only exception to these two types 21 is found in Tsuga, in which no infratectal layer is present between the tectum and the footlayer.

Gnetales - Electron micrographs of pollen walls for all genera of the Gnetales have been published as follows:

Ephedra (Erdtman, 1957; Ueno, 1960; Gullv&g, 1966a; Van

Campo & Lugardon, 1973; Hesse, 1984), Gnetum (GullvSg,

1966a; Hesse, 1980) and Welwitschia (Ueno, 1960; GullvSg,

1966a; Hesse, 1984). The exine is stratified into an outer ektexine, which consists of a tectum, a granular infratectal layer and a footlayer. The endexine is less electron dense than the ektexine and is lamellated. \ Summary - There are three basic groups of exine structural types within the extant gymnosperms (Fig. 4):

a. Ektexine tectate-alveolar Endexine lamellated - cycads - Ginkgo - saccate conifers

b. Ektexine atectate-granular Endexine lamellated - non-saccate conifers

c. Ektexine tectate-granular Endexine lamellated - G n etales

Exine development in extant gymnosperms

Sporoderm ontogeny has been published for selected members of the orders Cycadales, Ginkgoales and

Coniferales. In the following literature review, Ektexine tectate-alveolar

Endexine lamellated

'"Ektexine atectate-granular

Endexine lamellated

©

ta t Ektexine tectate-granular

I-Endexine lamellated

©

Fig. 4: Exine structural types within the extant gymnosperms (fl = footlayer; it = infratectum,; t = te c tu m ). 23 development of the exine in the non-apertural regions will be considered only.

Cycadales - Pollen wall development in Ceratozamia mexicana (Audran 1971, 1974, 1978b, 1979, 1980, 1981) and

Zamia floridana (Zavada, 1983) has been investigated ultrastructurally. These studies show that all the sporopolleninous wall layers in this group are deposited during the tetrad phase. In the tetrad phase, a dispersed fibrillar material is first deposited in the periplasmic space between the callose wall and the plasma membrane in the non-apertural zones. Where the plasma membrane remains

in contact with the callose wall, callosic extensions appear. They anastomose and are of various lengths. The appearance of osmophilic globules to the inside of the callose wall and next to the callosic extensions marks the initiation of the tectum and ipfratectum. The nexine is formed shortly before release from the tetrad by apposition of electron dense plates. These plates fuse to form a thin, lamellated layer around the spore body. In the free spore phuse no additional sporopolleninous wall layers are formed, but additional sporopollenin of tapetal origin is accumulated on the exine. This leads to a thickening of the exine at its outer surface but no modification of the infrastructure occurs.

Ginkgoales - Rohr (1977, 1980) investigated sporoderm ontogeny in Ginkgo biloba. He reported that the microspores are united in the tetrad for a long period of time after meiosis. The first deposition of the exine occurs in the proximal area of the microspores during the tetrad stage as recognized by the appearance of the tectum and an infratectal layer. The tectum is deposited to the inside of the callose wall and a fibrillar matrix is deposited into the periplasmic space. At the same time as the infratectal layer forms, the first indications of the nexine appear. The footlayer (nexine 1) shows the same structure as the tectal and infratectal elements. The endexine (nexine 2) is lamellated. Rohr (1977,1980), unfortunately, did not specify if the endexine forms in the tetrad or in the free spore phase, and this information cannot be determined from his illustrations.

Coniferales - Pollen wall developmental studies in genera of the Pinaceae, Podocarpaceae and Taxaceae have been published.

Pinaceae - The bi-saccate pollen of Abies pinsapo

(Lepousd, 1966, 1969a, 1969b, 1970, 1971), Pinus banksiana

(Dickinson, 1971, 1976; Dickinson& Bell, 1970, 1972,

1976a, b) and Pinus svlvestris (Willemse, 1971a-e) has been studied in detail at the ultrastructural level. In Abies pinsapo (Lepouse, 1970), a substance organized as so-called

'structural elements1 is deposited between the callose wall and the plasma membrane in the tetrad phase, both in the proximal and lateral areas. Where the plasma membrane 25

remains in contact with the callose wall, callosic

extensions appear which provide part of the template for

the alveolar layer. This is followed by deposition of

osmophilic globules marking the formation of the ektexine.

The lamellated endexine appears almost at the same time.

Dickinson (1971, 1976) and Dickinson & Bell (1970,

1972, 1976a, b) also reported that all the sporopolleninous wall layers in Pinus banksiana are formed during the tetrad

phase. The space between the early tectal and infratectal

elements, and the plasma membrane contains dispersed

fibrillar material and this space is much enlarged in the

sacci region. Early tectum and infratectum formation is

followed by the deposition of a lamellated layer.

Immediately after the formation of this lamellated layer,

callose dissolution takes place. Accretion of additional

sporopollenin to the already patterned exine occurs in the

free spore phase. An examination of the illustrations of

the mature sporoderm in Pinus banksiana shows the presence

of a "footlayer" between the alveolar infratectum and the

lamellated layer. However, there is no discussion in the

relevant papers as to how this layer forms. Dickinson

(1971) apparently considered it as part of the sexine.

Exine development in Pinus svlvestris (Willemse.

1971a, b) apparently differs somewhat from that in Pinus

banksiana. During the early tetrad phase, callosic

protrusions are formed on the inner surface of the callose 26 wall and these provide part of the template for the formation of the tectum and the infratectal layer. The periplasmic space is filled with a fibrillar material which gradually changes into a network structure. In the late tetrad phase, the footlayer (nexine 1) forms parallel to the plasma membrane by apposition of long plate- or tape­ like extensions originating from the microspore cytoplasm.

The nexine 2 has the same origin as the footlayer, but it appears in the early free spore phase after breakdown of the callose wall. In the mature exine, nexine 2 can no longer be distinguished from nexine 1.

Podocarpaceae - Vasil & Aldrich (1970, 1971) published accounts of pollen development in Podocarpus macrophyllus. They were especially interested in the organelles involved in the process of wall formation. The sexine and part of the nexine 1 (= endexine) are formed during the tetrad phase. Trilaminar tapes of unit membrane dimensions appear to be involved as sporopollenin deposition sites in the formation of these layers. After dissolution of the callose wall, in the free spore phase, nexine layers 2 and 3 are formed by coalescence of granular particles of sporopollenin.

Taxaceae - Pollen wall development in Taxus baccata has been investigated by Pennell & B ell.(1986) and Rohr

(1977, 1980). These two reports contradict each other in respect to the timing of wall layer deposition. Rohr 27

(1977, 1980) stated that the exine develops early in the tetrad phase. Initially, lamellate structures are produced outside the plasma membrane which constitute the nexine.

Simultaneously, osmophilic globules are deposited outside the nexine to form a granular layer. In the free spore phase, the granular layer becomes thicker as a result of further deposition of sporopollenin spherules. Pennell &

Bell (1986), however, reported that no wall deposition occurs during the tetrad phase. By mid-December the sporangia were filled with rounded microspores that show no exine and by the end of December the free microspores had developed a wall. This wall consists of an outer granular layer and an inner layer. This inner layer consists mostly of osmophilic material arranged in lamellae in which white lines are often evident.

Summary - There are variations in exine development among extant gymnosperms. In the alveolar exine types the tectum and infratectum are formed against the surrounding callose wall and the callosic extensions. In the granular exine types, the ektexine appears to be deposited mostly in the free spore phase. A fibrillar or network-like matrix that is always present in the alveolar exine types between the callose wall and the plasma membrane during the tetrad stage is not apparent in the granular exine types. The lamellated layers are formed on plates or tapes of unit membrane dimensions originating from the cytoplasm or the 28 plasma membrane, and depending on the exine type are deposited during the tetrad phase or after release from the t e t r a d .

Glycocalyx concept

In his study of pollen wall ontogeny in Silene,

Heslop-Harrison (1963) described the occurrence of a fibrillar matrix between the callose wall and the plasma membrane in the early stages of exine formation. Since this material acts as a template, or 'pattern-determinant1, for the exine, he referred to it as the primexine. This fibrillar material has been observed in most pollen grains during the early stages of exine formation and its chemical nature has been investigated with various techniques. In

Lilium (Heslop-Harrison. 1968a) and Nelumbo (Flynn, 1971) the primexine was shown to consist of cellulose. Waterkeyn

& Bienfait (1978) indicated that in Ipomoea, however, the endexine consists of noncellulosic polysaccharides and in

Teucrium (Nabli, 1979) it appears to be callosic. Rowley

(1973), using a variety of histochemical tests, demonstrated that the polysaccharidic coat in Epilobium and

Chamaenerion consists of rod-shaped units and is a specialization of the plasma membrane. He indicated that this matrix is present throughout the development of the sporoderm. On this basis, he proposed a new, broader concept: the plasma membrane - glycocalyx concept. 29

The term 'glycocalyx' was introduced by Bennett (1963) to describe the extracellular coating of many plant and animal cells. The glycocalyx always contains polysaccharides and a mixture of other substances (Bennett,

1969). The glycocalyx concept provides an integrated view of the organization of extracellular structures common to many types of cells. The structural characteristics of glycocalyces may show enormous variation (Bennett, 1969) and the pollen wall is but a special example.

Rowley and his collaborators elaborated on the chemical and structural nature of the glycocalyx in pollen exines (e.g. Rowley, 1973, 1976, 1978, 1981; Rowley& D ahl,

1977, 1982; Rowley et al., 1981a, b; Rowley& Skvarla, 1974;

1975; Rowley & Prijanto, 1977; Dunbar & Rowley, 1984). The glycocalyx of pollen exines consists of acidic and neutral polysaccharides, proteins and lipids. Based on their developmental studies of pollen walls and dissection of mature exines they described substructural units and proposed models for the formation of the exine (Fig. 5).

In Rowley's opinion (1981), the exine consists of rod-shaped units or tufts (ca. 50 - 100 nm in diameter and up to 1 pm or more in length). These tufts consist of two subunits, supercoiled helical subunits, called binders or gyres, and straight helical subunits, called cores (Fig.

5a). In the earliest phase of exine formation, the microspores are covered by a mat of coiled glycocalyx units or tufts (Fig. 5b). Then the probacules, tectum and nexine become differentially stainable and stabilized through envelopment with sporopollenin (Fig. 5c). After release from the tetrad, through the volume increase of the microspores, the surface may become distorted and gaps are thus created in the tectal and infratectal elements.

Distortion in the nexine is not apparent because addition of new glycocalyx units and their coating with sporopollenin fills the gaps. In dissected exines, the substructures of ektexine and endexine appear to be the same and the lamellation seen in untreated endexines is not present. The model accounts for these lamellae by alignment of the binders of the tufts as well as larger spaces between the subunits (Fig. 5d). This model provides an interpretation of pollen wall formation and furnishes a basis to discuss variation during ontogeny.

Southworth (1985a, b, 1986) also illustrated pollen exine substructures. Her micrographs, however, give no evidence of helical subunits. Dissection of exines with ethanolamine indicated that the substructure is formed of discrete, interconnected granules arranged in irregular polygons. Further extraction breaks the connections between the granules and leads to compound and open polygons. Her interpretation is that the substructure is a three-dimensional lattice without specific orientation. Fig. 5: Model of microspore surface coat and exine deposition (redrawn from Rowley, 1981). a. Glycocalyx unit or tuft consisting of core subunits and binder/gyre subunits. b. Glycocalyx covering the microspore surface before sporopollenin accretion begins, c. Exine in tetrad period; glycocalyx units become covered with sporopollenin. d. Exine in the early free spore phase.

/ 32

-,-core

iycocalyx

9V*/ binder

© ^tectum

infratectum

nexine

infratectum

foot layer end exine

© Chapter II

MATERIALS AND METHODS

MATERIALS - Microsporangiate stro b ili of Abies concolor (Gord. & Glen.) Lindl. (white fir), Tsuqa canadensis (L.) Carr, (eastern hemlock) and Taxodium d is tic h u m (L.) Rich, (bald cypress) were collected from trees in cultivated stands at the Mirror Lake area of The

Ohio State University campus, Columbus. Collecting was done over two successive years. During January and

February of the first year, each taxon was sampled at

2-week intervals. From the beginning of March until the appearance of free microspores, material was collected every third day, and subsequently at weekly intervals until shedding of the mature pollen grains. In the second year, material was also collected every third day, but beginning only two weeks before the anticipated date of the tetrad formation as determined from work on the previous year's material (Table 1), and ending once the microspores entered the free spore phase. Twigs bearing microsporangiate strobili were excised from both the lower and upper halves of the tree and taken to the laboratory. There they were

33 34 processed immediately for optical and electron microscopy according to the following procedures.

LIGHT MICROSCOPY - Developing buds were excised from

the shoots and the larger bud scales removed. The strobili were then cut in median longitudinal section and transverse

section with a razor blade and the segments were plunged

immediately into formalin - acetic acid - alcohol fixative

(FAA) for 48 hours. After this, the samples were

transferred to fresh FAA and stored until a number of

samples were available for further treatment. For such

further processing the fixed buds were dehydrated in a

tertiary butyl alcohol series and embedded in "Paraplast" according to standard procedures as outlined in Berlyn &

Miksche (1976). Sections were cut on a rotary microtome at

8 /rm and stained for two hours with 1% aqueous safranin

followed by 30 seconds of fast green. Permanent slides were prepared using Coverbond mountant (Hercules, Inc.) and

the sections were viewed and photographed with a Zeiss

U ltr a p h o t.

TRANSMISSION ELECTRON MICROSCOPY - The p o lle n cones were separated from the shoots and the bud scales removed as outlined above for light microscopy. In order to limit dessication, the material was immersed in cacodylate buffer at pH 6.8 containing 2% sucrose during dissection. With the aid of a scalpel and dissecting needles, microsporangia were isolated from the cone axis and cut into small pieces to ensure uniform fixation. These segments were fixed immediately for 2 hours at 4°C in a 5% solution of glutaraldehyde in cacodylate buffer at pH 6.8 containing 2* sucrose. The samples were subsequently rinsed twice with the buffer for 15 minutes and post-fixed in 1$ osmium tetroxide for 90 minutes at room temperature. The tissue was rinsed again in buffer (two 15 minute changes) and dehydrated in a graded ethanol series (10*, 25*, 40*, 50*,

60*, 75*, 85*, 95*) a t 20 minute in te rv a ls followed by two

20 minute changes in 100* ethanol and two 20 minute changes in 100* acetone, a l l a t room tem perature (approx. 24®C).

After dehydration the material was transferred to a 3:1 acetone and Spurr resin mixture (Spurr, 1969) and rotated on a "Roto-Torque" rotating apparatus for ten hours. This was followed by transfer to a 1:1 mixture of acetone and

Spurr resin for 10 hours, and subsequently to a 1:3 acetone and Spurr resin mixture, rotating for 10 hours in each mixture. The tissue was then placed in pure Spurr ('firm mixture1) and rotated for an additional ten hours. The samples were finally placed in fresh Spurr resin in flat aluminium dishes and polymerized in a vacuum oven for 12 -

20 hours (60°C, 380 mm Hg). Specimens selected for sectioning were cut out of the resin wafers with a jeweler's saw, oriented in the desired planes and cemented to Spurr blocks with Elmer's Wonderglue. The blocks were trimmed with a diamond mill cutter on an automatic block 36 trimmer (Reichert TM 60) and ultrathin sections in the pale gold range were cut on an AO ultracut microtome using a

DuPont diamond k n ife. S ections were c o lle c te d on formvar coated copper grids and stained with various combinations of 1.535 potassium permanganate (KMnO*) 235 uranyl acetate

("UA") and lead citrate ("Pb"). Details of the staining schedules are given in the figure captions since they vary for the various developmental stages. The sections were examined with a Hitachi H-300 transmission electron microscope operated at 75 kV.

Some of the mature pollen grains were acetolyzed according to the procedure of Erdtman (1960b) before embedding. Both acetolyzed and non-acetolyzed mature grains were drawn onto a 'Milipore' filter under suction in order to facilitate handling of the material. The filters with the adhering spores were coated on both sides with agar and the entire disc passed through the procedures outlined above. The acetone stage dissolved the filters so that the pollen entrapped within the agar discs was embedded in the Spurr resin .

SCANNING ELECTRON MICROSCOPY - This approach was employed to study the surface morphology of free microspores and mature pollen grains. One half of these grains was acetolyzed according to the procedure of Erdtman

(1960b) and the other half fixed with glutaraldehyde and osmium tetroxide as outlined above for transmission electron microscopy. In order to minimize handling such as centrifugation, the material was then enclosed in dialysis tubing, dehydrated in a graded ethanol series (25*, 40*,

50*, 60*, 75*, 85*, 95* and 100*; each for 30 m inutes) and critical point dried (CPD) in an Autosamdri-820 CPD apparatus. The material was then dusted on standard specimen stubs covered with high vacuum wax (Wachtel,

1980). The stubs were then coated with 10 - 20 nm of gold applied with a Hummer 8 III sputter coater and viewed in a

Hitachi HS—500 scanning electron microscope.

TERMINOLOGY - The terminological scheme as firs t defined by Faegri (1956) and the recommendations outlined by C erceau-Larrival et a l . (1975) and illu s tr a te d in Fig. 3 are adopted in this study. 38

Table 1: Time of appearance of tetrads in the microsporangia of Abies concolor. Tsuqa i canadensis and Taxodium distichum as observed in the spring of 1983 on The Ohio S tate U niversity campus, Columbus.

Taxa | Tetrads 1 1 - * ~ ~ - 1 Abies concolor | April 16 1 1 Tsuaa canadensis j March 22 ■ 1 Taxodium distichum | March 2 1

\ Chapter III

POLLEN WALL DEVELOPMENT IN ABIES CONCOLOR

The genus Abies comprises about 50 species, most of which are important forest trees (Dallimore & Jackson,

1967). Their wide distribution and commercial value is reflected in the large volume of literature concerning them. Many of these reports provide information about aspects of the reproductive cycle in various species of the genus (e.g., Owens & Molder, 1977b; Singh, 1978; Singh &

Owens, 1981, 1982).

Microsporogenesis and microgametogenesis in a number of species of Abies have been investigated, e.g. by

Hutchinson (1914), Mergen& Lester (1962), Kanter & Chira

(1965), Powell (1970), Owens & Molder (1977a, b), Konar &

Nagmani (1980), Singh & Owens (1981, 1982), and Owens&

Singh (1982). These studies have shown that microstrobili are differentiated during the summer and fall of the year before pollen development takes place, and that meiosis in the m icrosporangia of Abies is a postdormancy phenomenon.

After breaking of dormancy, the microsporangia enlarge, the microspore mother cells become separated from one another and they enter meiosis. Meiosis terminates with the

39 simultaneous formation of partitioning callose walls during telophase II. The microspores remain in tetrahedral tetrads for one to two weeks. After release from the tetrad, they undergo mitotic divisions and the pollen grains are shed at the 5-celled stage.

Mature pollen grains of Abies are bilateral, analept, and bi-saccate. Their pollen walls have been investigated by lig h t microscopy (e .g ., Wodehouse, 1935; Van

Campo-Duplan, 1950; Erdtman, 1957, 1965; Van Campo &. Sivak,

1972; Sivak 1975), scanning electron microscopy (e.g.

Martin & Drew, 1969; Ho, 1972; Van Campo& Sivak, 1972;

Bagnell, 1975; Sivak, 1975), and by transmission electron microscopy (e.g., Ueno, 1958, Lepous^, 1969a). The surface is coarsely granular and a faint triradiate mark is present on the proximal surface. The distal surface is smooth or slightly warty. The exine consists of a three-parted ektexine and a lamellated endexine. In the distal area the ektexine is reduced to a very narrow layer or is absent.

The sacci are rather small and they form through separation of the tectal and infratectal layer from the footlayer.

The development of the pollen wall in Abies is known for only one species at the ultrastructural level (Lepouse,

1966, 1970, 1971). In this species, A^_ pinsapo. pollen wall formation is initiated shortly after the deposition of the partitioning callose walls. So-called 'structural elements' (Lepouse, 1966) are deposited between the callo se 41 wall and the plasma membrane of the microspores and callosic extensions also form in the periplasmic space.

Elaboration of the exine occurs on these structures during the tetrad phase. The endexine appears almost at the same time as the ektexine, through the apposition of lamellae.

In the free spore phase, additional sporopollenin is accumulated on the exine, but no modification occurs of the structures established during the tetrad phase.

The purpose of this study was to describe in detail the pollen wall development in another species of Abies. A. concolor, at the ultrastructural level, and to compare the observed developmental patterns with those of other gymnospermous pollen w alls.

FORMATION OF THE TETRADS - The sporogenous tiss u e of dormant microsporangia in Abies concolor consists of uninucleate tapetal cells and microspore mother cells (MMC) in the premeiotic stage. The sporangial cavities are filled with compactly arranged MMC, surrounded by tapetal cells and by several layers of sporangial wall cells (Figs.

6, 7). Plasmodesmata interconnect the sporogenous cells

(Fig. 8). Dormancy is broken a t the beginning of March and mitosis is initiated in the tapetal cells. The MMC begin to separate as a result of enlargement of the microsporangia and by the breakdown of their primary cellulosic cell walls (Fig. 9). This separation destroys the already altered plasmodesmata (Fig. 10). The MMCs 42 produce a new, thin coating which is deposited around the I plasma membrane as is shown in two neighboring MMCs in Fig.

11. This surface coat consists of electron dense, fibrillar material. At this stage of development, the microspore mother cell cytoplasm is characterized by the presence of dilated endoplasmic reticulum and an abundance of dictyosomes (Figs. 12, 13). The nucleus enlarges and i t s envelope shows a large number of complex nuclear pores

(Fig. 14). Following the total lysis of the cellulosic wall the MMC become completely free within the enlarging microsporangial cavity and their organelle distribution is polarized (Fig. 15). Subsequently, additional material in the form of fine fibrils is deposited on the MMC surface external to the plasma membrane u n til a continuous, dense, fibrillar network is formed. During this secretion process, large vesicles with fine fibrillar material fuse / with the plasma membrane and deposit their contents in the perimembranous space (Fig. 17). This indicates that the fibrillar material is synthesized inside the MMC and transported by vesicles. The origin of the fibril-containing vesicles is unknown, but the presence of numerous dictyosomes and associated vesicles derived from them suggests involvement of the Golgi apparatus (Fig. 16).

The f i b r i l s become organized in to a perpendicular orientation relative to the cell surface (Fig. 17). 43

The tapetum in concolor is of the secretory type.

The tapetal cell walls (TC) begin to degrade at this stage and the tapetal protoplasts retreat from the wall. In the tapetal cytoplasm, large vesicles with granular contents are visible, and the same material is also found outside the ta p etal c e ll plasma membrane (Fig. 19). Osmophilic particles, which are most likely pro-orbicules, also appear outside the ta p e ta l c e ll plasma membrane (Fig. 18). With the appearance of pro-orbicules, osmophilic material is also deposited onto the middle lamella of the cells of the innermost wall layer, marking the initiation of the p e rita p e ta l membrane.

Callose production is initiated in the late meiotic prophase during which it is secreted between the plasmalemma and the fibrillar MMC surface coat. The surface coat is about 300 nm thick at the beginning of callose secretion. Synthesis of callose occurs inside the

MMC and the callose appears to be transported by vesicles.

Callose secretion continues until the meiotic products, the four daughter c e lls , become completely separated from each other. Meiotic divisions of the MMCs of A_j_ concolor are asynchronous. The formation of the callose wall at telophase II occurs simultaneously among the four daughter cells as a result of fusion of callose-containing vesicles.

The microspores are tetrahedrally arranged, with the 44 separating callose layer approx. 650 nm in thickness Fig.

20) .

TETRAD PERIOD - Sporoderm formation is initiated after the callose has completely separated the four daughter cells. At this stage dictyosomes again become more active and vesicles containing a granular-fibrillar material are especially abundant in the proximal regions of the microspore. Vesicles fuse with the plasmamembrane and deposit their contents onto the surface of the microspores, thereby coating them with a delicate, reticulate matrix.

This 'glycocalyx surface coat1 (Rowley, 1973) is made of two different structural units: (1) granular angular centers of approx. 40 nm diameter, which are interconnected by (2) granular f i b r i l s 50 nm in length (Figs. 21, 22).

This material is unevenly deposited over the microspore surface such that in the distal region (Fig. 23) the glycocalyx coating is quite thin or lacking while in the proximal area (Fig. 24) it reaches a thickness of about 600 nm. In the lateral regions, which are the areas where the sacci are la te r formed, i t may reach more than 1000 nm in thickness (Figs. 25, 28). During the deposition of this glycocalyx coating, callose deposition continues to occur where contact of the callose wall with the plasma membrane persists. In this way, callose protrusions into the glycocalyx are formed on the inner surface of the callose wall (Figs. 24, 25). The callose wall and its protrusions 45 appear to act in some degree as structural templates upon which part of the ektexine is later deposited. The formation of the ektexine .in Abies concolor is also dependent upon the glycocalyx material deposited into the perimembranous space. On the distal face, i.e. in the area of the incipient aperture and where very little or no glycocalyx is deposited, no ektexinous layer is formed

(Figs. 23, 26). In the proximal region, secretion of glycocalyx and callose occur at similar rates and both continue until the endexine is initiated (Figs. 24, 27).

On the lateral sides, i.e. the area of the sacci, the large amount of glycocalyx material deposited leads to the formation of a broad layer filled with fibrillar material, and the callose extensions protrude through only the outer part of this layer (Fig. 28).

Endexine initiation starts with the appearance of tripartite or so-called 'white-line-centered1 lamellae

(Rowley & Southworth, 1967) around the microspore (Figs.

26, 27). On the distal face, the endexine is the only wall layer differentiated. Adjacent lamellae are interconnected by radial units which produce a chambered infrastructure

(Figs. 29, 34). The lacunae thus formed within the endexine are filled with material of the same shape and size as the glycocalyx material deposited on the proximal and lateral sides. In the late tetrad stage, the endexine in the distal area consists of 10 to 11 white-line-centered lamellae, and is about 960 nm thick. The lumina in the outer p art of the endexine are somewhat sm aller than those closer to the plasma membrane (Fig. 29). In the proximal and lateral areas, lamellae are also present but they are closely appressed to each other in the early phases of endexine deposition (Figs. 28, 30). However, after about 7

- 9 white-line-centered lamellae have been formed, two additional lamellae are laid down that are in fact interconnected by radial units. The lacunae so created are filled with glycocalyx material as on the distal side.

Therefore, in the final tetrad phase, the endexine on the proximal and lateral faces consists of two structurally different zones. The outer is composed of 7 - 9 closely appressed white-line-centered lamellae and is approx. 200 nm thick. The inner zone consists of 2 - 3 lamellae that are separated from each other by radial units and is about the same thickness as the outer zone. Comparison between the microspores in the early tetrad and in the late tetrad phases shows that the swelling of the sacci is accommodated by the microspore, and that the callose wall is distorted remarkably little by sporoderm formation (Fig. 31). The endexine in the late tetrad phase undulates over the . microspore surface except in the distal area (Fig. 20, 31).

FREE SPORE PERIOD - Release of the microspores from the tetrad appears to be accomplished by enzymatic digestion of the callose wall and of the MMC surface coat. 47

Lysis of these layers apparently begins over the sacci

(Fig. 32). Release from the tetrad, as with the products of the meiotic divisions, is asynchronous within a microsporangium, such that free microspores as well as tetrads can be observed in the same sporangium (Fig. 33).

Microspore wails in the early free spore phase show the following characteristic features: (a) An endexine consisting of 9 - 11 white-line-centered lamellae forms the distal sporoderm (Fig. 34). These lamellae, as in the earlier period, are interconnected by radial units which gives the endexine a chambered appearance. The chambers created by these structures are filled with glycocalyx and the outermost lamellae are buttressing. (b) The proximal sporoderm is differentiated into an ektexine and an endexine (Fig. 35). The ektexinous elements appear rather delicate and are covered with the fibrillar glycocalyx network that forms the receptor sites for deposition of additional, most likely tapetal, sporopollenin. The ektexine is of variable thickness at this stage but is usually around 1000 nm. It consists of a tectum, an infratectal layer and a footlayer. The endexine consists of an outer zone of 7 - 9 compactly appressed white-line-centered lamellae and an inner irregularly chambered zone which resembles the distal endexine. (c) In the lateral area (Fig. 37), the floor of the saccus shows an endexine consisting of elements identical to those of 48

the proximal endexine (Fig. 35). Numerous glycocalyx

elements are accumulated to the outside of the endexine, which represents the footlayer of the ektexine. (d) The

distal corpus-saccus transition region is illustrated in

Fig. 36 which shows the closely appressed white-line-

centered lamellae of the lateral area continuous with the

separated and regularly chambered ones of the distal face,

(e) The endexine in the proximal corpus-saccus region is

unaltered. The ektexine, however, increases in thickness

toward the saccus (Fig. 38) .

Formation of the intine is initiated early in the free

spore phase. The intine is deposited between the endexine

and the plasma membrane and is ch aracterized by f i b r i l l a r

structures (Figs. 39 - 41). Initiation of the intine is

accompanied by a period of microspore vacuolization that

leads to an increase in volume. The increase in volume of

the protoplast leads first to a stretching of the formerly

undulated endexine and secondly to a stretching of the

endexine lamellae, especially in the distal area, resulting

in a decrease of its total thickness. To further

accommodate the volume increase, the saccus floor changes

from concave to convex (Fig. 42). As a result of

sporopollenin covering all the glycocalyx receptor sites,

the ektexine also becomes modified. It is not clear whether the sporopollenin originates from the tapetum or

from the microspore cytoplasm, but the tapetal residues contain a large number of orbicules and are in close

contact with the tectum at this stage (Fig. 43). Whatever

the case, this deposition of sporopollenin leads to a

thickening of the tectum and of the infratectal elements as well as to the formation of a clearly recognizable

footlayer (Figs. 40, 41). Some of the sporopollenin is also deposited onto the outermost buttressing lamellae of

the endexine on the distal face (Fig. 39). Therefore, in

the free spore phase, sporopollenin most likely originating

from the tapetum, is accumulated in two different ways by

the microspore. In the ektexine it is accumulated by the glycocalyx receptor sites while on the endexine it is deposited directly on lamellae without mediation of a glycocalyx. These structural changes of the exine seem to be linked to modifications of a chemical nature as evidenced by changing stain affinities of the various wall

layers. The affinity for uranyl acetate and lead citrate dim inishes in the ektexine, w hile the endexine becomes more electron dense (Figs. 47 - 49). This modification may well coincide with the transformation of the so-called

'proto-sporopollenin' (Heslop-Harrison, 1971) to the sporopollenin proper of the mature pollen wall.

After initiation of the intine and vacuolization of \ the microspore protoplast, the microspore undergoes mitotic divisions. At the time of shedding, the pollen grain is

5-celled (Fig. 44). The microgametophyte of the mature 50 pollen grains consists of two prothallial cells, a sterile cell, a spermatogenous cell and a tube cell (Sterling,

1963). During the formation of the microgametophyte, intine development continues so that in mature grains the intine is two-layered (e.g. Fig. 55b). In the final stages of pollen development, tapetal breakdown is completed and the peritapetal membrane becomes an acetolysis-resistant layer that lines the sporangial wall and envelops the pollen grains and orbicules (Fig. 51).

MATURE POLLEN WALL - The sporoderm of mature Abies concolor pollen is characterized as follows: (a) On the proximal side it consists of an ektexine, a two-layered endexine and an intine also made up of two zones (Fig. 47).

The ektexine measures up to 1.4 /im thick and consists of three structural regions. The outermost layer (tectum) is up to 200 nm in thickness. Its surface is scabrate and shows a faint triradiate mark that indicates the former contact position in the tetrad (Fig. 46). In oblique section it becomes clear that the infratectal layer has an alveolar infrastructure (Fig. 54). The footlayer is up to

200 nm thick. All the ektexinous layers show the same electron density. In contrast, the endexine is more electron dense than the ektexine in mature grains and consists of two layers that are difficult to distinguish in non-acetolyzed walls, but are more readily identifiable in acetolyzed sections (Fig. 53). The endexine in non-acetolyzed grains is approx. 200 nm thick while in acetolyzed ones it is up to 350 nm (Fig. 55). This reflects the deposition of the intine onto the innermost endexine elements which masks some of the lamellae.

Acetolysis removes the intine and thus exposes the entire endexine (Fig. 55a). The lamellated nature of the mature endexine is still apparent, but the white-line-centers cannot be recognized easily. This is most likely caused by the stretching of the lamellae and the deposition of additional sporopollenin. The intine consists of two zones which differ in electron density and is up to 700 nm in thickness. (b) In the lateral areas, the sporoderm consists of a saccus floor and a saccus roof (Figs. 49,

50) . The saccus roof consists of a tectum (approx. 200 nm thick) from which infratectal elements extend inwards into the saccus cavity forming an endoreticulum. The saccus floor consists of the footlayer and a two-layered endexine with the characteristic features as outlined for the endexine of the proximal side of the grain. The intine becomes thicker toward the distal area and is approx. 1400 nm thick in the lateral region. (c) In the distal area, the sporoderm consists of a lamellated endexine and a very thick intine (Fig. 48). This is the area of the leptoma, the surface of which is characterized by the presence of verrucose granules. These granules of sporopollenin are deposited on the outermost buttressing lamellae of the endexine (Figs. 45, 48). The endexine is approx. 700 nm thick, and the white-line-centered lamellae are easily recognized in the acetolyzed grains (Fig. 52) . The intine is two-layered and is 2700 nm in thickness (Fig. 48). PLATE I: Microspore mother cells of Abies concolor in premeiotic stages (Figs. 6 - 14). Scale bars = 0.5 ^im unless otherwise indicated. (ER = endoplasmic reticulum; ml = middle lamella; MMC = microspore mother cell; Pb = lead citrate; SC = microspore mother cell surface coat; T = tapetum; UA = uranyl acetate; WL = sporangial wall layer c e l l s ) .

Fig. 6: Cross section of dormant microsporangia showing microspore mother cells, tapetum and sporangial wall layers. X 123.

Fig. 7: Part of microsporangium with rounded microspore mother cells, tapetum and sporangial wall layer cells. 3 min. KMn04, 2 min. UA, 1 min. Pb. X 1080.

Fig. 8: Plasmodesmata (arrows) between sporogenous cells during dormancy. 3 min. KMnO*, 2 min. UA, 1 min. Pb. X 5400.

Fig. 9: End of dormancy as indicated by mitotic activity in the ta p e ta l c e lls . 10 min. KMnO*. X 1180.

Fig. 10: Breakdown of the primary c e ll wall leading to degradation of the plasmodesmata (arrows) between adjacent MMCs. 3 min. KMn04 , 2 min. UA, 1 min. Pb. X 5330.

Fig. 11: Lysis of the primary cell wall between adjacent MMCs and deposition of microspore mother cell surface coat. The middle lamella of the primary cell wall is still recognizable. 15 min. UA, 5 min. Pb. X 33300.

Fig. 12: Section through microspore mother cells showing dilated endoplasmic reticulum in the cytoplasm, microspore mother cell surface coat ancj degrading middle lamella. 15 min. UA, 5 min. Pb. X 12600.

Fig. 13: Dictyosomes with numerous vesicles (arrows) containing heterogeneous m aterial. 2 min. KMn04 , 2 min. UA, 1 min. Pb. X 38800.

Fig. 14: Periclinal section through nuclear envelope showing nuclear pores; microtubules are present in the cytoplasm near the nucleus (arrow). 2 min. KMnO*, 2 min UA, 1 min. Pb. X 23900.

53 PLATE I PLATE II: Meiotic stages of Abies concolor microspore mother cells (Figs. 15 - 20). Scale bars = 0.5 /um unless otherwise indicated. (C = callose wall; L = lipid globules; MMC = microspore mother cell; N = nucleus; Pb = lead citrate; S = starch grains; SC = microspore mother cell surface coat; T = tapetum; UA = uranyl acetate; V = v e s ic l e s ) .

Fig. 15: Microspore mother cell with polarized distribution of organelles. Lipid globules and starch grains are concentrated in one half of the cytoplasm, the nucleus in the other half. 10'min. KMn04. X 1360.

Fig. 16: Dictyosomes and dictyosome-derived vesicles (arrow) in the cytoplasm of the microspore mother cells may be the source of the fibrillar surface coat. Note the remnants of a plasmodesma (arrowhead). 8 min. UA, 4 min. Pb. X 28900.

Fig. 17: Fibrillar material apparently being exported by v e s ic le s becomes oriented perpendicular to the c e ll surface. 8 min. UA, 4 min. Pb. X 21000.

Fig. 18: Globules (arrow) coating the tapetal membrane. 8 min. UA, 4 min. Pb. X 7320.

Fig. 19: After lysis of the tapetal cell wall, granular material appears outside the tapetal plasmamembrane. Vesicles containing identical granular material are present in the tapetal cytoplasm. 8 min. UA, 4 min. Pb. X 2790.

Fig. 20: Microspore tetrad. Callose wall separates the four daughter cells from each other and the entire tetrad is surrounded by the microspore mother cell surface coat. 12 min. UA, 4 min. Pb. X 2600.

55 PLATE I I PLATE III: Early tetrad phase in Abies concolor (Figs. 21 -30). Scale bars = 0.5/um unless otherwise indicated. (C = callose wall; END = endexine; EKT = ektexine, GC = glycocalyx of microspore surface coat; Pb = lead citrate; SC = microspore mother cell surface coat; UA = uranyl a c e ta te ) .

Fig. 21: Glycocalyx of microspore surface coat inside callose wall before sporopollenin accretion. 12 min. UA, 4 min. Pb. X 14900.

Fig. 22: Structural organization of the microspore surface coat. The polysaccharidic glycocalyx consists of angular elements (arrowhead) that are interconnected by fibrils. Dispersed in the glycocalyx are osmophilic globules (arrow). 15 min. UA, 5 min. Pb. X 48900.

Fig. 23: On the distal side, in the area of the incipient aperture, very little or no glycocalyx material (arrowhead) is deposited inside the callose wall. 12 min. UA, 4 min. Pb. X 18200.

Fig. 24: Deposition of glycocalyx material (arrowhead) in the proximal area. Vesicles (arrow) transporting the material are abundant in the cytoplasm. 12 min. UA, 4 min. Pb. X 21800.

Fig. 25: Callosic protrusions (arrow) into the perimembranous space are formed where the plasmamembrane stayed in contact with the callosic wall layer. 12 min. UA., 4 min. Pb. X 18000.

Fig. 26: Initiation of the endexine on the distal surface of the microspore as lamellated structures (arrow) to the inside of the callose wall. 15 min. UA, 5 min. Pb. X 30100.

Fig. 27: Endexine lamellae (arrow) to the inside of the microspore surface coat on the proximal surface. 15 min. UA, 5 min. Pb. X 21600.

Fig. 28: Saccus area; callosic extensions (arrow) do not extend to the endexine lamellae (arrowhead). 12 min. UA, 4 min. Pb. X 12000.

57 PLATE III (cont.):

Fig. 29: Glycocalyx material (arrow) is deposited into the chambers of the endexine in the distal area. 12 min. UA, 4 min. Pb. X 12300.

Fig. 30: Proximal area showing ektexine and endexine. 12 min. UA, 4 min. Pb. X 6240.

58 PLATE I I I PLATE IV: Late tetrad phase in Abies concolor (Figs. 31, 32). Scale bars = 0.5/um unless otherwise indicated. (C = callose wall; END = endexine; EKT = ektexine; Pb = lead citrate; SC = surface coat; UA = uranyl acetate).

Fig. 31: Tetrahedrally arranged microspores still enclosed by the microspore mother cell surface coat and the callose wall. The ektexine and the endexine can be recognized on both morphological and staining differences. Note the structural differences of the exine layers in the distal and proximal areas. 2 min. KMnO*, 2 min. UA, 1 min. Pb. X 2500.

Fig. 32: Disaggregation of the surface coat and callose wall starting over the sacci (arrow). 8 min. KMnO*. X 2500.

60 PLATE PLATE V: Early free spore phase in Abies concolor (Figs. 33 - 38). Scale bars = 0.5/um unless otherwise indicated. (END = endexine; EKT = ektexine; Pb = lead citrate; UA = uranyl acetate).

Fig. 33: Microsporangium filled primarily with free microspores before volume increase. A few tetrads are still present (arrow). X 260.

Fig. 34: Exine on the distal side consisting of lamellated endexine only. All the chambers are filled with glycocalyx. 12 min. UA., 4 min. Pb. X 32600.

Fig. 35: Exine on the proximal side with delicate ektexinous elements and two-layered endexine. Glycocalyx material occurs around the ektexinous elements (arrow) and in the endexine chambers. 12 min. UA, 4 min. Pb. X 34300.

Fig. 36: Distal saccus-corpus transition region showing continuity of endexine lamellae. 12 min. UA, 4 min. Pb. X 27500.

Fig. 37: Sporoderm organization on the saccus floor: two-layered endexine and glycocalyx material (arrow) adhering to the outer side of the endexine. 12 min. UA, 4 min. Pb. X 35200.

Fig. 38: Proximal saccus-corpus region shows the same endexine organization as the proximal side; ektexinous elements of saccus. 12 min. UA, 4 min. Pb. X 33200.

62 PLATE V PLATE VI: Sporopollenin accretion in Abies concolor during the free spore phase (Figs. 39 - 43). Scale bars = 0.5 yum unless otherwise indicated. (END = endexine; EKT = ektexine; fl = footlayer; I = intine; it = infratectum; 0 = orbicules; t = tectum; T = tapetum; UA = uranyl acetate; Pb = lead citrate).

Fig. 39: Lamellae of endexine become le ss d is tin c t. Intine increasing in thickness. 12 min. UA, 4 min. Pb. X 29100.

Fig. 40: Sporoderm in the proximal area. Ektexine consisting of tectum, infratectum and distinct footlayer. Most of the glycocalyx is covered by sporopollenin. Lamellae in the endexine still distinct. 12 min. UA, 4 min. Pb. X 29300.

Fig. 41: Saccus floor also shows a distinct footlayer and a two-layered endexine. 12 min. UA, 4 min. Pb. X 31700.

Fig. 42: Unicellular microspores shortly before initiation of mitotic divisions. The exine is fully formed. 10 min. UA, 4 min. Pb. X 890.

Fig. 43: Tapetal protoplast, with few organelles still recognizable, is coated by numerous orbicules. 12 min. UA, 4 min. Pb. X 8330.

64 PLATE VI PLATE VII: Mature pollen grains of Abies concolor (Figs. 44 - 50). Scale bars = 0.5/um unless otherwise indicated, (be = body cell; END = endexine; EKT = ektexine; fl = footlayer; I = intine; it = infratectum; pc = prothallial cell; sc = stalk cell; t = tectum; tc = tube cell)

Fig. 44: Five-celled microgametophyte of mature pollen grain. Two prothallial cells, a stalk cell, a body cell and a tube cell. X 430.

Fig. 45: SEM of mature grain in e q u ato rial view. Note the difference in surface sculpturing in the proximal and distal areas. X 690.

Fig. 46: Proximal surface still showing faint trilete mark (arrowheads) from the tetrad position. X 610.

Fig. 47: Proximal exine with three-layered ektexine (tectum, infratectal layer and footlayer) and endexine. Note that the lamellated layer is considerably thinner than in earlier stages. The intine is two-layered. 12 min. KMn04. X 10700.

Fig. 48: On the distal face the exine consists of an endexine and some ektexinous elements (arrow) . The intine is two-layered and much thicker than on the proximal face. 12 min. KMn04 . X 10700. \ Fig. 49: Sporoderm in saccus-corpus region; fo o tla y e r and endexine forming the saccus floor. Intine is two-layered. 12 min. KMn04. X 10700.

Fig. 50: Tectum and infratectum forming end o reticu latio n s of the saccus. 12 min. KMn04. X 10700.

66 67 PLATE V II PLATE VIII: Peritapetal membrane (Fig. 51) and sporoderm of acetolyzed pollen of Abies concolor (Figs. 52 - 55). Scale bars = 0.5/um unless otherwise indicated. (END = endexine; EKT = ektexine; I = intine; PTM = peritapetal membrane; T = Tapetum).

Fig. 51: P e rita p e ta l membrane lin in g the m icrosporangial cavity. Degrading tapetal protoplasts and numerous orbicules. 10 min. KMnO*. X 18300.

Fig. 52: Distal sporoderm; lamellae and interconnecting radial structures are recognizable. 10 min. KMn04. X 31400.

Fig. 53: Proximal-lateral sporoderm; ektexine and endexine still exhibit differences in electron density. 10 min. KMn04 . x 12800.

Fig. 54: Oblique section through equatorial area of acetolyzed grain. The infratectal layer of ektexine shows alveolar infrastrucutre and the inner endexine has a honeycomb-type p a tte rn . 12 min. KMnO*. X 6230.

Fig. 55: Composite of acetolyzed (a) and unacetolyzed (b) pollen grain. The outer layer of the intine is deposited onto the innermost part of the endexine. 10 min. KMn0«. X 18800.

68 PLATE VIII C h a p te r IV

POLLEN WALL DEVELOPMENT IN TSUGA CANADENSIS

According to Sivak (1978), the genus Tsuga comprises

13 species, three of which are native to temperate North

America and ten to Asia. The genus has been the subject of various investigations and speculations concerning the origin, relationships and classification of its species.

Engelmann (1880) divided the genus into two sections,

Hesperopeuce and Eutsuga. This proposal was adopted by

Dallimore & Jackson (1967), but they substituted the name

Micropeuce for Eutsuga. Lemmon (1889) proposed a separation into two independant genera and Ueno (1957) suggested that, based on pollen morphology, two subgenera would be more appropriate than two sections or two independent genera.

Tsuga mertensiana (sect. Hesperopeuce) and T. heterophvlla (sect. Micropeuce) have received the most attention because of the supposed hybrid origin of T. m ertensiana (Van Campo-Duplan & Gaussen, 1948; D uffield,

1950; Van Campo-Duplan, 1950, 1955; Ho& S zik lai, 1972) and because of the occurrence of morphological intermediates between the two species (Ho, 1972; Taylor, 1972; Owens&

Blake, 1983). Sexual reproduction in these two species has

70 been extensively studied by Owens and his collaborators

(e .g ., Ho & Owens, 1974; Owens & Molder, 1974, 1975;

Stanlake & Owens, 1974; Owens & Blake, 1983; Owens, 1984).

Pollen cones are d iffe re n tia te d during the summer of the year before pollination occurs. The microspore mother cells enter meiosis in the fall and reach pachytene before entering dormancy. After the break of dormancy, meiosis is resumed and is completed rapidly. The microspores remain in the tetrad for about 10 days. After release from the tetrad, they undergo mitotic divisions and the mature pollen grains are shed at the 4-celled stage.

Mature pollen grains of mertensiana are bi-saccate while the species in the section Micropeuce are non-saccate

(Sivak, 1973) . The non-saccate grains are encircled by a puffy frill in the equatorial area which has been referred to as a rudimentary bladder by some authors (e.g.,

Wodehouse, 1935; B assett e t a l ., 1978). The exine is composed of an outer ektexine and an inner, lamellated endexine. In discussions of exine structural types, Tsuga is often mentioned as an exception since the infratectal layer is absent from the ektexine (Sivak, 1973; Van Campo &

Lugardon, 1973; Doyle et al., 1975).

Despite its unique morphology and exceptional exine structure, there have been few ultrastructural studies of the pollen of Tsuga (Ueno. 1958; Sivak, 1973, 1978). The purpose of the present study was to trace in detail the 72 development of the pollen wall in T. canadensis at the ultrastructural level in order to better understand its unique exine structure.

FORMATION OF THE TETRADS - At the beginning of March, the microsporangia of Tsuga canadensis are filled with angular microspore mother cells (MMCs), which are surrounded by a layer of tapetal cells and by layers of sporangial wall cells (Fig. 56). At this time dormancy is broken and the MMCs are in early meiotic prophase. The

MMCs show a characteristic polarized distribution of organelles in the cytoplasm (Fig. 57). The primary cell walls are broken down and a fibrillar surface coat surrounds the MMCs. Remnants of plasmodesmata are seen between adjacent MMCs (Fig. 58). By the middle of March, meiosis is concluded and tetrads are formed (Figs. 59, 60).

When dormancy is broken, the tapetal cells also become active and undergo mitotic divisions. These divisions are not always followed by cytokinesis, and as a result the ta p e ta l c e lls become binucleate (Figs. 57, 59, 61). In T . canadensis the tapetum is of the secretory type in which the tapetal cells mostly remain in their original position.

Their plasma membranes pull away from the cell wall, and the primary cell walls begin to degenerate.

The secretion of callose in canadensis is most likely initiated shortly after the break of dormancy. The callose is deposited between the MMC surface coat and the 73 plasma membrane. Cytokinesis is simultaneous, occurring in telophase II through the fusion of vesicles apparently containing callose. Callose secretion appears to continue until the four tetrahedrally arranged daughter cells in a tetrad are completely separated from each other. The callose layer is relatively thin and measures approx. 500 nm in thickness (Fig. 60).

TETRAD PERIOD - After the four microspores have been sequestered by the callose wall, deposition of the glycocalyx layer commences. This m aterial is deposited between the callose wall and the plasma membrane, and is recognizable as an electron-dense, fibrillar layer. The amount of glycocalyx deposited varies around the microspore

(Figs. 63 - 65). In the lateral areas (Fig. 64), the glycocalyx layer becomes the broadest while in the proximal area (Fig. 65) it appears as a relatively thin, uneven band. A large number of Golgi bodies and vesicles with fibrillar contents are present in the cytoplasm. These

Golgi-derived vesicles transport the glycocalyx to the plasma membrane where they fuse with it (Fig. 65). In this way, they empty their contents into the periplasmic space.

In the lateral areas, callosic protrusions extend into the glycocalyx (Figs. 68, 69). These are most likely formed in the initial phases of glycocalyx production while callose secretion is still occurring. Almost concomitantly with the appearance of the glycocalyx, globules accumulate to i 74 the inside of the callose wall (Figs. 63 - 65). This elaboration of presumed sporopollenin precursors marks the initiation of the first pollen wall layer. The pretectum consists of elongated elements that are about 35 nm thick

(Figs. 66, 69, 70).

After the glycocalyx layer reaches a certain thickness and the pre-tectum is laid down (Figs. 66, 69, 70), the endexine is initiated. At this stage, the glycocalyx layer measures approx. 200 nm in the distal area, 250 nm in the lateral region and 100 nm on the proximal side of the microspore. Endexine initiation is recognized by the appearance of white-line-centered lamellae outside the plasma membrane surrounding the entire microspore (Figs.

66, 69, 70). At the time of endexine deposition, the number of Golgi bodies is still very high. This may indicate that Golgi bodies are not only involved in the deposition of the glycocalyx, but also in the formation of both of the exine layers in T^ canadensis, i.e. the ektexine and the endexine. In the initial phases of its formation, the endexine does not vary around the microspore surface, but later, in the distal region, more lamellae are deposited and the outermost ones start to bulge outward

(Fig. 67). Here, the endexine lamellae are more loosely stacked and the spaces between them are f i l l e d compactly with glycocalyx material. No infratectal structures are deposited and the tectum is not continuous. The areas 75 between the tectum and the early endexine are also filled densely with fibrillar glycocalyx (Figs. 67, 68, 71). A footlayer cannot be recognized with certainty at this stage, but the outermost lamella of the endexine is bordered by a narrow, electron-dense band which lies in the position of the footlayer (Figs. 69, 70).

By the late tetrad phase, all the exine layers have formed (Fig. 72). Both the ektexine and the endexine are present around the entire microspore, but their composition v a rie s . In the d is ta l area, the exine is composed m ostly of endexine which is made of 10 - 12 white-line-centered lamellae (Fig. 67). These lamellae increase in thickness toward the ektexine, ranging from 25 to 40 nm in thickness.

The tectal elements are in contact with the outermost endexine lamellae in some places (Fig. 67). In the lateral region, the exine consists mostly of ektexine (Figs. 68,

69). The endexine is only up to 100 nm thick and the white-line-centered lamellae are closely appressed to each other. The tectal elements are completely separated from the lamellated layer by a broad glycocalyx layer. In the proximal area, the exine is rather thin (Fig. 71). The endexine is as on the equatorial area, but the ektexine exhibits structural differences. The glycocalyx layer separating the tectum from the endexine is much narrower than in the equatorial area. Some of the tectal elements 76 are in close proximity to the endexine lamellae as in the distal area (Fig. 71).

During the tetrad phase, most of the tapetal cells are b in u cleate and become in creasin g ly more secreto ry (Figs.

61, 62). Via vesicles, they secrete a fibrillar residue into the sporangial cavities. Large numbers of pro-orbicules are visible to the inside and outside of the ta p e ta l plasma membrane (Fig. 62).

FREE SPORE PERIOD - The breakdown of the c allo se wall and of the MMC surface coat is apparently effected by enzymatic activity and also by physical stress. At the beginning of the microspore release from the tetrads, the infratectal layer in the equatorial regions begins to swell. This expansion is most likely caused by the glycocalyx content of the sacci and may force the already partly digested callose wall to break. Most of the microspores in the early free spore phase show a crescent or half-moon-like outline with small, rudimentary, equatorial bladders (Figs. 72, 77). At the time of release from the tetrad, the ektexine still appears delicate, but the endexine is fully formed (Figs. 73 - 75). The free microspore at this stage shows the following features: (a)

In the distal region the endexine is the prominent layer of the sporoderm and is about 1300 nm thick (Fig. 73). (b)

The lateral areas are characterized by an enlarged infratectal space which is still filled with glycocalyx 77

(Fig. 74). This infratectal space is bordered by

convoluted tectal elements to the outside and an electron

dense, still narrow footlayer to the inside. The

glycocalyx is composed of angular and f i b r i l l a r stru c tu re s

(Fig. 76). The endexine is approx. 190 nm thick and

consists of closely appressed lamellae. (c) On the

proximal surface, a triradiate mark indicating the former

contact position in the tetrad can be recognized (Fig. 78).

The tectal elements are convoluted and rest on an electron

dense footlayer. Via orbicules, additional sporopollenin

is accumulated on the tectum all around the microspore

(Fig. 72).

After release from the tetrad, intine formation

commences and the microspores increase in volume as a

result of vacuolization. This volume increase is

accommodated by a change in microspore shape. The formerly

concave proximal face is pushed outwards (Fig. 80, arrow).

The tapetal cells have undergone almost total lysis by this

time, and sporopollenin also accumulates on the free cell wall of the innermost sporangial wall layer, forming the p e rita p e ta l membrane (Figs. 81, 87). The continuous accumulation of sporopollenin on the ektexine leads to an

increase in thickness of this layer (e.g., Figs. 79, 80).

The sporopollenin covers all the glycocalyx receptor sites

(Figs. 82 - 85). In the d is ta l region, the in tin e becomes much thicker than elsewhere on the microspore (Fig. 83). 78

There is no pre-formed, structurally recognizable aperture

(Fig. 83). '

After the volume increase of the microspore protoplast, the microspores undergo mitotic divisions (Fig.

87) such that at the time of shedding from the microsporangium the pollen grains are 4-celled (Fig. 86).

The microgametophyte of the mature pollen grain consists of a prothallial cell, a sterile cell, a generative cell and a tube cell. During microgametogenesis, intine development continues. The intine appears structurally homogeneous but shows two layers based on staining affinity (Figs. 88, 90,

92). When uranyl acetate and lead citrate are used as section stains, the outer layer is narrow and less electron dense than the broader, inner one. On the distal face the intine is undulating (Figs. 83, 88).

MATURE POLLEN WALL - The sporoderm of mature T. canadensis pollen is characterized as follows: (a) The proximal side is shown in Figs. 92, 93, 98 and 101.

Depending on the section stains employed, different numbers of wall layers can be recognized. In Fig. 92, where uranyl acetate and lead citrate were used, a two-parted ektexine, an electron dense endexine and a two-layered intine are recognized. The ektexine is up to 1700 nm thick and consists of a wavy tectum and a footlayer.No infratectal structures are present and the convolutions of the tectum rest on the footlayer. The endexine, approx. 120 nm in thickness, appears as a more electron dense layer and the lamellations can only be recognized at high magnifications.

The fibrillar intine is two-layered based on staining affinity and is up to 700 nm in thickness. When potassium permanganate is used as a section stain (Fig. 93), the differentiation between ektexine and endexine is based only on structural differences and the intine is homogeneous.

In these sections it is difficult to distinguish between the footlayer and the endexine because the white-line-centered lamellae are not always obvious in the endexine. In acetolyze

(Fig. 101). The tr i r a d ia t e mark th a t was v is ib le on the proximal surface in the early free spore phase is no longer seen in the mature grains. The continued accumulation of sporopollenin has evidently led to the covering of this earlier developmental feature.

(b) The equatorial areas are illustrated in Figs.

90, 91, 97 and 100. In Fig. 90, the sporoderm consists of a two-parted ektexine that is distinguished from the endexine by differences in stain affinities. The ektexine is much thicker (up to 3400 nm) and the tectal elements are much more convoluted than in the proximal region. In surface view, these convoluted projections in the equatorial areas appear as elongated, irregular ridges (Fig. 100). The intine increases in thickness toward the d is ta l area. (c) The d is ta l regions are shown in Figs.

88, 89, 96 and 99. The sporoderm consists of a gemmate ektexine, a lamellated endexine and a two-parted undulating

intine (Fig. 88). At first sight, the ektexine appears to be granular, but the tectal elements are convoluted as in the proximal and lateral areas (Figs. 89, 96). When uranyl acetate and lead citrate are used as section stains, the outermost w h ite -lin e-c en tered lam ellae show the same electron density as the ektexine. Only in the innermost portion, where the lamellae are closely appressed, is a difference in staining affinity from the ektexine seen

(Fig. 88). The undulating intine is quite thick (up to

2200 nm). The d is ta l surface is evenly gemmate (Fig. 99). PLATE IX : Formation of tetrads in Tsuqa canadensis (Figs. 56 - 60). Scale bars = 0.5/um unless otherwise indicated. (C = callose wall; L = lipid globule; MMC = microspore mother cell; N = nucleus; Pb = lead citrate; SC = microspore mother cell surface coat; T = tapetum; UA = uranyl acetate; WL = sporangial wall layer cells).

Fig. 56: Section of dormant microsporangium showing microspore mother cells, tapetal cells and sporangial wall layers cells. X 380.

Fig. 57: MMC in early meiotic prophase showing polarized distribution of organelles in the cytoplasm. Note the dividing tapetal cell. 6 min. KMnOd. X 1840.

Fig. 58: Degrading primary cell wall between two adjacent MMCs. Note the remnants of a plasmodesma (arrow). 1 min. KMn04 , 2 min. UA, 1 min. Pb. X 35100.

Fig. 59: Section through microsporangium after completion of meiosis. Tetrads are filled up the sporangial locule. Some of the tapetal cells are binucleate (arrow). X 290.

Fig. 60: Microspore tetrad. Callose layer separates the four daughter cells from one another. 10 min. UA, 4 min. Pb. X 1870.

81 PLATE IX PLATE X: Tapetal cells (Figs. 61, 62) and microspores (Figs. 63 - 65) of Tsuga canadensis in early tetrad period.. Scale bars = 0.5 yum unless otherwise indicated. (C = callose wall; GC = glycocalyx of microspore surface coat; Pb = lead citrate; SC = microspore mother cell surface, UA = uranyl acetate).

Fig. 61: Binucleate tapetal cell with degrading walls. 10 min. UA, 4 min. Pb. X 3300.

Fig. 62: Part of a tapetal cell showing pro-orbicular bodies (arrows) on both sides of the tapetal plasma membrane. Note the presence of vesicles with secretory products in the tapetal cytoplasm. 10 min. UA, 4 min. Pb. X 11800.

Fig. 63: Deposition of glycocalyx material in the distal area of the microspore, transported and deposited by vesicles (arrow)) in the periplasmic space. 20 min. UA, 6 min. Pb. X 46500.

Fig. 64: Glycocalyx deposition in lateral areas. Globules to the inside of callose indicating initiation of pre-tectum (arrowheads). 10 min. KMriO*. X 29100.

Fig. 65: Deposition of glycocalyx in proximal area via vesicles (arrow). Numerous vesicles with secretory contents are present in the cytoplasm. Pre-tectal elements outside the undulating plasma membrane (arrowheads). 20 min. UA, 6 min. Pb. X 31500.

83 PLATE X PLATE XI: Tetrad period in Tsuga canadensis,(Figs. 66 - 71). Scale bars = 0.5/um unless otherwise indicated. (C = callose wall; END = endexine; GC = glycocalyx of microspore surface coat; Pb = lead citrate; pt = pretectum; UA = uranyl acetate).

Fig. 66: Appearance of white-line-centered lamellae after deposition of the pre-tectum (arrowheads) and the glycocalyx (arrow). 10 min. UA, 4 min. Pb. X 37400.

Fig. 67: Distal area in late tetrad period. Pre-tectum (arrowheads) consisting of elongated granules and endexine of white-line-centered lamellae. Outermost lamellae embedded in dense glycocalyx m aterial (arrow ). 10 min. UA, 4 min. Pb. X 33400.

Fig. 68: Oblique section through microspore wall in lateral area showing callosic protrusions (arrow) into the glycocalyx. 10 min. UA, 4 min. Pb. X 12400.

Fig. 69: Endexine formation in lateral areas. Note the callosic protrusions into the glycocalyx (arrows) . Ektexine consisting of pretectum and narrow, electron dense footlayer (arrowhead) separated from each other by 1 glycocalyx. 10 min. UA, 4 min. Pb. X 16700.

Fig. 70: White-line-centered lamellae in proximal region forming endexine. Callose separating two neighboring microspores. Pretectum (arrowheads). Note Golgi-body in the cytoplasm. 10 min. UA, 4 min. Pb. X 29500.

Fig. 71: Proximal area in late tetrad phase. Pretectum (arrowhead) and endexine are separated by a thin region of glycocalyx material (arrow). Endexine consisting of white-line-centered lamellae. 10 min. UA, 4 min. Pb. X 30600.

85 PLATE X I PLATE XII: Early free spore phase in Tsuga canadensis (Figs. 72 - 76). Scale bars = 0.5/um unless otherwise indicated. (END = endexine; EKT = ektexine; fl = footlayer; I = intine; Pb = lead citrate; t = tectum; UA = uranyl acetate).

Fig. 72: Crescent-shaped microspore partially released from the degrading callose wall. Note structural differences of pollen wall in distal and proximal areas as well as the small saccus in the equatorial region (arrowheads). 10 min. UA, 4 min. Pb. X 3770.

Fig. 73: D istal sporoderm showing a few ektexinous elements and a well developed lamellated endexine. Glycocalyx remains only in the outer region of the endexine. 10 min. UA, 4 min. Pb. X 30000.

Fig. 74: Sporoderm in the saccus region. Ektexine consisting of tectum and footlayer, separated by glycocalyx material. Endexine consisting of white-line-centered lamellae. Intine forming to the inside of the endexine. 10 min. UA, 4 min. Pb. X 20100.

Fig. 75: Proximal sporoderm with convoluted tectum resting on footlayer. Infratectal area filled with glycocalyx. Lamellated endexine. 10 min. UA, 4 min. Pb. X 30400.

Fig. 76: Structural organization of glycocalyx. 10 min. UA, 4 min. Pb. X 106000.

87

PLATE XIII: Late free spore phase of Tsuga canadensis microspores (Figs. 77 - 80) and p e rita p e ta l membrane (Fig. 81). Scale bars = 0.5/am unless otherwise indicated. (ec = embryonal cell; END = endexine; 0 = orbicule; pc = prothallial cell; Pb = lead citrate; PTM = peritapetal membrane; T = tapetum; UA = uranyl acetate).

Fig. 77: Vacuolization of released microspores leading to change from crescent-shaped to spherical. Note small sacci in subequatorial position (arrows). The microspores are enveloped by tapetal cells. X 153.

Fig. 78: Trilete mark (arrowheads) on the proximal face of the microspore after release from the tetrad. X 2860.

Fig. 79: Deposition of tapetal material onto the sporoderm in the distal area. 20 min. UA, 6 min. LC. X 21300.

Fig. 80: Pollen grain after first mitotic division with 1st prothallial cell and embryonal cell. Note convex proximal face (arrowhead). 6 min. KMnO*. X 1370.

Fig. 81: Deposition of tapetal sporopollenin onto the p e rita p e ta l membrane. 6 min. KMn04. X 9680.

89 PLATE XIII 90

o PLATE XIV: Mature pollen grains of Tsuga canadensis (Figs. 82 - 85). Scale bars = 0.5 a 1"1 unless otherwise indicated. (END = endexine; EKT = ektexine; fl = footlayer; gc = generative cell; I = intine; Pb = lead citrate; pc = p r o th a llia l c e ll; PTM = p e rita p e ta l membrane; t = tectum; tc = tube cell; UA = uranyl acetate).

Fig. 82: Highly vacuolated pollen grain showing salient features of the sporoderm in the various regions. 10 min. KMnO*. X 2650.

Fig. 83: Distal sporoderm consisting of ektexine, endexine and intine. Ektexine divided into tectum and non-continuous footlayer. Endexine lamellate and intine undulating. 20 min. UA, 6 min. Pb. X 18700.

Fig. 84: Lateral area; highly convoluted tectal elements partly resting on footlayer are forming the ektexine. Endexine appears homogeneous and electron-dense. Intine undulating. 20 min UA, 6 min. Pb. X 18900.

Fig. 85: Proximal sporoderm consisting of ektexine, endexine and intine. Convoluted tectal elements resting on footlayer. Endexine very thin and underlain by undulating intine. 20 min. UA, 6 min. Pb. X 20000.

Fig. 86: Mature microgametophyte consisting of four cells (two prothallial cells, a tube cell and a generative cell). X 750.

Fig. 87: Section through microsporangium showing mature p o lle n grains enveloped by p e rita p e ta l membrane. X 132.

91 PLATE XIV PLATE XV: Sporoderm of mature pollen in Tsuga canadensis (Figs. 88 - 93). Scale bars = 0.5/am unless otherwise indicated. (END = endexine, EKT = ektexine, fl = footlayer; I = intine; Pb = lead citrate; t = tectum; UA = uranyl acetate).

Fig. 88: Distal sporoderm consisting of ektexine, lamellated endexine, and intine. Only the innermost lamellae show a difference in stain affinity from the ektexine. Undulating intine bi-layered based on difference in electron density. 10 min. UA, 4 min. Pb. X 12500.

Fig. 89: Distal sporoderm stained with potassium permanganate. Both exine layers of same electron density. Intine homogeneous and uni-layered. 6 min. KMnO*. X 12500.

Fig. 90: Lateral sporoderm consisting of ektexine, endexine and intine. Highly convoluted tectum partly resting on footlayer. Endexine more electron dense than ektexine. Intine bi-layered based on difference in electron density. 10 min. UA, 4 min. Pb. X 12500.

Fig. 91: Lateral sporoderm stained with potassium permanganate. Both exine layers show the same electro n density. Distinction between footlayer and endexine can not be made. In tin e homogeneous and u n i-lay ered . 6 min. KMn04 . X 12500.

Fig. 92: Proximal sporoderm consisting of ektexine, endexine and intine. Tectal convolutions resting on footlayer. Endexine more electron dense than ektexine. Intine bi-layered based on differences in electron density. 10 min. UA, 4 min. Pb. X 12500.

Fig. 93: Proximal sporoderm stained with potassium permanganate. Both exine layers show the same electro n density. A few white-line-centered lamellae (arrow) are seen in the position of the endexine. Intine homogeneous and uni-layered. 6 min. KMn04 . X 12500.

93 PLATE XV PLATE XVI: Sporoderm of acetolyzed pollen in Tsuga canadensis (Figs. 94 - 101). Scale bars = 0.5/um unless otherwise indicated. (END = endexine; t = tectum).

Fig. 94: Pollen grain showing distal surface and subequatorial saccus. X 2340.

Fig. 95: Section through whole grain showing salient features of the sporoderm: thick endexine in distal area and highly convoluted tectum in lateral area. 6 min. KMn04 . X 1670.

Fig. 96: Distal sporoderm with gemmate ektexine and lamellated endexine. Footlayer cannot be recognized with c e rta in ty . 6 min. KMn04. X 11600.

Fig. 97: Lateral sporoderm consisting of highly convoluted tectum in rudimentary saccus area. 6 min. KMn04. X 11600.

Fig. 98: Proximal sporoderm. Tectal convolutions resting on footlayer which cannot be clearly differentiated from the endexine. Note lamellae in the position of the endexine (arrow). 6 min. KMn04 . X 11600.

Fig. 99: Gemmate distal surface. X 12300.

Fig. 100: Equatorial surface. The convolutions form elongated, irre g u la r ridges. X 12300.

Fig. 101: Rugulate proximal surface. X 12300.

95 PLATE XVI C h a p te r V

POLLEN WALL DEVELOPMENT IN TAXODIUM DISTICHPM

The genus Taxodium was widely d is trib u te d in North

America and Europe during the Tertiary (Florin, 1963).

Today, however, only three species are known and they are native to the south-eastern United States and Mexico

(Fowells, 1965).

Coker (1903) and Vasil & Sahni (1964) reported on the embryology of Taxodium. They showed that the microstrobili begin to develop in the fall and that by the following

January the microspore mother cells are formed. According to them, form ation of the microspores occurs early in March and meiotic divisions are of the successive type. Shortly after release from the tetrad, the microspores undergo mitotic divisions and the pollen grains are shed at the

2-celled stage.

Pollen grains of the Taxodiaceae are morphologically uniform (Van Campo-Duplan, 1951). They are non-saccate and the exine consists of two layers, a granular ektexine and a lamellated endexine. The germinal zone has a central protrusion referred to as a papilla. With the aid of biometric analysis of pollen sizes, Van Campo-Duplan (1951)

97 was able to divide the genera in to two groups. Taxodium

falls in the group with 'small pollen'. Most descriptions

of Taxodium pollen have been based on light microscopical

studies (e.g., Wodehouse, 1935; Erdtman, 1965). The only

ultrastructural study is by Roscher (1975). She described

the sporoderm of T^ distichum as composed of a lamellated

layer, a footlayer, a granular layer and an orbicular

layer. The germinal zone is characterized by the absence of

the granular layer. In the region of the papilla, the

lamellae decrease in number and the tip of the papilla is t unilamellar.

Exine development in gymnospermous pollen with

granular infrastructure has been reported for only one

taxon of co n ifers, Taxus baccata. by Rohr (1977, 1980) and

Pennell & Bell (1985, 1986). These studies, however,

differ from each other in respect to the interpretation of

the timing of wall layer deposition. Rohr (1977, 1980)

stated that the exine develops while the microspores are

still in the tetrad, while Pennell & Bell (1986) reported

that it occurs in the free spore phase.

The purpose of the present study was to illustrate and discuss the deposition of the pollen wall layers in

Taxodium distichum and to compare th e timing of w all layer deposition in that taxon with that of other seed plants having granular pollen walls. 99

FORMATION OF THE TETRADS - In e a rly February, a

tran sv erse section through a microsporangium of Taxodium

distichum reveals densely packed microspore mother cells

(MMCs) surrounded by a layer of tapetal cells and two to

three layers of sporangial wall cells (Fig. 102). During

February the primary walls of the MMCs begin to break down

(Fig. 103). As the sporangia enlarge, the MMCs become

separated and begin to round off (Fig. 104). The MMCs

synthesize a fibrillar surface coat that is deposited outside the plasma membrane by v e s ic le s (Fig. 106). By the beginning of March, the MMCs are in m eiotic prophase. They

show a characteristic polarized distribution of organelles

in the cytoplasm. The nucleus is usually positioned in one half of the protoplast while lipid granules, starch grains

and other organelles are in the other half (Fig. 107). The

MMCs are surrounded by a fibrillar surface coat when

callo se deposition commences (Fig. 106). M eiotic d iv isio n s of the MMCs are asynchronous and of the successive type.

The first meiotic division is followed by the formation of a callose wall separating the two daughter cells. This is evidenced by the relatively large number of dyads present

in the sporangia (Fig. 108, arrows). The second meiotic division is again followed by callose deposition and leads to the formation of tetrads (Figs. 109, 110).

TETRAD PERIOD - The microspores in the tetrad are separated from one another by a thin layer of callose. At most this callose layer reaches 300 nm in thickness. The fibrillar MMC surface coat becomes granular and separates in places from the callose layer (Fig. 110). The nuclei of the microspores migrate toward the distal area where the callose wall is the thinnest (Fig. 111). Cytoplasmic preservation of distichum microspores was always very poor despite double fixation. This poor preservation makes

it impossible to assess and discuss further the involvement of various organelles in the processes of wall formation.

The cytoplasm is always densely populated by lipid g lo b u les.

Pollen wall formation is initiated after the microspores have been totally sequestered by the callose wall. The first indication of wall formation is seen in the center of the te tra d , where the plasma membrane becomes undulated and electron dense wedges and granules appear to the outside of the plasma membrane, against the callose wall (Fig. 112). Potassium permanganate staining reveals only iso la te d granules outside the plasma membrane a t th is stage (Fig. 113). The deposition of these granules continues until the proximal and lateral areas are covered by a granular layer consisting of widely spaced large granules that are interconnected by bridges formed of smaller ones. The large granules are in direct contact with the plasma membrane (Figs. 114, 115). In the d is ta l region, i.e. in the area of the future germinal zone, no granules are deposited. In this area, the nucleus is in

close contact with the plasma membrane (Figs. 121, 122).

The papilla that forms in the center of the germinal zone

is initiated as a protrusion of the plasma membrane in the

distal area (Figs. 121, 122).

At the time of callose dissolution, the exine of T.

distichum consists of a granulur ektexine (approx. 210 nm

thick). This layer surrounds the microspore except in the

germinal zone where no exine material is deposited during

the tetrad phase (Fig. 118). Electron dense material

appears interspersed between the granules and also on both

sides of the plasma membrane (Figs. 116, 119, 120). Small

packages of short lamellae also appear to the inside of the

plasma membrane (Fig. 120). No o th er evidence of endexine

\ formation during the tetrad phase was found.

FREE SPORE PERIOD - Microspores are released from the

tetrad as a result of the dissolution of the callose wall.

Once the microspores are so released, formation of the

endexine occurs rather rapidly and could not be traced in

detail with the samples available. One day after release

from the tetrad, 5 to 6 white-line-centered lamellae

forming the endexine were already encountered (Figs. 127, 128) .

Upon release from the tetrad, the microspores increase

in volume and f i l l the microsporangium (Fig. 123). Their volume increase, however, is much less than in Tsuga or 102

Abies. At most, they double their volume. At this stage, the microspores are still surrounded by a layer of secretory tapetal cells (Fig. 124). These cells have vesicles with electron opaque contents and tubular endoplasmic reticulum. As illustrated in Fig. 126, their cell walls are almost completely degraded and numerous electron dense orbicules surround the cells. A strongly electron dense layer is present outside the tapetal plasma membrane.

Some aborted tetrads are always present in the microsporangia (Fig. 125). These tetrads appear to have aborted after the deposition of the first granules but before a continuous granular layer was formed. After the dissolution of the callose wall, orbicules accumulate also on the surface of these aborted microspores, but no pollen wall layers are added.

Microspores in the early free spore phase always appear plasmolyzed (Fig. 124). In the germinal zone, the exine consists of a lamellated endexine with numerous orbicules adhering to the lamellae (Fig. 128). The proximal and lateral areas of the grain show a two-layered exine, composed of an outer granular ektexine and an inner, lamellated endexine. The lamellae of the endexine are fused to one another at various places (Fig. 127, arrows).

It appears that additional short lamellae are apposited from the microspore protoplast in the late free spore phase 103

(Fig. 128). After the endexine has reached a thickness of approx. 450 nm, i.e. 8 to 10 lamellae, intine deposition commences (Fig. 129).

MATURE POLLEN WALL - The salient features of mature

Taxodium distichum p o llen are il lu s tr a t e d in F igures 129 and 134. The germinal zone occupies approximately one

third of the surface area of the pollen grain in the distal area. In this region the exine consists only of a

lam ellated endexine. The number of lam ellae decreases

toward the papilla so that only one lamella covers its tip

(Fig. 136). Numerous orbicules adhere to the endexine. The rest of the pollen wall, i.e. in proximal areas, is characterized by a two-layered exine. The lamellated endexine is the same thickness as in the germinal zone and

the granular ektexine is up to 300 nm thick. The intine appears to be deposited evenly around the microspore. The size of the intine cannot be accurately determined because

it is not possible to assess the extent to which it has been altered by plasmolysis of the protoplast, but it does not exceed the thickness of the exine. The interlamellar and intergranular spaces in the mature pollen grains become partly filled with an electron-opaque material (Figs. 130 -

132). As also seen by scanning electron microscopy, the surface of the mature wall is granular and covered with orbicules (Fig. 133). The surface in the germinal zone is psilate (Fig. 135). The mature pollen grains are enclosed 104

in the p e rita p e ta l membrane (Figs. 137, 138), i.e . the acetolysis-resistant wall layer lining the innermost layer of sporangial wall cells. PLATE XVII: Microspore mother cells (MMCs) ’in Taxodium distichum (Figs. 102 - 107). Scale bars = 0.5/um unless otherwise indicated. (L = lipid globule; ml = middle lamella; MMC = microspore mother cell; N = nucleus; Pb = lead citrate; SC = microspore mother cell surface coat; T = tapetum; UA = uranyl acetate; WL = sporangial wall layer cells) .

Fig. 102: Section through a microsporangium showing microspore mother cells, tapetal cells and wall layer cells. The cells appear plasmolyzed. 20 min. UA, 6 min. Pb. X 1540.

Fig. 103: Degrading cell wall between microspore mother cells and deposition of fibrillar coat. Middle lamella of primary wall is still recognizable. 8 min. UA, 4 min. Pb. X 42600.

Fig. 104: MMCs are rounding off and becoming separated within the enlarging microsporangium. 20 min. UA, 6 min. Pb. X 3140.

Fig. 105: F ib r illa r surface coat of MMCs. 20 min. UA, 6 min. Pb. X 50800.

Fig. 106: Deposition of fibrillar surface coat material as a re s u lt of fusion of vesicles w ith the plasma membrane (arrow). Note remnants of former plasmodesma (arrowhead). 20 min. UA, 6 min. Pb. x 33500.

Fig. 107: MMC with polarized distribution of organelles. Lipid globules and starch grains are concentrated in one half of th e cytoplasm. 20 min. UA, 6 min. Pb. X 3090.

105 106

PLATE XVII PLATE XVIII: Dyads and tetrads in Taxodium distichum (Figs. 108 - 111). Scale bars = 0.5 /um unless otherwise indicated. (C = callose wall; L = lipid globule; N = nucleus; Pb = lead citrate; S = starch grains; T = tapetum; UA = uranyl acetate; WL = sporangial wall layer cells).

Fig. 108: Section through a microsporangium filled with numerous dyads (at arrows) and tetrads. X 360.

Fig. 109: Tetrads embedded in the tapetal residues. 20 min. UA, 6 min. Pb. X 1110.

Fig. 110: Young tetrad. The microspores are separated from each other by a callose wall and the tetrad is surrounded by the fibrillar MMC surface coat. 20 min. UA, 6 min. Pb. X 5730.

Fig. Ill: Microspore cytoplasm showing a dense population of lipid globules. Nuclei in distal positions within the c e lls . 20 min. UA, 6 min. Pb. X 6410.

107. 108 PLATE XVIII PLATE XIX: Proximal pollen wall formation in Taxodium distichum (Figs. 112 - 117). Scale bar = 0.5 /am u n less otherwise indicated. (C = callose wall; Pb = lead citrate; PM = plasma membrane; UA = uranyl a c e ta te ).

Fig. 112: In the cen ter of the te tra d , the plasma membrane undulates and granules (arrowheads) and electron dense wedges appear. 20 min. UA, 6 min. Pb. X 32300.

Fig. 113: Potassium permanganate s ta in in g of the same stage as in Fig. 112 reveals only the undulating membrane and granules (arrowheads). 6 min. KMn04. X -28000.

Fig. 114: Larger granules between callose wall and plasma membrane interconnected by smaller granules. 8 min. UA, 4 min. Pb. X 24000.

Fig. 115: Potassium permanganate s ta in in g of the same stage as shown in Fig. 114. 6 min. KMn0«. X 25000.

Fig. 116: In the late tetrad phase the proximal exine consists of granules (arrowheads) interspersed with dense, fibrillar material. 20 min. UA, 6 min. Pb. X 25700.

Fig. 117: Interspersed fibrillar material as seen in Fig. 116 not resolved when potassium permanganate is used as a section stain. 8 min. KMnO*. X 27800.

109 PLATE XIX I

PLATE XX: Late te tr a d phase in Taxodium distichum (Figs. 118 - 122). Scale bars = 0.5 Aim unless otherwise indicated. (EKT = ektexine, Pb = lead citrate; UA = uranyl a c e t a t e ) .

Fig. 118: At the time of callose dissolution, the germinal zones show no indication of pollen wall formation. Some electron-dense material is visible, adhering to the plasma membrane. 20 min. UA, 6 min. Pb. X 39500.

Fig. 119: In the lateral area, a granular ektexine is present. Electron-dense material is appressed to the plasma membrane. 1 min. KMnO*, 1 min. UA, 1 min. Pb. X 39200.

Fig. 120: Granular ektexine in the proximal area. In some places, short lamellae are seen to the inside of the plasma membrane (arrows) . 20 min. UA, 6 min. Pb. X 32100.

Fig. 121: No ektexine is formed in the germinal zone. The papilla is initiated as a protrusion formed by the plasma membrane (arrowhead). 10 min. UA, 4 min. Pb. X 3650.

Fig. 122: Potassium permanganate staining shows the exine to consist of a granular layer at the time of callose dissolution. No ektexine is present in* the germinal zone (between arrowheads). Note the position of the nucleus in relation to ektexine formation. 8 min. KMn04. X 4510.

Ill 112

PLATE XX PLATE XXI: Free spore phase in Taxodium distichum (Figs. 123 - 128). Scale bars = 0.5 /um unless otherwise indicated. (END = endexine; EKT = ektexine; M = microspore; 0 = orbicule; Pb = lead citrate; T = tapetum; UA = uranyl acetate; V = vesicle).

Fig. 123: Section through a sporangium with dividing microspores surrounded by a layer of partly degraded tapetal cells. X 350.

Fig. 124: Section through a microsporangium showing degrading tapetal cells and free microspores. All of the microspores appear to be plasmolyzed. 20 min. UA, 6 min. Pb. X 1790.

Fig. 125: Aborted tetrad showing no sporoderm formation. 20 min. UA, 6 min. Pb. X 3280.

Fig. 126: Tapetal cells characterized by vesicles containing secretory materials. Orbicules (arrows) surround the degrading tapetal cells. 20 min. UA, 6 min. Pb. X 9350.

Fig. 127: Exine in the proximal area consisting of a granular ektexine and a lamellated endexine. The granules of the ektexine are of various sizes, the smaller ones interconnecting the larger ones. Six to 7 white-line-centered lamellae form the endexine. 8 min. UA, 4 min. Pb. X 37000.

Fig. 128: Exine in the germinal zone consisting of endexine only. Five to 6 white-line-centered lamellae form the endexine. Orbicules are in close proximity to the endexine. 8 min. UA, 4 min. Pb. X 37000.

113 114 PLATE XXI PLATE XXII: Sporoderm of mature pollen in Taxodium distichum (Figs. 129 - 132). Scale bars = 0.5 /um unless otherwise indicated. (END = endexine, EKT = ektexine; P = papilla; Pb = lead citrate; UA = uranyl acetate).

Fig. 129: Section through whole grain showing salient fe a tu re s. Note the decreasing number of endexine lam ellae towards the germinal papilla. Sporoderm in germinal zone (between arrowheads) consisting of lamellae. Granular ektexine and lamellated endexine occur in the remainder of the grain. Protoplast plazmolyzed. 20 min. UA., 6 min. Pb. X 4800.

Fig. 130: Exine in proximal area. Spaces between the ektexine and endexine, as well as between the endexine lamellae become filled with electron dense material. 20 min. UA, 6 min. Pb. X 77600.

Fig. 131: Mature pollen wall in proximal area consisting of granular ektexine, lamellated endexine, and fibrillar intine. 20 min. UA, 6 min. Pb. X 50000.

Fig. 132: Mature pollen wall in germinal zone consisting of lamellated endexine and fibrillar intine. 20 min. UA, 6 min. Pb. X 50000.

115 116

PLATE XXII PLATE XXIII: Mature pollen (Figs. 133 - 136) and p e rita p e ta l membrane (Figs. 137, 138) of Taxodium distichum. Scale bars = 0.5/um unless otherwise indicated. (0 = orbicule; P = papilla; Pb = lead citrate; PTM = p e rita p e ta l membrane; UA = uranyl a c e ta te ) .

Fig. 133: Numerous o rb icu les adhering to the granular surface of the mature non-acetolyzed grains. X 4870.

Fig. 134: Section through mature grain showing position of papilla in the germinal zone (between arrows) and difference in exine composition between the germinal zone and the rest of the pollen grain. Intine electron transparent. The protoplast appears to be plasmolyzed. 1 min. KMn04 , 1 min. UA, 1 min. Pb. X 3970.

Fig. 135: Psilate surface of germinal zone and papilla. X 9760.

Fig. 136: Number of endexine lamellae in the papilla decreased to one in region of germinal papilla. 20 min. UA, 6 min. Pb. X 10100.

Fig. 137: Numerous o rb icu les s t i l l lin in g the p e rita p e ta l membrane after shedding of the pollen. 20 min. UA, 6 min. Pb. X 13200.

Fig. 138: P e rita p e ta l membrane in microsporangium a f te r shedding of pollen. 20 min. UA, 6 min. Pb. X 26100.

117 PLATE XXIII Chapter VI

DISCUSSION

Traditionally, pollen wall development in angiosperms has been divided into three phases: premeiotic, tetrad and

free spore phase (e.g. Heslop-Harrison, 1971). This temporal division of pollen wall development was later adopted by researchers investigating gymnosperms, and the i exact timing of wall layer deposition has been considered s ig n ific a n t (e.g. Audran, 1981; Zavada, 1983). In angiosperms, the ektexine is formed during the tetrad phase and the endexine, if present, in the free spore phase. In gymnosperms, however, both the ektexine and endexine are deposited during the tetrad phase.

In the following discussion of the gymnosperms investigated in this study, pollen wall development will also be divided into stages, but primary emphasis will be put on the deposition modes of the various wall layers and their sequential dependence (Figs. 139 - 141). On this basis, pollen wall development will be divided into six steps which coincide with the initiation of the various wall layers: microspore mother cell surface coat, callose

119 \

Fig. 139: Summary diagram of developmental stages of sporoderm of Abies concolor in distal, lateral and proximal areas. (C = callose wall; END = endexine; EKT = ektexine; fl = footlayer; GC = glycocalyx of microspore surface coat; I = intine; it = infratectum; pit = pre-infratectum; pt = pretectum; SC = microspore mother cell surface coat; t = tectu m ). a. at completion of cytokinesis b. glycocalyx deposition and initiation of ektexine c. initiation of endexine d. at dissolution of callose wall e. initiation of intine and elaboration of ektexine f . mature sporoderm

120 121

DISTAL LATERAL PROXIMAL Fig. 140: Summary diagram of developmental stages of sporoderm of Tsuqa canadensis in distal, lateral and proximal areas. (C = callose wall; END = endexine; EKT = ektexine; fl = footlayer; GC = glycocalyx of microspore surface coat;" I = intine; pt = pretectum; SC = microspore mother cell surface coat; t = tectum). a. at completion of cytokinesis b. glycocalyx deposition and initiation of ektexine c. initiation of endexine d. at dissolution of callose wall e. initiation of intine and elaboration of ektexine f . mature sporoderm

122 123

wmmmm Fig. 141: Summary diagram of developmental stages of exine of Taxodium distichum in d is ta l and proximal area. (C = callose wall; END = endexine; EKT = ektexine; 0 = orbicule; SC = microspore mother cell surface coat). a. at completion of cytokinesis b. glycocalyx deposition and in itiation of ektexine c. elaborattion of ektexine d. at dissolution of callose wall e. elaboration of endexine f . mature exine

124 125

DISTAL PROXIMAL

© 126 wall, microspore surface coat, ektexine, endexine, and i n t i n e .

Microspore mother cell surface coat

In all three taxa investigated, the primary cell walls of the MMCs are broken down before the onset of meiosis.

The plasma membrane appears to withdraw from the degrading w all and becomes undulating or wavy. The MMCs produce a new, thin coating which is deposited between the plasma membrane and the remnants of the primary c e ll w all. This surface coat consists of electron dense fibrils, oriented perpendicularly to the cell surface.

The production of such MMC surface coats has also been observed in other gymnsosperms. Willemse (1971a) reported the presence of a thin, electron dense wall between the MMC wall and the plasma membrane in the early meiotic stages of

Pinus s v lv e s tris MMC. Rowley & Walles (1985) described the

MMC surface coat in P_;_ svlvestris as consisting of an irregular filamentous mesh. In Podocarpus macrophvllus, the dissolution of the primary cell wall is accompanied by the appearance of a loose fibrillar material. Vasil &

Aldrich (1970) suggested that this material may represent breakdown products of the MMC wall. This interpretation was a lso offered by Lepouse (1966, 1971) for the dark staining layer outside the callose wall in tetrads of Abies / 127

pinsapo. Figures 17 and 106 however, provide evidence that

the MMC surface coat consists not only of remnants of the

original, primary cell wall but also includes newly

deposited materials. Large vesicles containing a fibrillar

material are seen in the cytoplasm and they evidently fuse

w ith the plasma membrane, thereby emptying th e ir contents

into the periplasmic space and coating the MMC surface.

The deposition of perpendicularly oriented fibrils on

the MMC surfaces in Ceratozamia mexicana during meiotic

prophase was described by Audran (1973). His histochemical

investigations showed that the primary MMC walls as well as

the MMC surface coats consist mostly of hemicellulose.

Even though the MMC surface coats in conifers appear

structurally similar to those in the cycads, it cannot be

inferred that they are of similar chemical composition,

since the primary MMC walls in conifers are cellulosic and

not hemicellulosic in composition as in cycads. Only

histochemical analyses could provide an answer as to what

type of polysaccharides are present in the MMC surface coat

of coniferous taxa.

As c a llo s e deposition commences, the MMC surface coat

becomes partly covered with callose. The MMC surface coat

may serve as a precursor for callose deposition as well as

providing a protection for the MMC protoplast, since the 128

prim ary MMC w all becomes degraded before c a llo se deposition

begins.

Callose wall

The callose wall is deposited between the microspore

surface coat and the plasma membrane during meiosis of the

MMCs. In Abies concolor and Tsuqa canadensis, callose wall

formation between the meiotic products occurs

simultaneously at telophase II leading to the formation of

tetrads. In Taxodium distichum, however, wall formation is

of the sucessive type. The first meiotic division is

followed by deposition of callose, leading to the formation

of dyads, and continuing depositon of callose following the

second meiotic division gives rise to tetrads.

Huynh (1976) showed that there is a causal

relationship between the type of callose wall formation

during microsporogenesis and the symmetry of the mature

pollen grains in angiosperms. In taxa with simultaneous

callose wall formation, the mature pollen grains are

radially symmetrical while in taxa with successive type of wall formation, the mature grains exhibit bilateral

symmetry. He also indicated that this might not hold true

for gymnosperms, citing pollen of Pinus and Sequoia as examples. The taxa investigated in this study support his conclusion for gymnosperms. The pollen of Abies concolor 129 and Tsuqa canadensis is bilaterally symmetrical despite the simultaneous type of callose wall formation, while mature pollen of Taxodium distichum is ra d ia lly symmetrical following successive callose wall formation during microsporogenesis.

The callose in all three taxa investigated forms a homogeneous layer around the microspores during the tetrad phase. In Abies concolor it is up to 650 nm thick, in

Tsuqa canadensis up to 500 nm and in Taxodium distichum up to 350 nm. In comparison to gymnosperms, callose walls are much more massive in angiosperms. In Heliconia (Stone et al., 1979), for example, it is around 1200 nm, in Leontodon

(El-Ghazaly, 1982) also around 1200 nm and in Gibasis

(Owens & Dickinson, 1983) around 1100 nm.

Various functions have been ascribed to the callose wall layer during microsporogenesis. Heslop-Harrison

(1968b, 1971) suggested th a t i t provides an is o la tin g mechanism in that i t e s ta b lis h e s a b a rrie r between the genetically haploid microspores and the surrounding diploid tissues. He also assumed that it may provide a carbon source for the development of the microspore surface coat.

Brooks & Shaw (1968) in d icated th a t the c a llo se w all may protect various enzymes involved in the development of the microspore surface coat and also prevent random oxidation of sporopollenin precursors. Waterkeyn& Bienfait (1970, 130

1971) demonstrated that the callose wall plays a role in the establishment of the exine pattern, in Ipomea.

El-Ghazaly & Jen sen (1986) suggested that the callose wall is an energetically inexpensive temporary wall that can accommodate changes in size and shape while the complex permanent pollen wall is laid down. This temporary wall protects the microspores from changes in osmotic pressure in their immediate environment.

Bajaj et al. (1975) isolated microspore protoplasts of

Nicotiana from the surrounding callose wall in the early tetrad stage when the callose wall was still in contact with the plasma membrane, and at a later stage, during deposition of the microspore surface coat. The cultured protoplasts isolated at the early stage produced only a simple wall consisting of a fibrillar network while the ones isolated at the later stage showed deposition of sporopollenin. Their experiments show that the callose wall plays an important role in the establishment of a complex pollen wall. According to Jain& Shah (1985), callose is absent during microsporogenesis in Naias marina, and the mature pollen grains lack exines. In their view, the absence of callose is correlated with the absence of the exine, and this supports the hypothesis that callose protects enzyme systems responsible for exine deposition and that it provides a template for exine establishment. 131

In other exineless pollen, however, a callose wall is

formed during during microsporogenesis (e.g., Stone et al., I 1979; Pettitt et al., 1984). This seems to emphasize that

the callose wall has both structural and physiological

fu n c tio n s.

In Abies concolor and Tsuga canadensis, callose

deposition continues selectively after the microspores are

completely separated from each other in the tetrad by the

callose wall. In this way, callose protrusions are formed

and these protrusions act as structural templates against

which microspore surface material coat and the alveolar

ektexine is later deposited. In Taxodium distichum the

mature ektexine is granular and no calloslc extensions are

present. Depending on the type of ektexine to be formed,

the "structural template" function of the callose wall may

be more or less important. Therefore, in pollen with

alveolar exines, callose provides a template for exine

deposition, while in pollen with granular exines the

structural function of the callose is less important.

Microspore surface coat

After the callose wall completely separates the meiotic products, the microspore surface coat is deposited.

The deposition of this coat is initiated before or is

concurrent with the appearance of ektexinous elements. 132

This surface coat has been called the primexine based on its function as an exine template (Heslop-Harrison, 1963) and Rowley (1973) termed it a glycocalyx based on its chemical composition.

The microspore surface coat in Abies concolor forms a reticulate network between the callose and the plasma membrane. I t does not change in appearance during the course of ektexine development and it provides receptor sites for the sporopollenin forming ‘the tectum, infratectum and foot layer. In Tsuga canadensis, the microspore surface coat is deposited during the early tetrad phase in the form of short fibrils which are oriented parallel to the plasma membrane. I t changes in s tru c tu re during the course of ektexine development and appears as a loose, reticulate network by the end of the tetrad period. The microspore surface coat in Taxodium distichum consists of very densely packed material and appears concurrently with the granular ektexine.

The deposition of a microspore surface coat is not only a uniform feature of pollen wall development in angiosperms, but appears also to be so in gymnosperms. The microspore surface coat in Abies pinsapo is structurally identical to that in A^_ concolor (Lepouse, 1966, 1971). In

Podocarpus macrophvllus (Vasil & Aldrich, 1970), the fibrillar microspore surface coat consists of two zones. 133

In the inner zone, the fibrils are oriented parallel to the plasma membrane, while in the outer zone, they form a loose, reticulate network. In Pinus banksiana (Dickinson,

1971) the microspore surface coat forms a dispersed fibrillar layer, while in £\_ svlvestris it is initiated in a granular form which changes into a fibrillar network

(Willemse, 1971a). A fibrillar microspore surface coat has also been reported in Ginkgo biloba (Rohr, 1980). Audran

(1981) reported that a fibrillar substance is laid down in a delicate network between the callose wall and the plasma membrane in Ceratozamia mexicana. and in Zamia floridana

(Zavada, 1983), a dispersed fibrillar surface coat appears concurrently with the tectum.

The glycocalyx is transported to the plasma membrane via Golgi-derived vesicles and is deposited unevenly over the microspore surface. Heslop-Harrison (1971) pointed out that the establishment of apertures in angiosperm pollen is correlated with the absence of the surface coat material or glycocalyx. In angiosperms, there is evidence that the deposition of glycocalyx in the area of future apertures is blocked through the apposition of cisternae of endoplasmic reticulum to the plasma membrane. In gymnosperms, the microspore surface coat is also absent or is very thin in the region of future apertures, but there is no evidence that endoplasmic reticulum cisternae block its deposition. 134

It is not clear what controls the deposition of the surface coat material. In Taxodium distichum, however, it appears that the position of the nucleus inhibits glycocalyx deposition in the distal region by preventing the organelles involved in its secretion from reaching the plasma membrane in this area. The amount of glycocalyx deposited in gymnosperms also seems to be correlated with the thickness of the ektexine. In the lateral region, if sacci will later be formed, the glycocalyx layer becomes t very thick.

The glycocalyx is a general component of the cell surface, but its structural organization shows enormous variation (Bennett, 1963, 1969). No helical subunits as identified by Rowley (1981) in Artemisa have been seen in the microspore surface coats of the gymnospermous taxa here investigated. This might be due to the different preparation techniques employed, but more likely it underlines the inherent structural variations of glycocalyces. Different types of ektexines may well be elaborated on structurally different microspore surface coats. The structure of the glycocalyx in the taxa investigated is more similar to three-dimensional lattice described by Southworth (1986) in Juniperus. 135

E k te x in e

Ektexine formation is characterized by the deposition of sporopollenin precursors into the periplasmic space occupied by the microspore surface coat. The microspore surface coat provides the receptor sites for the sporopollenin as mentioned above.

In Abies concolor, ektexine initiation is indicated by the appearance of osmophilic globules in the microspore surface coat. These appear to accumulate against the callose wall and against the branching callosic protrusions during the tetrad phase. Continuous accumulation of these globules leads to the formation of the pretectum and preinfratectum. The footlayer, which is present in the mature pollen wall, is not recognizable during the tetrad period. It forms in the early free spore period and like the tectum and infratectum is also deposited with the mediation of glycocalyx. At the time of callose dissolution, the ektexinous elements are very delicate and the microspore surface coat material is still visible. It appears to condense against the ektexinous elements formed during the tetrad phase and also against the outermost endexine lamella. Additional sporopollenin is accumulated in the free spore period until all the glycocalyx receptor sites are covered. This leads to a thickening of the tectum and infratectum and to the formation of the footlayer. 136

Ektexine is only formed where surface coat material has been deposited. Therefore, no ektexine is formed on the distal face of the pollen grains.

Tsuga canadensis, ektexine formation is similar to that observed in A_^ concolor. Elongated, osmophilic globules are deposited against the callose wall and the callose protrusions. Since these protrusions in T. canadensis are short and unbranched, only a continuous pretectum is formed and no in f r a te c ta l elements are elaborated. The pretectum is also formed on the distal surface since a microspore surface coat was earlier deposited also in this region. Upon release from the tetrad, the glycocalyx appears to condense against the ektexinous elements formed during the tetrad phase and against the outermost lamella of the endexine as in A. concolor. Continuous sporopollenin accretion leads to a thickening of the tectum and the formation of the fo o tla y e r.

In Taxodium distichum the ektexine is in itia te d through the deposition of osmophilic globules between the callose wall and the plasma membrane, concurrently with the deposition of the microspore surface coat. Granules of two sizes are deposited, and the smaller ones appear to form bridges between the larger ones. In the distal region, i.e. the area of the future germinal zone, no ektexine is 137 deposited. After release from the tetrad some additional sporopollenin is accumulated on the ektexine and numerous orbicules adhere ot the outer surface of the mature pollen w all.

Ektexine formation in other gymnosperms is similar to

the modes observed in the study. Lepouse (1971) reported

that electron dense material is deposited against the

callose wall and callose extensions forming the tectum and

infratectum in A^ pinsapo. He did not discuss the

involvement of the microspore surface coat in this process, but he remarked that the footlayer forms at a later stage, shortly before the disappearance of the fibrillar material during the free spore phase. In Pinus banksiana. ektexine

i n i t i a l s emerge at the plasma membrane through the fusion of vesicles (Dickinson, 1971). Deposition of the microspore surface coat between these initials and the plasma membrane displaces the p ro to p la s t. In P. sylvestris. the tectum and infratectum are formed by condensation and precipitation of the fibrillar surface coat against the callose wall and its protrusions

(Willemse, 1971a, d). In Podocarpus macrophvllus. the pretectum is laid down on tapes of unit membrane dimensions between the callose wall and the plasma membrane. In later stages of development these membranes disappear and the tectum becomes electron dense (Vasil & Aldrich, 1970). Short rod-like structures that extend inwards from the tectum form the infratectum and the periplasmic space becomes f i l l e d w ith a f i b r i l l a r network. Rohr (1977, 1980) also indicated that the tectum and infratectum are initiated prior to the deposition of the microspore surface coat in Ginkgo biloba. In Ceratozamia mexicana, the fibrillar surface coat is deposited before the osmophilic granules appear against the callose wall and its extensions

(Audran, 1981). The fibrillar surface coat in Zamia floridana appears concurrently with the tectum (Zavada,

1983).

When the formation of ektexine initials in these taxa is compared, it appears at first sight that they are different. Some reports indicate that the tectum and infratectum are initiated before glycocalyx material is deposited. In others, however, a relatively large amount of glycocalyx is deposited before the ektexine initials appear. This apparent difference might be due to the fact that some of these studies do not provide sufficient detail of the earliest stages of glycocalyx deposition. In Abies concolor and Tsuga canadensis, for example, deposition of the fibrillar glycocalyx of 'the microspore surface coat continue while the tectum and intratectum are elaborated.

The first ektexine initials appear while the microspore surface coat is not very thick. Therefore, if 139

sporopollenin precursor deposition is relatively rapid it

may appear in some taxa that the tectum and infratectum are

formed prior to any glycocalyx deposition. If this is

considered in the interpretation of less detailed studies,

it is clear that the entire formation of the ektexine in

gymnosperms i s mediated by a glycocalyx, i.e . sporopollenin

accumulation in this wall layer occurs within the

glycocalyx of the microspore surface coat.

Ektexine elaboration within a glycocalyx of the

microspore surface coat is also a characteristic feature of

angiosperm pollen walls. Abadie & Hideux (1980) recognized

three principal modes of ektexine development in

angiosperms. In each, a fibrillar microspore surface is deposited, but there is a difference in the sequence in which the structures appear within it. In the first type,

infratectal initials are deposited before other ektexinous

layers are formed (e.g. in Silene and Lilium) . The second

type produces tectal initials first (e.g. Artemisia) and in the third type, tectal and infratectal initials are elaborated concurrently (e.g. Saxifraga) .

Sacci formation - The sacci are formed while the microspores are still enclosed in the callose wall. In the area of the future sacci, the periplasmic space between the callose w all and the plasm a membrane becomes much enlarged due to the increased amount of glycocalyx deposited in these areas. The sacci walls are apparently formed in the same manner as the re s t of the ektexine, except th a t the space between the callo se w all and the plasma membrane is much wider. In Abies concolor, two sacci are formed in a lateral position while in Tsuga canadensis, one saccus surrounding the entire microspore in the subequatorial area is initiated. The saccus in T^. canadensis remains small most likely because of spatial constraints within the tetrad. A small saccus surrounding the entire microspore may indeed occupy the same area as two larger lateral sacci. In A^ concolor and T^ canadensis release from the tetrad is marked by an increase in saccus volume.

Additional sporopollenin accretion in the free spore phase leads to a thickening of the tectum and of the elements forming the reticulate infrastructure. The distinct footlayer forming the saccus floor is deposited on glycocalyx materials at the same time just as in the remainder of the ektexine.

Vasil & A ldrich (1970, 1971) considered th a t the reticulate matrix material in the sacci of Podocarpus macrophvllus represents sporopollenin precursors which are later lost. In Pinus banksiana. the indication of sacci formation is also a broad space between the plasma membrane and the callose wall in the lateral areas (Dickinson &

Bell, 1970). This space usually contains a fibrillar 141 network which reacts positively to treatment with periodic acid Schiff's reagent. Dickinson & Bell (1970) suggested that saccus expansion in the free spore phase may be due to swelling as a consequnce of osmotic forces, or more likely that the polysaccharidic material in the sacci is capable of swelling by imbibition. Willemse (1971a) indicated that the area of the future sacci provide an excellent opportunity to study ektexine development because the structures involved are enlarged.

E n d e x in e

Endexine initiation is recognized by the appearance of tripartite lamellae ('white-line-centered lamellae' of

Rowley & Southworth (1967)) which are oriented parallel to the cell surface. The endexine is deposited over the entire microspore surface and sporopollenin is accumulated directly on the lamellae without the mediation of a glycocalyx.

In Abies concolor, the endexine lamellae on the distal surface are broadly spaced and all adjacent lamellae are interconnected by radial units which produce a distinctly chambered infrastructure. In the lateral and proximal areas, however, the outer lamellae are closely appressed to one another and only the innermost ones are interconnected by radial units. The endexine is elaborated during the 142

tetrad phase and its structural characteristics are most

distinct before the microspores increase in volume during

the early free spore period. The volume increase of the protoplast is accommodated by a lateral stretching of the endexine. This leads to a thinning of the endexine

lamellae and to a large decrease in total endexine

thickness. Increase in turgor pressure in the protoplast may also press the lamellae closer to one another.

Therefore, the lamellated appearance of the endexine is not as distinct in mature pollen walls as it is during earlier developmental stages.

In Tsuga canadensis. the endexine also shows structural variation over the microspore surface. In the distal area, the endexine is composed of widely spaced lamellae which decrease in thickness towards the plasma membrane. In the la te ra l and d is ta l regions, the endexine lamellae are closely appressed to each other. No radial units interconnect the endexine lamellae. As in Abies concolor. the endexine of Tj. canadensis is elaborated during the tetrad phase, and its structural characteristics are most d is ti n c t in the e a rly free spore phase. Volume increase of the protoplast is accommodated by a change in microspore shape. This affects mostly the proximal and lateral regions and, therefore, the endexine lamallae in 143

these areas are difficult to resolve in the mature pollen w a ll.

Germination in Abies concolor and Tsuga canadensis pollen will take place through an area on the distal face

termed the leptoma (Erdtman & Straka, 1961). In A.

concolor the leptoma is characterized by an elaborate endexine and the absence of an ektexine. In T_^ canadensis,

it also has a thick endexine but a reduced ektexine is also p re s e n t.

In Taxodium distichum . the f i r s t t r i p a r t i t e lam ellae appear in the late tetrad period but the endexine is elaborated during the free spore phase. Volume increase in

Taxodium is,much smaller than in Tsuga and Abies, and the endexine retains its distinctively lamellated appearance in the mature pollen wall. In Taxodium distichum pollen, germ ination takes place through a p a p illa . The number of endexine lamellae decreases towards the papilla and only one lamella covers its tip.

Tripartite lamellae are also involved in the elaboration of the endexine in other gymnospermous taxa.

In Abies pinsapo. the lamellated endexine shows the same characteristic features as A_j_ concolor (Lepous^, 1966,

1971). Tripartite lamellae are also deposited during endexine formation in Pinus banksiana, but according to

Dickinson (1971) sporopollenin is accumulated only on one 144 side of these lamellae. Willemse (1971a) observed electron dense tapes outside the plasma membrane and a ls o in the microspore cytoplasm of Pinus svlvestris. In these tapes lamellae of unit-membrane size are present. Tripartite lamellae or tapes are also involved in the deposition of at least part of the endexine in Podocarpus macrophyllus

(Vasil & Aldrich, 1970). Rohr (1977, 1980) showed that the endexine in Ginkgo b ilo b a and Taxus baccata is also formed by the apposition of lamellated structures. In Ceratozamia mexicana, the endexine is initiated with the appearance of electron dense plates and joining of these plates leads to the elaboration of the endexine (Audran, 1981). In Zamia floridana, the endexine is also accumulated on unit-membrane like structures and appears lamellated at maturity (Zavada, 1983).

In contrast to gymnosperms, the endexine in angiosperms is elaborated during the free spore phase, but accumulation of sporopollenin on tripartite lamellae is also well documented (e.g., Heslop-Harrison, 1971; Nabli,

1979; Hideux& Abadie, 1985). In some taxa, the lamellated appearance is retained in the mature sporoderm (e.g.,

Horner & Pearson, 1978). In others, however, the mature endexine appears homogeneous. Heslop-Harrison suggested that continuous sporopollenin deposition may cover the lamellated structures so that in the mature endexine the 145 lamellae become obscured and the endexine appears homogeneous. Loss of the lamellated appearance may also, at least in part, be caused by stretching and thinning of the lamellae during the volume increase of the microspores in the free spore phase.

In tin e

The intine is the last wall layer to be formed and is deposited between the endexine and the microspore plasma membrane. In Abies concolor. its formation is initiated early in the free spore phase by the appearance of fibrillar structures. Intine development continues through microgametogenesis and in mature pollen grains the intine is two-layered. The two zones are recognizable because of differences in electron density. The intine varies greatly in thickness around the pollen grain. In the proximal region it is 700 nm thick, gradually increasing towards the distal area where it is some 2700 nm thick. Intine formation in Tsuga canadensis is similar to that of A. i concolor. It also commences in the early free spore phase by the appearance of fibrillar material to the inside of the endexine and in mature pollen is two-layered. It is up to 700 nm thick in the proximal area, increasing to 2200 nm towards the distal region where it undulates. Problems with fixation of mature Taxodium distichum pollen render 146 discussion of its intine difficult. It appears, however, that it is evenly deposited over the microspore surface.

In discussions of pollen waJ.1 layers in the literature, the intine is usually little noted, since no sporopollenin is accumulated in this wall layer. Its chemical properties are similar to those of cellulosic cell w alls (Buchen & Sievers, 1981) and its growth involves the activity of dictyosomes (Heslop-Harrison, 1971). The intine is not acetolysis resistant and is usually not preserved during fossilization.

The intine plays an important role in germination and pollen tube development. Pettitt (1985) demonstrated a structural relationship of the pollen tube wall and the intine in germinating conifer pollen. In the early stages, the wall of the pollen tube corresponds to the wall of the intine, while in more mature pollen tubes only the inner layer is continuous.

Tapeturn

Tapetum development has not been followed in detail in this study. However, the formation of a peritapetal membrane has been observed in a l l three taxa in v estig ated .

This structure is deposited to the inside of the innermost layer of sporangial wall layer cells and lines the microsporangial cavity containing the pollen grains. 147

Dickinson (1970) and Dickinson & Bell (1972, 1976a, b) traced the development of the tapetum in Pinus banksiana from the initiation of the microspore mother cells to the shedding of the mature pollen grains. Tapetal cells are similar to microspores in that they also synthesize sporopollenin-containing structures, i.e. tapetal orbicules and peritapetal membranes.

Homology of pollen wall layers

With the initiation of phylogenetically oriented studies utilizing pollen wall characters, exine stratification was used as a tool in elucidating differences and/or sim ilarities among the major groups of seed plants. In 1971, using ektexine/endexine terminology,

Van Campo established two structural types of exines for the seed plants as a whole (in order to avoid confusion based on terminology, she referred to the non-sculptured layer of the exine as the endexine sensu lato (s .1.) and in the cases where a footlayer is recognized, she divided the endexine s .1. into an endexine sensu stricto (s .s .) and a footlayer). These two structural types are that the ektexine in gymnosperms shows an alveolar in fra s tru c tu re while in angiosperms the infratectal layer is columellate.

The endexine s .1. in gymnosperms is ty p ic a lly lam ellate.

Division into a footlayer and endexine s.s ■. according to

Van Campo, is not pronounced clearly in gymnosperms. In 148 angiosperms, however, she reports that the endexine s .1. can be more readily divided into a footlayer and an endexine s . s . . The footlayer is of identical chemical nature to the ektexine and, in electron micrographs, does not show the same density as the endexine. The endexine s .s ., if present, exhibits considerable variation in width and structure. Mature endexines in angiosperms are, with a few exceptions, not lamellated. In 1973, Van Campo&

Lugardon added a third exine structural type to the two proposed by Van Campo (1971), based on the suggestion th a t the most remarkable part of the exine in terms of phylogenetic significance is the infratectal layer, i.e. the layer between the tectum and the endexine s .1.. The infratectal layer can be alveolar, columellar or granular.

Columellar infrastructure is found only in angiosperms, with the sole exception of the pollen of the Mesozoic gymnospermous family Cheirolepidiaceae (Taylor & Alvin,

1984). Alveolar infrastructure is restricted to gymnosperms, and within them, to the cycads and saccate conifers. Granular infrastructure is present both in gymnosperms (non-saccate co n ife rs and Gnetales) and in certain angiosperms generally considered primitive. Tsuga is mentioned in the literature as the one exception among gymnosperms, in having an exine without an infratectal layer. Other exceptions also occur among angiosperms, e.g. the exineless pollen of many aquatic angiosperms. V

149

Doyle et al. (1975) further considered exine

structural characters in discussing the systematics and

phylogeny of seed plants, and elaborated on the potential

of the exine structural characters of pollen in order to

discriminate early angiosperm pollen from that of gymnosperms in the fossil record. The major character they

referred to is the endexine s.1. (=nexine). They stated

th a t the endexine s . 1. in angiosperms is composed e ith e r of

an inner endexine s.s . together with a footlayer of

ektexinous material or it may be entirely ektexinous. In

seed plants having pollen with granular infrastructure, the

structure of the endexine takes on considerable importance.

In those gymnosperms with granular infratectum, for

instance, the endexine is relatively thick and is clearly

lamellated in structure. In angiosperm pollen with a

granular infratectum, however, the endexine, if present, is

typically non-lameHated except in the apertural regions.

The hypothesis of Doyle et a l. (1975) is that the

'typical', lamellated endexine of gymnosperms was lost

during the evolution of angiosperms from gymnosperms and

then "regained" with a different structure in the

angiosperms.

Hill & Crane (1982) also incorporated the structure of

the pollen endexine in their preliminary phylogenetic

analysis of the extant seed plants. They discussed the

problem of homology of the endexine in gymnosperms vs. 150 angiosperms, referring to the supposedly primitive angiosperms that have uniformly lamellated 'footlayers'

(sensu Faegri), and pointing out that the comparison of developmental sequences in pollen wall formation may be equally or more useful than staining criteria in order to discriminate which layers are homologous. This idea was also elaborated by Guedes (1982) and by Zavada (1984b) who discussed wall layer homologies in angiosperms and gymnosperms using developmental criteria. Their interpretations, however, are quite different from one a n o th er.

Guedes (1982) stated that the endexine is deposited on periclinal membranes, while the ektexine is deposited within a polysaccharidic matrix or glycocalyx, usually without mediation of membranes (if membranes are present they are not periclinal). He interprets this as evidence that the endexine in angiosperm pollen is homologous with the morphologically identical layer in gymnosperms. On this basis, he rejects the hypothesis of Doyle et al.

(1975) by pointing out that some angiosperms may simply no longer develop an endexine. According to Guedes, there is no evidence that these taxa gave rise to others with a "new endexine".

Zavada (1984b) s ta te d th a t in most gymnosperms no additional sporopolleninous wall layers are formed in the free spore phase. In contrast, in angiosperms only the 151 ektexine is formed in the tetrad phase and the development of a sporopolleninous endexine is delayed until the free spore phase. He interprets this as evidence that the endexine in angiosperm pollen and the morphologically id e n tic a l layer in gymnosperm pollen are not homologous, and on this basis he rejects the morphological criteria used by Doyle et a l. (1975) to d istin g u ish f o s s il pollen as angiospermous versus gymnospermous.

The concept of homology is fundamental to the comparative understanding of organisms and their s tru c tu re s. Homology of organization has become an important line of evidence for the existence of evolutionary processes. There are basically two components of homology (e.g. Wiley, 1981).' One, essential similarity, is the morphological criterion by which homologies may be recognized while the other, common ancestry, is the phylogentic, partly theoretical component, which provides an interpretation of what homologies mean. The parallel recognized between ontogeny and phylogeny suggests that the study of ontogeny may provide valuable insights into phylogenetic h is to ry (e.g. Nelson, 1978, 1985; Gould,

1977).

Alberch (1985) discussed the informational value of developmental sequences and pointed out that there are two types of sequence: a temporal one and a causal one. A temporal sequence is simply a series of events while in a 152

causal sequence the expression of a certain stage is a

prerequisite for the expression of the next. The lack of

causality makes temporal sequences of lesser value for

systematic and phylogenetic purposes than causal ones.

In considering criteria of homology for pollen wall

layers, the deposition modes of the various layers is

clearly more important than their timing of deposition.

The ektexine is formed within the glycocalyx of the

microspore surface coat in both gymnosperms and

angiosperms. The deposition of the microspore surface coat

is thus a prerequisite for the formation of the ektexine.

The endexine, however, is elaborated on lamellated

structures without the mediation of glycocalyces. The

deposition of the ektexine is not a prerequisite for the

formation of the endexine as is clearly seen in the distal

areas of coniferous pollen grains. Therefore, the shift in

timing of endexine deposition from gymnosperms to

angiosperms cannot sensibly be used to refute homology of

pollen wall layers as done by Zavada (1984b). Moreover, it

suports the interpretation of Gued&s (1982) that the

endexine in angiosperms is homologous to that of

gymnosperms and re fu te s the hypothesis of Kress (1986) th at

the endexine may have evolved independently several times

in angiosperms. The discussion of the deposition modes of

the various wall layers presented above and their evident sequential dependence show that the ektexine and endexine 153 in gymnosperm pollen are homologous w ith the ektexine and endexine in angiosperm pollen.

\ Chapter VII

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