BEVELOPMENTAL HFE HlSTORY 0F P221211 Q‘UELEPEBOTH KORF 8| O’DONNEH. Thesis for “to Bergman of" pk. D. MICEFGAN STATE UNIVERSITY Kerry L O’Donnell 1975 . _., may LIBRA R Y “7 Michigan State University I} This is to certify that the thesis entitled Developmental Life History of Pezize Quelepidotia Korf, & 0' Donnell presented by Kerry L. 0' Donnell has been accepted towards fulfillment of the requirements for PhD degree in BOtm (“F010”) gimcwe W Everett 8. Beneke, Professor Major professor Date August 5. 1975 0-7 639 A“ ABSTRACT DEVELOPMENTAL LIFE HISTORY OF PEZIZA QUELEPIDOTIA KORF & O'DONNELL By KERRY L. O'DONNELL This thesis on Peziza quelepidotia Korf & O'Donnell, a homothallic operculate Discomycete (Pezizales, Peziiaceae), presents the first detailed developmental life history of any species in this genus. This investiga- tion was possible because Peziza quelepidotia, unlike other Peziza spp., produces fertile ascomata in pure culture. Correlative light microscopic and scanning ultrastructural ontogeny studies were conducted on the following fungal structures: conidiophores, germinating conidia, apothecia, germinating ascospores, and excipuloid stroma. The asexual phase is pleomophic with the development of chlamydo- spores and a Botrytis-like imperfect state. The conidiophores, which belong to Hughes' (l953) conidiogenetic section lB, bear determinate retrogressive conidiogenous cells, single holoblastic conidia and multiple synchronous conidiogenous loci. However, unlike other members of the Botryoblastosporae (Barron, l968), the conidia are not borne on well differentiated, swollen conidiogenous cells (i.e. ampullae). A simple time-lapse technique for following conidium ontogeny in Peziza quelepidotia is described using differential interference phase- contrast illumination and an inexpensive commercially available slide culture chamber. It has the following advantages over previously described chambers: higher resolution, no problem with condensation or dessication, Kerry L. O'Donnell and most of the conidiophores can be photographed. Subsequent to time- lapse photomicrography, the conidiophores can be removed from the culture chamber in any stage of development and easily prepared without damage for viewing in the transmission or scanning electron microscope. Germination of deciduous conidia is generally by means of two succes- sive germ-tubes. lg_§jtg_germination of conidia is also by two successive germ-tubes. A complete structural and functional image of apothecium morphogenesis was obtained by correlating scanning electron micrographs with light micro- graphs of whole mounts and thick-sectioned (i.e. l.5-2.0 um), plastic- embedded apothecia in all stages of development. The natural relationship of the ascogonia, conidiophores and investing hyphae are resolved and accurately recorded with the scanning electron microscope. The temporal-spatial relationship between the ascogonia and the Botrytis-like imperfect state suggests that the ascogonial coils may be under chemotrophic control of the conidiophore. Since the incipient para- physes are never completely closed by the overarching ectal excipular margin, hymenial development is classified as paragymnohymenial. Although functional ascogonia are present, sexuality is apparently parthenogenetic since antheridia were never observed. Development of the gametophytic and sporophytic portions of the ascomata conforms to the familiar asco- mycetous pattern. The microanatomy of the excipula, as revealed by median longitudinally cryofractured ascocarps, is characteristic for the genus. The behavior of nuclei and spindle-pole bodies was investigated by light microsc0py from ontogeny of the crozier up to ascosporogenesis. All nuclear divisions appeared to be intranuclear. Plaque-shaped Kerry L. O'Donnell spindle-pole bodies were discernible during the synchronous mitotic divi- sion in the crozier, the reductional division in the ascus and the mitotic division preceding sporogenesis. The intranuclear reductional division was of the 'Neurospora type' with no time interval between karyogamy and the onset of meiosis. This type of meiosis is further characterized by the presence of highly contracted homologues at synapsis and the absence of leptotene. Transmission electron microscopic observations revealed a globular, electron-dense spindle-pole body in an indentation of a meiotic prophase nuclear envelope. Plaque-shaped spindle-pole bodies were dis- cernible during mitotic divisions in the multinucleate excipular cells. Transmission electron microscopy was used to study ascosporogenesis. Primary and secondary walls are formed successively and centrifugally between two limiting membranes. As a result of water imbibition, asco- spores triple in volume and then typically produce germ-tubes successively at the poles. Germ-tube emergence results in the rupture of the ascospore wall. Light microscopic observations on thick-sectioned, plastic-embedded excipuloid stroma of all stages are correlated with scanning electron micrographic observations on whole and cryofractured stroma. A comparison of the structural and functional relationship between excipuloid stroma and sclerotia is presented. DEVELOPMENTAL LIFE HISTORY OF PEZIZA QyELEPIDOTIA KORF & O'DONNELL _B.Y KERRY L. O'DONNELL A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1975 ACKNOWLEDGEMENTS The author wishes to express his appreciation to his major professor, Dr. Everett S. Beneke, for his support and for the independence he encour- aged in me in pursuing this research. Special thanks are due Dr. William G. Fields for his good humor and many thoughtful criticisms, mycological and otherwise. The many interesting conversations with Dr. Fields, both in the laboratory and field, have had a profound influence on my mycological training. It is a pleasure to acknowledge Dr. William Tai for generously making available his supplies and equipment and for expertly supervising all light microscopic observations. Sincere appreciation is expressed to Dr. Gary R. Hooper, Director of the Center for Electron Optics, for super- vising all scanning electron microscopic observations and to Art 0. Ackerson for his patience, interest, and expert instruction in the use of the scanning electron microscope and preparatory equipment. The encourage- ment and material assistance received from Dr. Hooper and Art Ackerson will not be soon forgotten. Likewise, the friendly advice and interest of William S. McAfee, Wayne S. Johnson, Dr. Paul H. Rasmussen, and Vivion E. Shull in different aspects of the scanning electron microsc0pic study is appreciated. It is a pleasure to acknowledge the assistance of Dr. Gordon C. Spink, Mrs. June P. Mack, and Dr. Robert Glick with the transmission electron microscopy. Thanks are due to Dr. Clarence W. Minkle for allowing me to attend the University of Michigan's Biological Station (Summer l970, l973) under the C.I.C. Traveling Scholar Program. 1'1 I am deeply honored for having received the annual E.A. Bessey Memorial Graduate Scholarship for 1974. It is with great pleasure that I acknowledge the receipt of this award. During my graduate training at Michigan State University, I was alpparted by graduate teaching and research assistantships administered through the Department of Botany and Plant Pathology. The encouragement and material assistance of my parents, Mr. and Mrs. John M. O'Donnell, is gratefully acknowledged. Plates 19, 22-26 are reproduced by permission of the National Research Council of Canada from the Canadian Journal of Botany, Volume 52, 1974. pp. 873-876. TABLE OF CONTENTS Page LIST OF PLATES ................................................... vi INTRODUCTION ..................................................... l LITERATURE REVIEW: ............................................... 2 A. Conidium Ontogeny ...................................... 2 B. Conidium Germination ................................... 4 C. Apothecium Ontogeny .................................... 5 D. Vegetative Nuclear Condition ........................... 9 E. Septa of Paraphyses .................................... lO F. Ascosporogenesis and Ascospores ................ ' ........ l0 G. Ascospore Germination .................................. ll H. Excipuloid Stroma ...................................... l2 MATERIALS AND METHODS ........................................... 13 A. Source of Culture ...................................... 13 B. Media and Cultural Conditions .......................... l3 C. Apothecium, Conidium, and Excipuloid Stroma Ontogeny: _ Scanning Electron Microscopy ........................ 14 D. Conidium Ontogeny: Light and Scanning Electron Microscopy l5 E. Conidium and Chlamydospore Germination ................. l6 F. Apothecium and Excipuloid Stroma Ontogeny: Light Microscopy .......................................... l6 G. Transmission Electron Microscopy ....................... l7 H. .Vegetative Nuclear Condition ........................... 18 I. Ascospore Germination .................................. l8 J. Cryofractury of Apothecia and Excipuloid Stroma ........ 19 RESULTS ......................................................... 20 A. Imperfect States. ....................................... 20 B. Apothecium Ontogeny ......... . .......................... 37 C. Cryofractured Apothecia ................................ 79 D. Vegetative Nuclear Condition ........................... 87 E. Septa of Paraphyses .................................... 87 F. Ascosporogenesis and Ascospores ........................ 92 G. Ascospore Germination ..... ............................. 97 H. Excipuloid Stroma ...................................... lOO iv Page DISCUSSION ...................................................... 110 A. Imperfect States ....................................... 110 B. Apothecium Ontogeny .................................... 117 C. Cryofractured Apothecia ................................ 122 D. Vegetative Nuclear Condition ........................... 122 E. Septa of Paraphyses .................................... 123 F. Ascosporogenesis and Ascospores ........................ 124 G. Ascospore Germination .................................. 125 H. Excipuloid Stroma ...................................... 126 BIBLIOGRAPHY .................................................... 129 APPENDIX ........................................................ 140 LIST OF PLATES Plates 1-37. Micrographs of developmental life history of Peziza 10. ll. 12. 13. 14. quelepjdotia Korf & O'Donnell. Magnifications are approximate. Page Scanning electron and light micrograph of chlamydospores ..... 21 Time-lapse sequence of conidium formation using differential interference phase-contrast illumination .................... 24 Time-lapse sequence of conidium formation using differential interference phase-contrast illumination (continued from PLATE 2) .................................................... 26 Scanning electron and light micrographs of conidiophore from time—lapse sequence (continued from PLATE 3) ................ 28 Scanning electron and light micrographs of identical conidiophores ............................................... 31 Scanning electron and light micrographs of conidiophores with conidium initials ........................................... 33 Scanning electron and light micrographs of conidiophores with maturing conidia ............................................ 35 Scanning electron micrographs of conidia germinating jg_situ. 38 Germination of deciduous conidia ............................ 4O Scanning electron and light micrographs of ascogonial coils developing near conidiophores ............................... 43 Scanning electron and light micrographs of ascogonia and conidiophores ............................................... 45 Scanning electron and light micrographs of ascogonia and conidiophores ............................................... 47 Scanning electron micrographs of ascogonia with investing hyphae ...................................................... 49 Scanning electron micrographs of ascogonia with investing hyphae ...................................................... 52 vi Plate 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. Light micrographs of thick-sectioned, plastic-embedded archihymenial and prohymenial phase apothecia .. ............. Light micrographs of thick-sectioned, plastic-embedded prohymenial apothecia ................ 5 ....................... Light micrographs of thick-sectioned, plastic-embedded prohymenial apothecia ....................................... Light micrograph of a thick-sectioned, plastic-embedded early mesohymenial phase apothecium ............................... Scanning electron micrographs of early prohymenial to early mesohymenial phase apothecia ......................... . ....... Light micrographs of thick-sectioned, plastic-embedded mesohymenail phase apothecia ................................ Light micrographs of thick-sectioned, plastic-embedded mesohymenial phase apothecia ................................ Scanning electron micrographs of lateral view of mesohymenial phase apothecia ............................................. Scanning electron micrographs of late mesohymenial to early telohymenial phase apothecia ................................ Scanning electron micrographs of late meso- to telohymenial apothecia ................................................... Scanning electron and light micrographs of asci ............. Scanning electron micrographs of telohymenial to posthymenial phase apothecia ............................................. Scanning electron micrographs showing cryofractured and razor blade sectioned apothecia ................................... Scanning electron micrographs showing longitudinally cryofractured late mesohymenial phase apothecia ............. Light micrographs showing nuclear condition of vegetative cells ....................................................... Transmission electron micrographs showing septa in paraphyses Scanning electron micrographs of whole and cryofractured ascospores .................................................. Transmission electron micrographs of ascospores and mitotic prophase nucleus ............................................ vii Page 54 56 58 61 63 65 68 7O 72 .75 77 80 82 85 90 93 95 Plate Page 33.’ Scanning electron and light micrographs showing ascospore germination ....... ........................................... 98 34. Scanning electron and light micrographs of germinating ascospores ................................................... 101 35. Scanning electron and light micrographs of excipuloid stroma.. 103 36. Scanning electron and light micrographs of maturing excipuloid stroma ....................................................... 106 37. Scanning electron micrographs of cryofractured excipuloid stroma ....................................................... 108 viii INTRODUCTION In May, 1971, Dr. D.T.A. Lamport of the A.E.C. Plant Research Laboratory at Michigan State University brought an operculate Discomycete growing on Jiffy-7-Pellets in for identification. The fungus was tenta- tively placed in the genus Pegjgg, Dr. Richard P. Korf, to whom a culture was sent, assigned this discomycete to the species Peziza quelepidotia Korf & O'Donnell (Korf, 1973). Due to the inability of previous investi- gators to obtain fertile ascomata of Eegjgg_in pure culture (Arx, 1970), there are no detailed studies dealing with the developmental life history of any species of Eegjgg, As apothecia, conidiophores and excipuloid stroma of P. quelepidotia readily develop in pure culture (O'Donnell and Beneke, 1973), this study was initiated with the aim of obtaining correla- tive light microscopic and scanning electron microscopic observations on the ontogeny of these structures. LITERATURE REVIEW A. Conidium Ontogeny In the past few years, imperfect states in the Pezizales have been the subject of excellent reviews by Eckblad (1968), Kimbrough (1970), and Paden (1972). About 60% of the reported imperfect states in Eegjgg_produce Oedocephalum (Tulasane, 1853, 1865; Vuillemin, 1886; Brefeld, 1891; Dodge, 1937; Wolf, 1958; Berthet, 1964a; Webster et a1, 1964; and Paden, 1972, 1973) with the remainder producing either Chromelosporium (Schneider, 1954; Rieth, 1957; Wolf, 1958; Fergus, 1969; Korf, 1961; Paden, 1972) or chlamydospores (Berthet, 1964a; Paden, 1972). Tubaki (1958) discussed the imperfect-perfect relationship within the Pezizales and noted that most belong to Hughes' (1953) conidiogenetic section 18. Similar observations have been made by Paden (1972), Arx (1970) and Hennebert (l973). Oedocephalum and Chromelosporium imperfect states are holoblastic, with determinate retrogressive conidiogenous cells, single conidia, and multiple synchronous conidiogenous lock (Kendrick, 1971). In recent years, conidium ontogeny has been the subject of numerous correlative light microscopic and electron microscopic studies (Cole & Kendrick, 1968; Kendrick & Cole, 1968; Kendrick et a1, 1968; Cole, 1969; Kendrick and Cole, 1969; Cole and Kendrick, 1969a,b; Cole and Aldrich, 1971; Kendrick, 1971; Dixon, 1971; Cook, 1972, 1974; Campbell, 1972; Cole, l973a,b, 1974; Hughes and Bisalputra, 1970; Harvey, 1974). However, only a few hyphomycetes in Hughes' conidiogenetic section 18 have been studied at the light and ultrastructural levels. Conidium ontogeny of the botryose blastospores of the ampullate hyphomycete Gonatobotrym apiculatum has been followed by time-lapse photomicrography with the light microscope (Cole, 1969; Kendrick et a1, 1968) and at the ultrastructural level by scanning and transmission electron microscopy (Cole, 1973b). Hughes and Bisalputra (1970) studied conidium ontogeny in the Chromeloa sporium state of Peziza ostracoderma with the optical and transmission electron microscope. They found that conidium ontogeny in this species does not involve rupture of the ampulla wall. Rather, the denticles and botryose solitary blastoconidia are produced by an outgrowth of the ampulla wall. As a result, "the ampulla wall remains continous and expands to form the integument of the new conidium" (Hughes and Bisalputra, 1970). Similar results were reported by Cook (1974) for Oedocephalum roseum. She found that "the denticle develops to its maximum length prior to conidium forma- tion, and nuclei enter the conidium late in development and before a sep- tum develops in the conidium stalk". Peziza quelepidotia produces an imperfect state with holoblastic, determinate retrogressive conidiogenous cells, single conidia, and multiple synchronous conidiogenous lock (Kendrick, 1971). This would place it in Hughes' (1953) conidiogenetic section 18 together with Oedocephalum and Chromelosporium. However, unlike these conidial states, the conidia in E, quelepidotia are not borne on well differentiated swollen conidiogenous cells (i.e. ampullae). Prof. Hennebert has indicated that the imperfect state of E, quelepidotia probably belongs to an undescribed form genus (Korf, 1973). The uniqueness and enigmatic nature of this conidial state prompted an investigation of conidiophore-conidium ontogeny in this fungus by correlative time-lapse photomicrography and scanning electron microscopy. Although numerous elaborate time-lapse culture chambers for viewing conidium ontogeny have been developed in recent years (Cole and Kendrick, 1968, Harvey, 1971; Madelin, 1969; Cook, 1974; Cole, 1969), all of these have one or more disadvantages. The chambers used by Cole and Kendrick (1968), Cole (1969), and Cook (1974) do not achieve the highest resolution attainable because the cells are surrounded by air. Madelin (1969) described a slide culture technique in which conidia were grown in a thin layer of liquid paraffin. However, none of the species that produce botryose solitary blastospores sporulated in her chamber. In addition, the chambers developed by Cole (1969) and Harvey (1971) are not commercially available. A simple time-lapse technique for following conidium ontogeny in Pezigg_ quelepidotia using differential interference phase-contrast illumination and an inexpensive commercially available slide culture chamber is described. Subsequent to time-lapse observations, techniques are described for viewing these identical conidiophores in all stages of development in the scanning electron microscope. B. Conidium Germination Although species of Peziza are known to produce chlamydospores, Oedocephalum and Chromelosporium imperfect states (Berthet, 1964a, Paden, 1972), conidium germination in Pegigg_has been the subject of only a few light microscopice and no ultrastructural studies. Fergus (1971) germinated the conidia of Peziza ostracoderma on a number of agar media and in citrate- phosphate buffers from pH 3 through 8. He found that the conidia swell to twice their original size within 2 hours. 92-98% of the conidia germinated by one germ-tube within 10 hours. Hawker (1966) hypothesized that "spores which normally produced germ-tubes only after considerable swelling are unable to germinate in the absence of glucose, whereas those which can germinate in water undergo little or no swelling before germ-tube emer- gence". The conidia of P, ostracoderma do not fit this hypothesis since they do swell and germinate in distilled water. Light microscopic observations on the Oedocephalum imperfect states of several pezizaceous fungi (Brefeld, 1891; Berthet, 1964a, Gamundi and Ranalli, 1964; Cook, 1973) indicate that the conidia have the capacity to germinate jngiicg. Bartnicki-Garcia (1968) distinguished two types of germination in mycelial fungi. In one type the germ-tube wall is derived from an extension of all or part of the spore wall. The second type is characterized by the ge_ggyg_formation of a vegetative wall under the spore wall. Although this spore germination scheme was originally thought to be of taxonomic significance, recent work (Hawker et a1, 1970; Gull and Trinci, 1971) does not support this hypothesis. A scanning ultrastructural study of conidium germination in Pegigg_ (quelepidotia was undertaken to determine what role these spores may play in the life cycle. C. Apothecium Ontogeny Fifty years ago ontogenetic studies of ascocarp development in the Pezizales were very much in vogue. The salient aspects of some of the more important developmental studies are treated in extensive reviews by Gaumann and Dodge (1928), Gwynne-Vaughan (1922), Gwynne-Vaughan and Barnes (1937), Gaumann (1952), Moreau (1953), Chadefaud (1960), and Kimbrough (1970). Although Corner's classical publications (1929a, 1929b, 1930a, 1930b, 1931) laid the foundation for comparative morphology of the operculate Discomycetes, interest in apothecium ontogeny has been quiescent until the last twenty years. In recent years, there have been a number of light microscopic studies of ascocarp ontogeny in the Ascobolaceae by Bistis (1956, 1957), Gamundi and Ranalli (1963, 1966, 1969), Conway (1973), Paden and Stanlake (1973), and Brummelen (1967, 1972); Thelebolaceae by Kimbrough (1966a, 1966b, 1974), Durand (1970, 1974), Wicklow and Malloch (1971), Jain and Morgan-Jones (1973), and Conway (1973); Pyronemataceae by Rosinski (1956), Moore (1963), Cain and Hastings (1956), Durand (1968), Larsen (1973), and Kish (1974); and Pezizaceae by Gamundi and Ranalli (1964), and Milam (1971). As no members of the Morchellaceae, Sarcoscyphaceae, Sarcosomataceae, and Helvellaceae produce fertile ascomata in pure culture (Arx, 1970; Kimbrough, 1970), there are no detailed studies dealing with apothecium ontogeny in these four operculate families. Although earlier investigators thought the results on sexuality might yield taxonomically significant patterns (Gwynne-Vaughan, 1922; Gwynne- Vaughan and Barnes, 1937), this information has proven to be of little taxonomic value (Gaumann, 1964; Eckblad, 1968; Kimbrough, 1970). These studies do show, however, considerable variation in the morphology and development of the apothecium in the Pezizales. At the Symposium on the Taxonomy of Operculate Discomycetes held at the First International Mycological Congress (Exeter, England 1969), Drs. Brummelen, Kimbrough, and Korf emphasized the value of developmental studies within the Pezizales. The ontogenetic studies on apothecia by Brummelen (1967) within the Ascobolaceae and Kimbrough within the Thelebolaceae (1966a, 1966b, 1969, 1970, 1972, and 1974) provide foraimore natural classification of these families. In order to obtain a complete structural and functional image of apothecium ontogeny, three methods should be employed: study of initiation, microtome sections of the different stages, and Corner's hyphal analyses. However, with these techniques, the intri- cate structures and natural relationships of the initial and subsequent stages are sometimes difficult to resolve with the light microscope. Corner (1929b) differentiated three developmental types within the Pezizales: angiocarpic, hemi-angiocarpic, and gymnocarpic. Brummelen (1967) rejected these terms as ambiguous in favor of a more precise set of terminology. With respect to hymenial maturation, he was able to dis- tinguish the following five chronological phases: 1) archihymenial phase (before paraphyses and croziers), 2) prohymenial phase (paraphyses present but not croziers), 3) mesohymenial phase (croziers through ascospore ripening), 4) telohymenial phase (mature asci through ascospore discharge), 5) posthymenial phase (senescent apothecia). Additionally, Brummelen (1967) distinguished ascomata with regard to hymenial development. These types are as follows: 1) cleistohymenial ascoma (hymenium enclosed, at least during early development, 2) gymnohy- menial ascoma (the hymenium is enclosed from the first until the maturation of asci). Gymnohymenial ascoma are subdivided into paragymnohymenial ascoma in which the ascogonium is overarched by investing hyphae of limit- ed growth, and eugymnohymenail ascoma in which the ascogonium is not over- arched by investing hyphae. Our knowledge of apothecium ontogeny in the genus Pezjg§_is limited to three scanty reports. Vuillemin (1886) studied pleomorphism (although he incorrectly referred to it as polymorphism, Hennebert, 1971) in Eegjgg_ Asterigma (Boudier, 1904-1911) but gave no cultural data other than it was a coprophile. He figured two stages in ascogonia coil formation as well as an Oedocephalum imperfect state. In a study of ascosporogenesis in Peziza vesiculosa, Fraser and Welsford (1908) only indicated that they were not able to recognize an ascogonium. Gwynne-Vaughan (1922) observed that apothecium ontogeny takes place in Peziza tectoria without the forma- tion of sexual organs. Peziza bolarioides, P, Subumbrina, and E, egg_are incorrectly reported to be somatogamous in review articles by Gwynne- Vaughan (1922), Gwynne-Vaughan and Barnes (1937), Gaumann and Dodge (1928), and Kimbrough (1970) since the original studies (Bagchee, 1925; Matsuura and Gondo, 1935; and Schultz, 1927) did not deal with apothecium ontogeny. This study was initiated with the aim of obtaining correlative light microscopic and scanning ultrastructural observations on apothecia in Peziza quelepidotia by: A) making detailed light microscopic observations on whole mounts of ascocarp initials using differential interference phase- contrast illumination, 8) conducting light microscopic observations on thick-sectioned, plastic-embedded apothecia in all stages of development. Critical microanatomical study of these sections is made with special emphasis of the ental (medullary) and ectal excipula (Eckbald, 1968; Korf, 1972a, Brummelen, 1967), ascogonial coil, ascogenous hyphae, investing hyphae, paraphyses, and the developmental type as proposed by Brummelen (1967). Paraffin embedded apothecia proved to be of little value as they could not be microtomed thin enough and all elements differentiate before the fruit bodies are over 1 mm tall, C) obtaining scanning electron micro- graphs of the surface features of the ascogonial coils up to spore dis- charge. The natural relationships of the initial stages are best resolved with this technique, D) perfecting a technique for viewing cryofractured (=freeze-fractured) apothecia in various stages of development, and E) developing techniques for studying the behaviour of nuclei and spindle- pole bodies from ontogeny of the croziers up to ascosporogenesis (see published account in Appendix, O'Donnell, Tai, and Beneke, 1974). Attempts to look at sectioned fungal cells and tissues in the scann- ing electron microscope have been met with limited success (Kinden and Brown, 1975; Littlefield, 1974; Jones et a1, 1974; Elliott and Corlett, 1972; Zeyen and Shearer, 1974; Silverberg and Morgan-Jones, 1974; Blanchard, 1972; Laane, 1974a, 1974b). More recently (Nei et a1, 1974) described a method for viewing freeze etched yeast cells in the scanning electron microscope. A recent report (Humphreys et a1, 1974) describes a simple procedure for critical point drying cryofractured tissue. I have modified this technique for viewing freeze-fractured fungal tissues in the scanning electron microscope. As the excipula tissues of most operculate Discomycetes are in need of critical study (Korf, 1972a), this technique could prove valuable in elucidating the microanatomy of the apothecium. D. Vegetative Nuclear Condition Studies of the nuclear numbers of vegetative cells have proved to be of some taxonomic importance in the Pezizales (Korf, 1972, Kimbrough, 197D; Eckblad, 1968; Berthet, 1964b). Aside from the uninucleate members of the Thelebolaceae (Kish, 1974; Conway, 1973; Kimbrough, 1974), the cells of the mycelium of all other operculate Discomycetes are generally multinucleate. Zickler (1971) studied mitosis in three ascomycetes and found typical mitosis. Mitosis is, however, extremely rapid (only 6-7 minutes) and the nuclei are capable of redivision in about 3 to 4 hours. She found plaque-shaped spindle-pole bodies at prophase and metaphase of mitosis in Ascobolus stercorarius. Nuclear numbers of the excipular cells as well as mitosis were studied in Peziza quelepidotia. 10 E. Septa of Paraphyses Although the ascomycetous septa are relatively simple, elaborate striate and laminar structures have been observed in the pores of para- physes (Bracker, 1967; Schrantz; Schrantz, 1970b; Pepin, 1971) and vegeta- tive hyphae (Hughes, 1971; Pepin, 1971; Schrantz, 1964; Brenner and Carroll, 1968; and Zickler, 1973). Hughes (1971) found elaborate striate "peristome teeth pores“ in the septa of a Chromelosporium. According to Bracker (1967), this apparatus, three-dimensionally is a series of concentric cylinders. Hughes (1971) also looked at the septa of Oedocephalum, another imperfect state of Pegigg, but did not find “peristome teeth pores.“ How- ever, he did find Woronin bodies near the septal pores. A preliminary transmission electron microscopic study of the septal pore in the paraphyses of Peziza quelepidotia was undertaken to determine what septal elaborations might be present. F. Ascosporogenesis and Ascospores Le Gal (1974) detailed optical observations on ascospore ornamentation have been of immense importance in the taxonomy of the operculate Disco- mycetes. A number of recent scanning electron microscopic investigations on ascospores demonstrate that this instrument is able to resolve more details than with the light microscope (Dissing, 1972; Elliott and Kaufert, 1974; Malloch, 1973; and McKnight and Batra, 1974). Ascospore ontogeny in the Pezizales has been the subject of a number of transmission electron microscopic studies (for a review see Wells, 1972). In all Ascomycetes investigated, the ascospore wall is deposited between a pair of ascospore-delimiting membranes. The inner membrane becomes the sporoplasmalemma while the outer becomes the investing 11 membrane. In an attempt to bridge the_gap between Le Gal's classic light microscopic observations on the patterns of ascospore ornamentation in the Pezizales, and the more recent transmission electron microscopic studies, Merkus (1973, 1974) studied the development of wall layers and ascospore ornamentation in members of the Pyronemataceae (gen§g_Eckblad, 1968) with the transmission electron microscope. Only one transmission electron micro- scope study has been made on ascospore wall ontogeny in a Pegjgg_($chrantz, 1970b). The results of a preliminary study dealing with spore wall onto- geny in Peziza quelepidotia, using two types of fixatives, are correlated with previous studies. Spindle-pole bodies are conspicuous features of actively dividing mitotic and meiotic fungal nuclei. In Ascomycetes, they are typically rod- shaped (O'Donnell, Tai and Beneke, 1974) whereas they are globular in the Basidiomycetes. The light microscopic features of spindle-pole bodies during mitosis and meiosis in Peziza quelepidotia have been reported else- where (O'Donnell et a1, 1974). Transmission electron microscopic observa- tions on spindle-pole bodies are presented and compared with other reports. G. Ascospore Germination Ascospores in the Pezizales are either constitutionally or exogenously dormant (terminology of Sussman, 1965). Reports of ascospore germination are scattered throughout the literature. The coprophilic operculate Disco- mycetes are constitutionally dormant (Brummelen, 1967; Kish, 1974; Conway, 1973). These spores require physical (usually heat) /or chemical factors to trigger germination. Most operculate Discomycetes appear to be exo- genously dormant (Berthet, 1964b; Paden, 1973, 1974, 1975; Milan, 1971). Most Eegjgg_spp. appear to be endogenously dormant (Berthet, 1964b; Paden, 12 1973). Unfortunately, ultrastructural aspects of germinating ascospores are limited to the Pyrenomycetes (Lowry and Sussman, 1968; Melendez-Howell and Cailleux, 1969; Melendez-Howell, 1970; Cailleux and Melendez-Howell, 1970; Seale, 1973). This study was undertaken to elucidate the morphological aspects of ascospore germination in Peziza quelepidotia. H. Excipuloid Stroma According to Ainsworth (1961) a sclerotium is a 'firm, frequently rounded, mass of hyphae with or without the addition of host tissue or soil, normally having no spores in or on it." Townsend and Willetts (1954) were able to distinguish three basic types: 1) the loose-type, 2) the terminal- type, and 3) the strand-type. Discomycetes are known that produce each of these types. Trichophaea bullata (Whitney and Parmeter, 1964) and Morchella hortensis (Moreau and Moreau, 1956) produce the loose-type (or Rhizoctonia- type). The terminal or Botrytis-type is known for Pyronema domesticum (Moore, 1962). Species of Sclerotinia produce the strand-type (Townsend and Willetts, 1954). The surface features of these three types of sclerbtia, as viewed in the scanning electron microscope, enabled Willetts (1969) to distinguish these basic types. In an earlier report (O'Donnell and Beneke, 1973), Peziza quelepidotia was reported to have produced sclerotia-like structures on enriched media. The ontogeny of these excipuloid structures was studied from the initials up to maturation in an attempt to elucidate their structure and function. This stromatic structure is compared with the sclerotial types of Townsend and Willetts (1954). 13 MATERIALS AND METHODS A. Source of Culture In May, 1971, Dr. D.T.A. Lamport of the A.E.C. Plant Research Laboratory at Michigan State University brought a fungus in for identifi- cation that he had found growing on Jiffy-7-Pe11ets in his home greenhouse. This operculate Discomycete, Peziza quelepidotia Korf & O'Donnell (Korf, 1973), readily produced fertile ascomata in pure culture (O'Donnell and Beneke, l973). Cultures of this fungus have been maintained throughout this investigation on Jiffy-7-Pellet infusion agar under continuous white light supplied by fluorescent tubes at 2511°C. Air-dried, mature apothecia stored aseptically at -20°C for 30 months have produced fertile ascomata within two weeks when placed on the infusion medium. 8. Media and Cultural Conditions Mature apothecia were allowed to "puff" onto sterile 4% water agar in 100x15 mm plastic petri dishes. Germinating ascospores were isolated by Dr. William G. Fields (Department of Botany and Plant Pathology) and my- self and these single spore isolates were placed on a fruiting medium called Jiffy-7-Pellet infusion agar (JPA). This medium is prepared by homogoniz- ing one Jiffy-7-Pellet per liter of double distilled water plus 209 Difco agar. The pH was adjusted to 6.5 with NaOH prior to autoclaving. The honothallic nature of this fungus is demostrated by the fact that mycelium derived from single, uninucleate ascospores is able to complete the life 14 cycle and form mature apothecia on JPA. On this medium mature fruit bodies are produced within two weeks. The Botrytis-like conidia and chlamydospores of Peziza quelepidotia were produced on one quarter strength JPA. These cultures Were started with hymenial pieces. The excipuloid stroma were produced on yeast-malt agar (YMA) composed of 39 yeast extract. 39 malt extract, 59 peptone, 109 glucose and 209 Difco agar per liter of distilled water. These cultures were inoculated with either ascospores or hymenial pieces. All cultures were grown under continuous white light supplied by fluorescent tubes at 251l°C. C. Apothecium, Conidium and Excipuloid Stroma Ontogeny: Scanning Electron Microscopy Agar squares bearing various stages of apothecium, conidium or stroma development were fixed in 0.2M phosphate-buffered 3 or 6.25% glutaraldehyde at pH 6.5 for 3-5h. The initial fixation was followed with a wash in the same phosphate buffer for 30 min. The fungal material was then postfixed with 1 or 2% 0504 in the same buffer for 2-4h. Postfixation was followed with a 30min wash in the same buffer. All previous steps were carried out at 25tl°C. The buffer-washed material was then dehydrated with ethanol at O-4°C using the following series: l0,20,30,40,50,60,70,80,90% ethanol, 5 min at each step; 100% ethanol twice, 10 min each time. The dehydrated material was then carried through a graded iso-amyl acetate-ethanol series at 2511°C as follows: 30,50,60,70,80,90% iso-amyl acetate-ethanol, 3 min at each step; 100% iso-amyl acetate twice, 5 min each time. Sometimes the iso-amyl acetate series was omitted and the ethanol dehydrated material was carried directly to the critical-point dryer. The specimens were dried in a Bomar-9OO critical-point dryer 15 (Bomar Co., Tacoma, Wa.) using C02 as the carrier gas. The critical-point- dried specimens were mounted on stubs with double-stick Scotch Brand Tape. The stub surrounding the specimen was then painted with television Tube Koat (GC Electronics) to prevent charging. The specimens were coated with 200-3OOA of gold using an EMS-41 mini-coater (Film-Vac Inc., Englewood, N.J.) and viewed in an AMR-9OO scanning electron microsc0pe. Photographs were obtained using Polaroid Type 55 positive-negative film. D. Conidium Ontogeny: Light and Scanning Electron Microscopy The ontogeny of the Botrytis-like conidia and chlamydospores was followed on one-quarter strength JPA inoculated with either ascospores or hymenial pieces. At the first signs of differentiation, small agar squares (about 5 mm in diameter) bearing the various stages were cut out of the petri dishes. The squares were trimmed parallel to the colony so that the resulting squares were about Sum square and 0.5-0.8 lllll thick. These agar squares were immediately placed colony-up in a polished, single depression (18 mm in diameter and 0.8 mm deep) 76x25 mm microscope slide (Sargent- Welch S-58815) and then the depression and agar square was flooded with 0.2M phosphate buffer pH 7.2 After air bubbles were gently teased away from the agar, this spherical depression time-lapse microchamber was covered with a No. 1 22x22 mm cover slip. All time-lapse slides were examined and photographed with differential interference phase-contrast illumination on a Zeiss Photomicroscope II fitted with an apochromatic 63x/1.4 objective and an achromatic-aplanatic phase condenser (numerical aperature = 1.4). Photographs were taken with a built-in 35 mm camera on Kodak Panatomic-X film exposed at ASA 25 and developed in Kodak Microdol-X (1:3). After time-lapse 16 observations had been conducted, the agar squares were fixed immediately in 6.25% glutaraldehyde and prepared for the scanning electron microscope as detailed above. E. Conidium and Chlamydospore Germination JPA cultures bearing Botrytis-like conidia /or chlamydospores were flooded with 3 m1 of sterile double distilled water, agitated with a sterile glass rod, and aliquots were pippeted aseptically onto one-quarter strength JPA dishes and then streaked with a bent glass rod. Conidia and chlamydo- spores in various stages of germination were fixed, dehydrated and critical point dried for viewing in the scanning electron microscope as detailed above. F. Apothecium and Excipuloid Stroma Ontogeny: Light Microscopy Apothecia and stroma in various stages of development were fixed ya §jtg_in 0.2M phosphate-buffered 6.25% glutaraldehyde at pH 6.5 for 3-5h. The initial fixation was followed with a phosphate buffer wash for 30 min. The specimens were then postfixed with 0504 (same buffer) for 2 h. Post- fixation was followed with a 30 min wash in the same buffer. All previous steps were carried out at 2511°C. The buffer-washed material was then de- hydrated with ethanol at O-4°C using the following series: 10,20,30,40,50, 60,70,80,90% ethanol, 5 min at each step; 100% ethanol twice, 10 min each time. The specimens were then infiltrated with E.R.L. epoxy resin (Spurr's standard medium, A firm; Polysciences, Inc.; Harrington, Penn.) on a Labline Unimixer (National Scientific; Cleveland, Ohio) using the following graded Spurr's-ethanol series: 33% Spurr's 24h, 66% Spurr's 24h, 100% Spurr's 24h. The apothecia and stroma were placed in flat-embedding moulds with 17 fresh 100% Spurr's and polymerized in a 60°C oven for 8-10h. Thick-sections (about 1.5-2.0 um) were obtained using glass knives on a Porter-Blum MT-2 ultra-microtome. These sections were transferred from the boat to a dr0p of distilled water on a clean microscope slide. Slides were routinely cleaned with Formula '40' Glass Cleaner (Sprayway Inc.; Chicago, 111.). These slides were placed on a hot plate (about 50-60°C) and xylene-soaked Q-tips (Chesebrough Pond's Inc.; Greenwich, Conn.) were waved over the sections to flatten and spread the sections out. Sections were stained for 15-25 min with 1% basic fuchsin in 50% ethanol, rinsed with 70% ethanol and then dried on hot plates at 50-60°C. Sections were covered with 22x22 mm, No. 1 cover slips using a mixture of Spurr's-Xylene (3:1) as the mountant. This mounting medium was stored in the freezer when not in use. The slides were individually covered with a lead weight (about 1409) and allowed to harden at room temperature. This procedure greatly reduced section wrinkling. All slides were examined and photographed with phase-contrast illumina— tion on a Zeiss Photomicroscope II fitted with apochromatic phase-contrast objectives and an achromatic—aplanatic phase condenser (numerical apera- ture = 1.4). Photographs were taken with either a built-in 35mm camera on Kodak Panatomic-x film exposed at ASA 25 and developed in Microdol-X (1:3) or with an attached Kardan Color 4"x5" Linhof Camera on either Kodak Ektapan or Kodak Plus-X Pan film. G. Transmission Electron Microscopy Maturing apothecia were harvested and cut longitudinally into thin pieces (about l-2mm in diameter) with a razor blade. The fertile slices were immediately fixed in either: 18 1) cold (O-4°C), unbuffered 2% KMno4 for 2h, or 2) cold (o-4°c) 2% glutaraldehyde in 0.1M phosphate buffer with 10‘3 M CaClz, pH 7.4 for 2.5h. The hymenial slices were washed three times in the same cold buffer, the first two times 30 min each and left over- night at O-4°C in the third change. The material was postfixed for 5h in 1% 0504 (same buffer) at pH 7.4 with 10"3 M CaClz. The first three hours were at 0-4°C, the last 2h at room temperature. Both fixations 1 and 2 were followed by a rapid alcohol dehydration and embedded in epoxy resin (same infiltration schedule as for light microscopy). Ultrathin sections were mounted on uncoated 300 mesh copper grids and the tissue fixed by method 2 was then double stained with uranyl acetate followed by lead citrate. All sections were viewed and photographed in a Philips 100 electron microscope operating at an accelerating voltage of 60-80KV. H. Vegetative Nuclear Condition The methods for studying nuclear behavior were identical to those used in a previous communication (see Appendix, O'Donnell et a1, 1974). I. Ascospore Germination Mature apothecia were allowed to "puff" their ascospores onto one- quarter strength JPA. Agar squares bearing ascospores with various stages of germination were viewed and photographed in the light microscope. Other squares were prepared for the scanning electron microscope as de- tailed above. 19 J. Cryofractury of Apothecia and Excipuloid Stroma Specimens were fixed in 0.2M phosphate-buffered 6.25% glutaraldehyde at pH 6.5 from 2h to overnight. The initial fixation was followed with a phosphate buffer wash for 15-30 min and then the material was postfixed with 1% 0504 (same buffer) for 2h. Postfixation was followed with a 30 min wash in the same buffer. All previous steps were carried out at 251100. The buffer-washed material was then dehydrated with ethanol at O-4°C using the following series: l0,20,30,40,50,60,70,80,90% ethanol, 5 min at each step; 100% ethanol twice, 10 min the first time, and 10 min to overnight at O-4°C the second change. Small dehydrated pieces (5mm or less) were immersed in liquid Freon-12 (Ucon Refrigerant, Union Carbide, N.Y., N.Y.) for 10-15 min and then transferred to liquid nitrogen where they were left at least 3 min prior to freeze-fracturing. The tissue was cryofractured with a precooled single-edge razor blade attached to a pair of insulated pliers. All tissue fractures were made under liquid nitrogen. The fractured tissue was placed in metal baskets under liquid nitrogen and transferred to a Denton critical point drying apparatus that had been precooled by pouring liquid nitrogen into the bomb. Critical point dry- ing and subsequent procedures were the same as in "C" above. 20 RESULTS A. Imperfect States Chlamydospores (aleurospore-like conidia sen§g_Paden, 1972) and a Botrytis-like imperfect state (sen§g_Hennebert, 1973) are produced by Peziza quelepidotia in pure culture on JPA at 24-25°C. The chlamydospores are 5.8-13.5x4-10.8um, smooth, hyaline, l-celled. obovate (Plate 1, Figures 1,2,5), spherical (Plate 1, Figures 3,6), ob- pyriform (Plate 1, Figure 3), to elliptical (Plate 1, Figure 4), with truncate bases. Chlamydospores are occasionally produced at the apices of hyphal tips (Plate 1, Figures 1,2) or intercalarly on broad denticles (Plate 1, Figure 3) on the germ tubes of germinating ascospores. Although obovate to spherical forms are most common, the shape and size are other- wise variable. Chlamydospores normally germinate within 36h on JPA at 24-25°C by either 1 or 2 germ-tubes. The Botrytis-like imperfect state of Peziza quelepidotia produces determinate retrogressive conidiogenous cells, single holoblastic conidia and multiple synchronous conidiogenous loci. Conidiophores are 25-170um in length, often procumbent and generally curved when associated with ascogonial coils or other conidiophores. Conidiophores are smooth, hyaline, septate, 3.0-8.0um broad and uniform in diameter above and below. The conidiogenous cells are not discernibly swollen. Conidia are sphaero- pedunculate, globose, subglobose, to ampulliform, smooth, hyaline, and (4.6-9.6)7.3x(4.6-9.3)6.7um. 21 PLATE 1 Figs. 1-6. Peziza_guelepidotia. Scanning electron and light micrographs Fig. Fig. Fig. Fig. Fig. Fig. of chlamydospores. 1. Scanning electron micrograph of germinated ascospore with apical chlamydospore. x 510. 2. Magnified portion for Fig. 1 showing two obovate chlamydospores. x 1870. 3. Scanning electron micrograph of ruptured ascospore with germ-tube bearing obpyriform and spherical chlamydospores. x 1720. 4. Scanning electron micrograph of an apical, elliptical Chlamydospore x 2520. 5. Light micrograph of obovate chlamydospores. x 740. 6. Light micrograph of spherical chlamydospores. x 740. 22 Plate 1 23 Time-lapse photomicrography of a single conidiophore is illustrated in Plates 2-5. The first discernible sign of conidiophorogenesis in the dichotomous branching of the side brances of the conidiophore-producing - hyphae. The conidiophore apex may branch dichotomously with each dichotomy broadly rounded. Concomitant with the attenuation of these rounded apices is the cessation of vegetative growth and the simultaneous appearance of denticles over the conidiogenous cells (Plate 2, Figure 1). The denticles double in length every 30 min (i.e. exhibit linear growth) until they reach their maximum length of about 4um at 90 min (Plate 2, Figures 1-4). The apices of these denticles swell synchronously revealing the spherical conidium initials (Plate 2, Figures 4-6; Plate 3, Figures 1-6). Vacuoli- zation of these conidiogenous cells is a corollary of conidium ontogeny. Small vacuoles coalesce forming larger vacuoles which may play an active role in pushing cytoplasm into the conidium initials. At maturity, septa are formed in the denticle (Plate 3, Figures 4-6; Plate 4, Figures 1-4). Although three septa appear to be formed in some denticles (Plate 3, Figure 4), this has not yet been confirmed in the transmission electron microscope. Large vacuoles are conspicuous features of the conidiogenous cells at maturity. The mature conidia contain small vacuoles and lipid globules (Plate 4, Figure 2). Mature conidiophores were fixed jn_§jtg, prepared for and examined in the scanning electron microscope (Plate 4, Figures 1,3,4). Arrows (Plate 4, Figure 4) indicate the septa in one of the denticles. Also note that conidia may arise on or just to one side of cross-walls. Although conidium ontogeny is a continuous process in Peziza. qgelepidotia, for convience it can be divided into the following stages: I) formation of conidiogenous cells and cessation of vegetative growth 24 PLATE 2 Figs. 1-6. Peziza quelepitodita. Time-lapse sequence of conidium forma- tion using differential interference phase-contrast illumination. Figs. 1-6 at O,35,75,90,125,l80 minutes, respectively. All x 570. 25 Plate 2 26 PLATE 3 Figs. 1-6. Peziza quelepidotia. Time-lapse sequence of conidium formation using differential interference phase-contrast illumination (continmn from PLATE 2). Arrow in Fig. 4 indicates septa in denticle. Figs. 1-6 at 210,225,240,315,325,26O minutes, respectively. All x 570. 27 Plate 3 28 PLATE 4 Figs. 1-4. Peziza quelepidotia. Scanning electron and light micrographs of conidiophore from time-lapse sequence continued from PLATE 3. Fig. 2 at 390 minutes. Figs. 1,3,4. Scanning electron micrographs at x 510, x 1150, and x 2500, respectively. Arrows in Fig. 4 indi- cate septa in denticle. Fig. 2. Light micrograph using differential interference phase-contrast illumination. x 570. 29 Plate 4 30 II) denticle ontogeny and maturation III) swelling of the conidium initials IV) septum formation and conidium maturation. Conidiophores in these stages of development were examined with differential interference phase-contrast illumination followed by obser- vation of these identical conidiophores in the scanning electron micro- scope (Plate 5, Figures 1-6; Plate 6, Figures 1-6). Light microscopic observations reveal that the conidiophore apices are devoid of conspicuous vacuoles in stage I (Plate 5, Figures 2,4). In stage II, numerous vacuoles are evenly distributed throughout the conidiogenous cells. However, vacuoles are not discernible in the denticles (Plate 5, Figure 6). The surface features of these stage I and II conidiophores is revealed in the scanning electron microscope (Plate 5, Figures 1,3,5). Conspicuous denticles are not always formed at the conidiophore apices (Plate 6, Figures 1-4). Rarely, stage II and III conidia may be found on the same conidiophore (Plate 6, Figure 3). Where this aberrant situation occurs, the stage III conidia are always at the conidiophore apices while the denticles are in a lateral position on the conidiogenous cells. Likewise, stage III and IV conidia may rarely be found on the same conidiophore (Plate 6, Figures 5,6; Plate 7, Figure 4). Aberrant subterminal protru- berances give these conidiophores the false appearance of sympodial proliferation. Observation in the scanning electron microscope revealed that these aberrant denticle primordia are broadly rounded as in stage I. The mature conidia are aberrant, multifarious and are not borne on conspicuous denticles (Plate 6, Figures 5,6). Rather, they are broadly attached either apically or laterally on the conidiogenous cells. Typically the conidia are borne on conspicuous denticles (Plate 7, 31 PLATE 5 Figs. 1-6. Peziza quelepidotia. Scanning electron and light micrographs of identical conidiophores. Figs. 1-2. Scanning electron and light micrographs of conidiophore initial x 1110 and x 680, respectively. Figs. 3-4. Scanning electron and light micrographs of conidiophore initial x 970 and x 640, respectively. Figs. 5-6. Scanning electron and light micrographs of conidiophore with denticles. x 550 and x 590, respectively. 32 Plate 5 33 PLATE 6 Figs. 1-6. Peziza quelepidotia. Scanning electron and light micrographs of conidiophores with conidium initials. Fig. 1. Scanning electron micrograph of conidiophore with conidium initial. x 1300. Figs. 2-3. Scanning electron micrographs of conidiophore in Fig. 1 showing magnified portion. x 2260 and x 2710, respectively. Fig. 4. Light micrograph of conidiophore shown in Figs. 1-3. x 1215. Figs. 5-6. Scanning electron and light micrographs of aberrant conidiophore. x 1280 and x 1185, respectively. 34 Plate 6 35 PLATE 7 Figs. 1-4. Peziza quelepidotia. Scanning electron and light micrographs of conidiophores with maturing conidia. x 1030. Fig. 1. Scanning electron micrograph of mature conidiophore. x 1030. Fig. 2. Light micrograph of mature conidiophore with empty conidiogenous cells with adjacent maturing, coiled conidiophore. x 1000. Fig. 3. Mature conidiophore with integrated vegetative cells. x 1000. Fig. 4. Scanning electron micrograph of aberrant conidiophore. x 1650. 36 Plate 7 37 Figure l). The conidia borne at the conidiophore apices are on denticles up to 5um in length. If conidiophores are formed successively in close proximity, the younger conidiophores have a tendency to curve around the straight, older conidiophores (Plate 7, Figure 2). The conidiophores are largely (or entirely) composed of conidiogenous cells. Occasionally, vegetative cells adjacent to the characteristic conidiogenetic side brandies are integrated into the conidiophore by the synchronous prolifera- tion of their conidiogenous cells (Plate 7, Figure 3). Rarely, a con- idiophore produces but a single conidium (Plate 10, Figure 3). These conidia may be within the normal size range (9.3x9.3um shown here) or up to 15.5x13.7um. Collapse of the highly vacuolated conidiogenous cells is discernible in some of the conidiophores (Plate 8, Figures 1,2). If mature conidia are left undistrubed, they may germinate jn_§itg_(Plate 8, Figures 1-4). The first visible sign of germination is an increase in size of the conidia. Germ-tubes arise distally and rupture the outer layer of the original parent conidium. A septum is generally produced at the base of the germ-tube. A second germ-tube frequently is formed at the base of the conidium next to the denticle (Plate 8, Figures 1,4). Approximately all of the deciduous conidia germinate by one or two germ-tubes within 36h on dilute JPA at 24-25°C. Aside from the conspicuous, truncate dentical scar, germination of these conidia is similar to those that germ- inate jn_situ (Plate 9, Figures 1-4). B. Apothecium Ontogeny The discriptive terms used in this study to distinguish the five phases of ascocarp ontogeny with respect to hymenial development were 38 PLATE 8 Figs. 1-4. Peziza quelepidotia. Scanning electron micrographs of conidia germinating in situ. Fig. l. Conidiophore with jn_situ germination. Note collapsed conidiogenous cells. x 730. Fig. 2. Sonidiophore with conidia germinating 1n_situ and collapsed coni iogenous cells. x 1460. Fig. 3. Conidium germinating jn_situ by single distal germ-tubes. x 3645. Fig. 4. Conidium germinating jn_situ with 2 germ-tubes. x 3655. 39 Plate 8 40 PLATE 9 Figs. 1-4. Peziza quelepidotia. Germination of deciduous conidia. Fig. Fig. Fig. Fig. 1. #00“) Conidium with distal germ-tube and truncate conidium stalk. x 25mm Conidium with distal germ-tube. x 2500. Clumped conidia with single germ-tubes. x 2000. Conidium with two germ-tubes. x 3600. 41 Plate 9 42 adopted from Brummelen (1967). To obtain a complete structural and functional image of the dynamic aspects of ascocarp ontogeny, whole mounts and microtome sections of the different stages were correlated with the scanning electron micrographs. I Archihymenial Phase Twenty-four to thirty-six hours after the cultures are inoculated, numerous ascogonial coils begin to appear on the parent hyphae as short lateral, recurved branches. A coil is easily distinguished since it is up to twice the diameter of the ordinary vegetative hyphae. In addition, ascogonial coils invariably develop in close proximity to the conidio- phores (Plate 10, Figures 1—6, Figures 1-6; Plate 11, Figures 1-4; Plate 12, Figures 1-6) that have generally reached maturity (Note the mature conidia and the highly vacuolated conidiogenous cells). Also note that the conidiogenous cells of some of the conidiophores have collapsed (Page 12, Figures 1,3,6; Plate 13, Figure 2). The mean conidiophore- ascogonium distance, as measured from their respective bases, is approx- imately 12um. An ascogonial coil initial (Plate 10, Figures 1-6), which measures about 3.5um in diameter at its base, doubles in diameter near its apex. As these coils differentiate, they generally become more uniform in diameter (Plate 11, Figures 1-4). On rare occasions, ascogonial coils developed on the basal cell of the conidiophore stalk (Plate 12, Figures 1-2). Conidiophores and ascogonial coils frequently intertwine (Plate 12, Figure 4). Scanning ultrastructural (Plate 12, Figures 1,5; Plate 13, Figures 1,3; Plate 14, Figures 1-6) and light microscopic observations (Plate 13, Figure 4) demonstrate that slender investing hyphae (about 4-5um in diameter) arise from one or more cells of the ascogonial stalk and from ordinary vegetative hyphae near the base of the ascogonium. 43 PLATE 10 Figs. 1-6. Peziza quelepidotia. Light micrographs of ascogonial coils near conidiophores. Fig. Fig. Fig. Fig. Fig. Fig. 1. 2. Ascogonium initials near mature conidiophore. x 590. Ascogonial coil near mature conidiophore with vacuolated conidiogenous cells. x 575. 3. 4. 5. Ascogonium near conidiophore with single conidium. x 590. Ascogonium coiling around conidiophore. x 625. Maturing coil and conidiophore. x 615. Mature conidiophore with basal ascogonium. x 585. 44 Plate 10 45 PLATE 11 Figs. 1-4. Peziza quelepidotia. Scanning electron and light micrographs of ascogonia and conidiophores. Fig. 1. Scanning electron micrograph of ascogonium near conidiophore. x 1200. Fig. 2. Scanning electron micrograph of ascogonial coil. x 2000. Fig. 3. Scanning electron micrograph of ascogonium and conidiophore. x 1500. Fig. 4. Light micrograph of ascogonium and conidiophore. x 780. 46 Plate 11 47 PLATE 12 Figs. 1-6. Peziza qgelepidotia. Scanning electron and light micrographs Fig. Fig. Fig. Fig. Fig. Fig. of ascogonia and conidiophores. 1. Scanning electron micrograph of ascogonium on conidiophore stalk. x 600. 2. Light micrograph of ascogonial initial on conidiophore stalk. x 580. 3. Scanning electron micrograph of ascogonium at base of conidiophore. x 600. - 4. Light micrograph of ascogonium coiling around conidiophore. x 691 5. Scanning electron micrograph of ascogonium and investing hyphae. x 1400. 6. Scanning electron micrograph of conidiophore and ascogonium with investing hyphae arising from stalk of coil. x 600. 48 Plate 12 49 PLATE 13 Figs. 1-4. Peziza quelepdetis. Scanning electron micrographs of ascogonha Fig. Fig. Fig. Fig. with investing hyphae. 1. Scanning electron micrograph showing investing hyphae arising from ascogonial stalk and conidiophore. x 1100. 2. Scanning electron micrograph showing investing hyphae arising from conidiophore stalk. x 1090. 3. Scanning electron micrograph of ascogonium with investing hyphae. x 1250. 4. Light micrograph of ascogonium with investing hyphae. x 1200. 50 Plate 13 51 Investing hyphae may also arise from the conidiophore stalk (Plate 13, Figures 1,2). Within the next 24h, these slender investing hyphae branch, anastomose and form a complete prosenchymatous sheath around the archicarp (Plate 14, Figures 3-6). With further growth and differentiation, this sheath becomes pseudoparenchymatous with angular to globose cells (Plate 15, Figures 1-2). A large central vacuole is a conspicuous feature of these primordial excipular cells. Archihymenial ascomata are generally about 0.1 mm in diameter (Plate 15, Figures 1-2). Prohymenial Phase In the early portion of this phase, which is characterized by the presence of paraphyses but no croziers, a pulvinate to ovate ascocarp about 0.2-0.3mm in diameter is formed (Plate 15, Figures 3-4; Plate 16, Figures 1-4; Plate 17, Figures 1-6; Plate 19, Figures 1-3). The margin of the ectal excipulum is initially disorganized (Plate 15, Fig. 3; Plate 19, Figure 1). However, upon further development, the ectal excipular hyphae becomes radially arranged, and overarches the paraphyses (Plate 15, Figure 4; Plate 19, Figures 2-3). As the incipent hymenium is never completely closed, hymenial development in Peziza quelepidotia is para- gymnohymenial. Concomitant with stipe initiation (Plate 19, Figure 3) is the elevation of the hymenial elements by the excipula (Plate 15, Figure 4). The ascogonial coil, which occupies a central position above the excipula, is characteristically highly vacuolated and thick-walled. The nuclei, in the ascogonium which have densely staining nucleoli, appear to be paired. Some of the ascogonial coil cross-walls break down (Plate 15, Figure 4; Plate 16, Figures 1-4) partially or completely. Septal dissolutionment is thought to faciliate nuclear migrations (note nucleus in septal pore Plate 17, Figures 2-3). 52 PLATE 14 Figs. 1-6. Peziza quelepidotia. Scanning electron micrographs of ascogonia with investing hyphae. Fig. Fig. Fig. Fig. Fig. Fig. 1. 2 3 4. 5 6 Ascogonium with investing hyphae. x 910. Ascogonium with investing hyphae. x 1420. Ascogonium with investing hyphae. x 1135. Ascogonium with investing hyphae. x 910. Ascogonium with prosenchymatous sheath of investing hyphae. x 910. Ascogonium with prosenchymatous sheath of investing hyphae. x 910. 53 2%,, o A... .. ... , ‘ . a. L .er N). cvaaelt' ’- 2.... L x ., .« 3 4r. , _. / 2v Plate 14 54 PLATE 15 Figs. 1-4. Peziza quelepidotia. Light micrographs of thick-sectioned, Fig. Fig. Fig. Fig. plastic-embeddedarchihymenial and prohymenial phase apothecia. l. Archihymenial phase apothecium showing prosenchymanous sheath and ascogonium. x 550. 2. Archihymenial phase apothecium showing prosenchymatous sheath and ascogonium. x 455. 3. Prohymenial phase apothecium showing globose-angular cells of excipulum, ascogonial coil, and initials of paraphyses. x 300. 4. Prohymenial phase apothecium showing centrally located ascogonium paraphyses and excipulum. Note that septa have broken down in coil. x 300. ‘ 55 56 PLATE 16 Figs. 1-4. Peziza gyelepidotia. Light micrographs of thick-sectioned, plastic-embedded prohymenial apothecia. Fig. 1. Arrow indicates origin of paraphyses bearing branch from angular excipular cells. Note centrally located coil. x 960. Fig. 2. Note phototrophic response of apothecium. x 300. Figs. 3-4. Adjacent sections of prohymenial apothecium showing paraphyses and ascogonia with degenerated septa. Both x 830. 57 . a)». w s A‘s“, . .fififi; . ‘% Oils a» v..." v.1 Teflon. o .. -, )8Jol.. .n.\a.~v—u. . _ ......._.......a.u..s . .. . I ~ . ' .le A I: .. v...Al. .K...‘l.4 0N.o§n\‘lfl... .\.. h a...“...\...sm.....~.o.\&n . .9 . .i .4. ”our“! 5.. , no.) way x Mfitfll~fiq. . n... 1W6: Plate 16 58 PLATE 17 Figs. 1-6. Peziza quelepidotia. Light micrographs of thick-sectioned, plastic-embeddedprohymenial apothecia. Fig. 1. Apothecium with ascogonium. Note presumptive investing hyphae arising from coil. x 300. Figs. 2-3. Note nuclear migration and vacuolization in ascogonium. Also note ascogenous hyphae proliferating from basal ascogonial cell. x 235 and x 650, respectively. Figs. 4-5. Different magnifications of an apothecium showing ascogonial stalk, ascogonium, ascogenous hyphae, and paraphyses. Arrow in Fig. 5 indicates ascogonial stalk. x 240 and x 435, respectively. Fig. 6. Section adjacent to Fig. 4. showing similar features. x 240. 59 u...\fi :m Véa... Plate 17 60 The paraphyses develop from plasma rich, angular excipular cells around the ascogonium. Broad plasma rich hyphae (about Sum in diameter) proliferate from these angular excipular cells and branch monopodially at regular intervals. These branchesgive rise to the paraphyses which are about 2um in diameter (Plate 16, Figure 1). Investing hyphae derived from the ascogonial stalk also proliferate around the coil and give rise to paraphyses (Plate 17, Figures 4-6). Numerous Woronin bodies are discernible near the septa of the exci- pular cells (Plate 16, Figure l), ascogonial coils (Plate 16, Figure 3), and ascogonial stalk cells (Plate 17, Figure 5). Ascogenous hyphae develop from one or more ascogonial coil cells (Plate 16, Figures 3—4; Plate 17, Figures 1-6). Ascogenous cells may be filamentous (Plate 17, Figure 1) or pseudoparenchymatous (Plate 17, Figure 2-3). Mesohymenial Phase The croziers, which are formed in the early portion of this phase, are exceeded by the paraphyses (Plate 18, Figures 1-7). Continued mono- podial growth of the paraphyses from plasma-rich cells beneath and around the ascogonium results in the expansion of the hymenium (Plate 19, Figure 4). In addition to the centrally located paraphyses, the radial, thick- walled ectal excipular marginal cells are conspicuous feature. Croziers develop from a dense mound of anastomosing, septate, ascogenous hyphae that proliferate from one or more ascogonial coil cells (Plate 18, Figures 1-7). The hymenium and ascogonial coil are further elevated from the sub- strate by stipe elongation during the early-mesohymenial phase (Plate 18 Figure 1; Plate 20, Figure 1). The surface features of a stipe in lateral 61 PLATE 18 Figs. 1-7. Peziza quelepidotia. Light micrographs of a thick-sectioned, plastic-embedded early mesohymenial phase apothecium. Fig. 1. Note proliferation of paraphyses and ascogenous hyphae. x 155. Figs. 2-7. Sections throuch same coil in Fig. 1. All x 615. sectfc'a e. 3"? 62 a .. -- \ . ." .Q " . fire °- 331‘? \._k’ '4 M ‘. 63 PLATE 19 Figs. 1-4. Peziza quelepidotia. Scanning electron micrographs of early prohymenial to early mesohymenial phase apothecia. Fig. 1. Surface features of ascocarp in early prohymenial phase. x 170. Fig. 2. Apothecium in prohymenial phase showing radial arrangement of ectal excipular margin. x 180. Fig. 3. Prohymenial phase apothecium showing stipe initiation. x 170. Fig. 4. Early mesohymenial phase apothecium showing paraphyses and radial, thick-walled ectal excipular cells. x 200. \!:!:‘19:V“"K‘I ' .‘ .g. Q Plate 19 65 PLATE 20 Figs. 1-6. Peziza quelepidotia. Light micrographs of thick-sectioned, plastic-embedded mesohymenial phase apothecia. Fig. 1. Early mesohymenial phase apothecium showing elevated ascogonium and stipe. x 155. Figs. 2-3. Midmesohymenial phase apothecium. Both x 150. Fig. 4. Igadmesohymenial phase apothecium. Arrow indicates ascogonium. Fig. 5. Magnified ascogonium from Fig. 4. x 75. Fig. 6. Giant ascogonial cell (about 53.5x28.5um) in subhymenium of midmesohymenial phase apothecium. x 560. 66 Plate 20 67 view reveals an undifferentiated stipe (Plate 22, Figure 1). The mid- mesohymenial phase is characterized by stipe elongation, differentiation of the ectal excipulum, and the initial stages of ascosporogenesis (Plate 20, Figures 1-6; Plate 21, Figures 1-3,5; Plate 22, Figure 2). Sympodial crozier development and centrifugal maturation of the asci is concomitant with the monopodial proliferation of the paraphyses. The ascogonial coil may be positioned midway up the stipe (Plate 20, Figures 3-4, about 0.2- 0.25mm above the substrate) or in the subhymenium (Plate 20, Figures 1,6, about 0.3-0.6 mm above the substrate). In the latter case, an abnormally large ascogonial coil cell was observed (about 50x28um) that contained numerous irregular vacuoles and dense cytoplasm with paired nuclei. As the stipe elongates, the ectal excipular margin is easily distin- guished by the annular aggregation of cemented hyphoid processes. The tips of the paraphyses interspersed with a few centrally located maturing asci are discernible in surface view (Plate 22, Figure 2). However, the centrifugal maturation of the asci is best seen in sections (Plate 21, Figures 1-4). At the point of maximum stipe elongation (i.e. late meso- hymenial phase) numerous squamules composed of subglobose to irregular cells are discernible on the upper part of the stipe (Plate 22, Figure 3). Sections of the stipe reveal that the ental excipulum is primarly textura intricate whereas the ectal excipulum is textura angularis. The surface features of the hymenium and ectal excipular margin are partially obscurbed due gelatinous material which may function in impeding dessica- tion in the incipient hymenium (Plate 22, Figure 3). At a subsequent stage, this gelatinous cover appears as membranous material at the margin of the hymenium. (Plate 23, Figure 1) In addition, operculate asci pro- trude as much as 50 um (i.e. about 25-35% of their length) above the 68 PLATE 21 Figs. 1-5. Peziza quelepidotia. Light micrographs of thick-sectioned, plastic-efibeddéd’mesohymenial phase apothecia. Fig. l. Midmesohymenial phase apothecium. x 120. Fig. 2. Midmesohymenial phase apothecium. x 110. Fig. 3. Midmesohymenial phase apothecium. Note protruding asci at center of hymenium. x 85. Fig. 4. Telohymenial phase apothecium showing portion of hymenium, subhymenium and ental excipulum. x 135. Fig. 5. Late mesohymenial phase apothecium showing protruding asci. x 100. 69 sci at WA , (43:; . k}; .19”. figs: 'h'v‘. ,‘_{_v¢ (J' 9' :j':l: ._ .1 I“ a v Plate 21 7O PLATE 22 Figs. 1-3. Peziza quelepidotia. Scanning electron micrographs of latenfl Fig. Fig. Fig. view ofmesohymenial phase apothecia. 1. Lateral view of early mesohymenial phase apothecium showing further stipe elongation. x 180. 2. Laterial view of midmesohymenial phase apothecium showing differentiation of the ectal excipulum. x 65. 3. Lateral view of midmesohymenial phase apothecium showing further differentiation of ectal excipulum. Note the squamules on upper half of the stipe. Also note that the excipular margin and the hymenial elements appear to be enveloped with a .mucilaginous material. x 40. s 0115:» s'nhi'; owing h'ng ‘f- on WC? and it inous Plate 22 72 PLATE 23 Figs. 1-4. Peziza quelepidotia. Scanning electron micrographs of late Fig. Fig- Fig. Fig. mesohymenial to early telohymenial phase apothecia. 1. Late-mesohymenial phase apothecium with asci protruding above level of paraphyses. Note that opercula are developing on most of the asci. The arrow indicates membranous material at the edge of the hymenium. x 160. 2. Early telohymenial phase apothecium showing expanding hymenimn and ectal excipular margin. x 66. 3. Magnified view of the edge of the apothecium in Fig. 2. showing asci and paraphyses to the interior (left) and hy hoid processes in the ectal excipular margin at the exterior (right . x 444. 4. Portion of the hymenium from Fig. 2. showing asci in various stages of operculum development and paraphyses with recurved tips. x 1400. 73 Plate 23 74 level of the paraphyses and a number of scales composed of cemented, dark-brown hyphoid processes are discernible on the excipular margin. Telohymenial Phase Hymenium expansion between the late-mesohymenial and early-telohymenial phase results in the formation of a shallow cupulate ascocarp (Plate 23, Figure 2; Plate 24, Figure l). A magnified view of the edge of a telo- hymenial ascocarp (Plate 23, Figure 3) reveals thick-walled, hyphoid pro- cesses in the ectal excipular margin and the tips of the cylindric asci (170-190um long) interspersed with septate paraphyses (1.5-2.0um in diameter) with recurved tips (Plate 23, Figure 4). The paraphyses may be simple or bifurcately branched below. As the opercula develop, the ascus tip becomes zonate and the center slightly concave. At maturity the operculum, which is about 3.5um in diameter (Plate 23, Figure 4; Plate 25, Figures 2-4), must be stretched considerably to allow for the discharge of spores 7um broad. Median longitudinally sectioned ascocarps viewed in the scanning electron microscope (Plate 24, Figure 1) show that the ental (medullary) excipulum is composed of inflated vescicular cells interspersed with connecting hyphae. The ectal excipulum is composed of globular to angular cells intermixed with ordinary hyphae. The surface of the ectal layer is subtomemtose. As the hymenium expands, as a result of the development of new hymenial elements, the ascocarps generally becomes convex with the margin reflexed (Plate 24, Figure 3; Plate 26, Figures 2,4). In the telohymenial phase a number of depressions develop through the hymenium as a result of the collapse of clusters of dehisced asci (Plate 24, Figure 2; Plate 25, Figure 1). If spore discharge is gradual (i.e. if the cultures are left undisturbed), irregular patches of hyphoid 75 PLATE 24 Figs. 1-4. Peziza quelepidotia. Scanning electron micrographs of late Fig. Fig. Fig. Fig- meso- to télohymenial phase apothecia. 1. Median longitudinal section of a late mesohymenial to early telohymenial phase apothecium showing the microanatomy of the ectal and ental excipula. x 20. 2. Telohymenial phase apothecium showing depressions in the hymenhm resulting from the collapse of clusters of dehisced asci. x 20. 3. Telohymenial apothecium with large irregular hyphoid patches throughout the hymenium. x 20. 4. Magnified hyphoid patch from Fig. 3. showing hyphoid elements similar to those found in the margin of the ectal excipulum. x 180. 76 “W Plate 24 77 PLATE 25 Figs. 1-4. Peziza quelepidotia. Scanning electron and light micrographs Fig. Fig. Fig. Fig. of asci. 1. Scanning electron micrograph of depression in hymenium of telo- hymenial phase apothecium in PLATE 24, Fig. 2. x 190. 2. Scanning electron micrograph of dehisced asci with opercula attached interspersed with recurved paraphyses. x 1300. 3. Light micrograph of nature ascus. Note crown of globules on either side of nucleus in each ascospore. x 1300. 4. Light micrograph of dehisced asci with opercula attached. x 1400. 78 Plate 25 79 processes frequently grow up into the hymenium (Plate 24, Figures 3-4; Plate 26, Figure 1). These patches may be broadly concave with a distinct margin (Plate 24, Figures 3-4), or isolated hyphoid elements (Plate 26, Figure l). Occasionally these patches are contiguous with the margin of the ectal excipulum (Plate 24, Figure 3). Aberrant, bicupulate ascocarps have been observed on rare occasions (Plate 26, Figure 2). A magnified portion of a median longitudinally sectioned ascocarp (Plate 26, Figure 2; Plate 21, Figure 4) reveals a differentiated hymenium (170-190um in thickness), subhymenium (50-90um in thickness) composed of filamentous, plasma-rich cells, and part of the ental excipulum with vescicular cells interspersed with ordinary hyphae. Posthymenial Phase During this phase, the old ascocarps no longer discharge spores and they have changed from yellow-brown to olive-brown. A senescent apothecium is characterized by depressions throughout the hymenium, resulting from the collapse of the ascal walls and paraphyses. C. Cryofractured Apothecia The cellular definition of ethanol-dehydrated longitudinally cryo- fractured critical point dried apothecia (Plate 27, Figures 1, 3-6; Plate 28, Figures 1-6) is far superior to free-hand, razor blade sections that are ethanol dehydrated and critical point dried (Plate 24, Figure 1; Plate 26, Figure 2; Plate 27, Figure 2,4). The excipular and hymenial elements of cryofractured apothecia are devoid of conspicuous compression artifacts and the various tissue types (Eckbald, 1968; Korf, l973b) are clearly discernible. The margin of the ectal excipulum is primarly composed of textura angularis interspersed with some textera intricate (Plate 27, 80 PLATE 26 Figs. 1-4. Peziza quelepidOtia. Scanning electron micrographs of telo- Fig. Fig. Fig. Fig. hymenial'to posthymenial phase apothecia. 1. Isolated group of hyphoid processes in hymenium from PLATE 24, Fig. 3. x 840. 2. Median longitudinal section of a bicupulate apothecium in the late telohymenial phase. Arrow indicates the juncture of the two hymenia. x 20. 3. Magnified view of Fig. 2 showing the hymenium, subhymenium, and a portion of the ental excipulum. x 95. 4. Posthymenial phase apothecium showing the arrangement of the collapsed paraphyses and discharged asci. x 20. 81 . ,0}? 4 (\u 1 . I. 11.0 ,v. ’7 \ v e #1 W ~.n r 1.... A. \I\.I., ; 1 ..v Plate 26 Figs. F19- Fig. Fig. F19. Fig. Fig. 82 PLATE 27, 1-6. Peziza quelepidotia. Scanning electron micrographs showing cryofractured and razor blade sectioned apothecia. 1. m-fi (0N 5. Cryofractured late mesohymenial phase apothecium. x 55. Razor blade sectioned midmesohymenail phase apothecium. x 20. Cryofractured late mesohymenial phase apothecium. x 40. Cryofractured late mesohymenial phase apothecium showing ectal xcipular margin and hymenium. x 285. Cryofractured late mesohymenial phase apothecium showing cells of ectal excipular margin. x 1070. 6. Razor blade sectioned late mesohymenial phase apothecium showing ectal excipular margin and hymenium. x 125. 83 Plate 27 84 Figure 4). Paraphyses may develop from some of the marginal cells at the interface of the hymenium and ectal excipulum. The textura angularis cells of the ectal excipulum are also characterized by a single large central vacuole filled with fibrous material (Plate 27, Figure 5). The textura angularis and textura intricate interconnect (Plate 27, Figure 5). Note that none of these features is discernible in a free-hand, razor blade sectioned apothecium (Plate 27, Figure 6). The ental excipulum is composed of textura globosa (about 20-40um in diameter) intermixed with connecting hyphae about 3.5-7um in diameter (Plate 28, Figures 1, 5,6). These highly vacuolated globose cells are occasionally l-septate and contain numerous spherical membrane-bound bodies about 0.5—1.5um in diameter. The ental excipulum may form cortical patches in the hymenium (Plate 28, Figure 1) compare with Plate 24, Figure 3). A view of the hymenium reveals the relationship of the croziers, young asci and paraphyses (Plate 28, Figures 2-4). The basal region of the corzier stalk cells and maturing asci are vacuolated. Fibrous material is located within these vacuoles. Some fractures (Plate 28, Figure 2) also reveal that the paraphyses branch below. Young asci (Plate 28, Figure 4) are characterized by numerous vesicles (about 0.5um‘ in diameter) located in the midregion of the ascus. These vesicles are aggregated on either side of the meiotic nucleus with its laterally situated nucleolus. Numerous subspherical vacuoles are in the basal region of the asci. Interstitial gelatinous material is discernible as fibrous material traversing the hymenial elements (Plate 23: Figures 3'4)° 85 PLATE 28 Figs. 1- 6. Peziza quelepidotia. Scanning electron micrographs showing longitudinally cryofractured late mesohymenial phase apothecia. Fig. 1. Cortical patch in hymenium. x 115. Fig. 2. Hymenium showing paraphyses, croziers and asci. x 1170. Fig. 3. Hymenium showing paraphyses, young ascus and asci. x 2750. Fig. 4. Hymenium showing paraphyses, crozier and asci. x 1100. Fig. 5. Ental excipulum of textura globosa and textura intricata. x 580. Fig. 6. Globose cell in ental excipulum with numerous spherical, membrane- bound bodies (about 0. 5- 1. Sum in diameter). x 1165. 86 Plate 28 87 D. Vegetative Nuclear Condition The textura globosa cells (Plate 29, Figure 1) and connecting hyphae (Plate 29, Figure 2) of the ental excipulum are multinucleate. Woronin bodies, which function as septal plugs, appear to be interconnected by a membranous structure (Plate 29, Figure 2, note arrow). Mitotic prophase (Plate 29, Figure 3) and metaphase (Plate 29, Figure 5) nuclei from squashed excipular cells reveal typical chromosome morphology. At mitotic metaphase, spindle-pole bodies (about 0.6-O.9um in diameter) are located at the poles (note arrows, Plate 29, Figure 5). The nucleolus appears as a small dot about 0.2um in diameter near one spindle-pole body. Chromo- some morphology of mitotic (Plate 29, Figure 5) and meiotic metaphases (Plate 29, Figure 4) are typical. The textura intricata cells are also multinucleate (Plate 29, Figure 6-7). The behaviour of nuclei and spindle- pole bodies during ascosporogenesis is presented in the Appendix. E. Septa of Paraphyses Elaborate laminar and striate structures are associated with the pore rim of paraphyses (Plate 30, Figures 1-4). When they are of the striate "peristome teeth" type sen§g_Hughes (Plate 30, Figures 1-2), they originate at the pore rim and extend out into the cytOplasm on just one side of the septum. Woronin bodies are typically near these septal pores. Obovate Woronin bodies about lum in length are discernible as septal plugs (Plate 30, Figure 2). Osmiophilic bilaterally symmetric, omegoid, laminar structures are associated with the basal septa of paraphyses (Plate 30, Figures 3-4). These structures, which are about 0.75-0.95um long by 0.1um, are always inserted proximal to the hyphal apex and adjacent to the pore rim. The central portion of this structure curves back through 88 PLATE 29 Figs. 1-7. Peziza quelepidotia. Light micrographs showing nuclear condi- Fig. Fig. Fig. Fig. Fig. Fig. Fig. tion of vegetative cells. 1. Multinucleate textura globosa. x 1440. 2. Multinucleate textUra intricata. x 1290. 3. Isolated mitotic nuclei from squashed excipular cell. x 1690. 4. Isolated meiotic metaphase nucleus showing 14-15 bivalents. x 2100. 5. Isolated mitotic metaphase nuclei. Arrows indicate spindle-poke bodies. x 1770. 6. Multinucleate connecting hyphae. x 1500. 7. Multinucleate connecting hyphae. x 1560. 89 9 I”. 3' g , L .3.’ , u '19 ~ .~:‘E"'~’ l‘ki. .o e "f‘.‘l‘fl’ U ' . ‘V.."" “ '. 3 ~, tu'\\~‘ O Ve- \.. . .P ‘. .2 1 ‘ . '4‘; r . .. H J .. (I 1. ‘ . a! e . ~ . 0 g Q. I “ a ., . v e 6" M“ '2 fi "‘6 ‘2— 11 ‘ ,' fi- 0 *3. ‘Q ”a u , 4" 0 ' s 59‘ ' P b. ., 9. ® . z o . . O .. 0“ .0. ‘ ’fi‘ . r9 ' ' ‘ré \ e . Plate 29 90 PLATE 30 Figs. 1-4. Peziza quelepidotia. Transmission electron micrographs smnnng septa in paraphyses. Fig. Fig. Fig. Fig. 1. 2 3. 4 Striate structure on pore rim. x 11,600. Striate structure on pore rim and Woronin body in pore. x 21,291 Omegoid laminar structure on pore rim. x 10,700. Omegoid laminar structures on pore rim. x 10,700. 91 Plate 30 92 the pore. The p1asma1emma and mu1tivescicu1ar bodies are found adjacent to these structures and proxima1 to the hypha1 apex. F. Ascosporogenesis and Ascospores Cryofractured asci revea1 the ca11ose-pectic reticu1ations of the mature ascospores (P1ate 31, Figures 1-2). Interstitia1 ge1atinous materia1, which appears as membranous materia1 (P1ate 31, Figure 2) s1ight1y obscures the surface features of the ascospores. Numerous spherica1 g1obu1es about 1um in diameter are discernib1e in freeze- fractured maturing ascospores (P1ate 31, Figure 3). A1though these . g1obu1es are more numerous on either side of the nuc1eus, they may a1so be found in the median and adjacent to the 1atera11y disposed nuc1eus. The comp1ementary face of cryofractured ascospores revea1s spherica1 cavities in the sporop1asm. These g1obu1es are dense1y stained in Me1zers-IKI (1:1, see Figure 39 in Appendix). When viewed in the trans- mission e1ectron microscope, these g1obu1es are osmiophi1ic (P1ate 32, Figure 1) and are removed by KMnO4 (P1ate 32, Figure 2). Ascospore wa11s are deposited in centrifuga1 succession between two 1imiting membranes. The inner membrane becomes the sporop1asma1emma and the outer one forms the investing membrane (or perispora1 sac membrane). 0ccasiona11y, the investing membranes of adjacent ascospores are initia11y conf1uent (P1ate 32, Figure 2). The first wa11 to be deposited is the inner thick (about 300-500um in g1utara1dehyde-osmium),e1ectron-transparent endospore (P1ate 32, Figures 1-3). The next 1ayer is the thinner (about 50-75um thick in g1utara1dehyde-osmium)epispore wa11. In g1utara1edehyde- 0504 fixed materia1, this wa11 is e1ectron-dense (P1ate 32, Figure 1). Whereas in KMn04, the epispore appears three-1ayered. The inner and 93 PLATE 3] Figs. 1-4. Peziza que1epidotia. Scanning e1ectron micrographs of whoha and cryOfractured ascospores. Fig. 1. ‘Mature ascospores in cryofractured ascus. Note continuous reticu1ations on exospore. x 3330. Fig. 2. Mature ascospore. x 4000. Fig. 3. Cryofractured ascospore showing g1obu1es. x 4000. Fig. 4. Cryofractured ascospore spherica1 cavities of comp1ementary face. x 4000. 94 Plate 31 95 PLATE 32 Figs. 1-4. Peziza que1epidotia. Transmission e1ectron micrographs of Fig. Fig. Fig. Fig. ascospores and meiotic prophase nuc1eus. 1. G1utara1dehyde-osmium fixed mature ascospore showing nuc1eo1us (NU), g1obu1es, mitochondria, endospore wa11 (ENW), epispore wa11 EPN), and exospore wa11 (EXN). x 20,000. 2. Permanganate fixed ascospore showing ascospore (ASP), endospore wa11 (ENW), epispore wa11 (EPW), exospore wa11 (EXN), spore matrix (SM), investin membrane (IM), tonop1ast (T), vacou1es (V), and ascus wa11 (AH . x 6000. 3. G1utara1dehyde-osmium fixed maturing ascospore. Note 1omasome on sporop1asma1emma. x 9000. 4. Spind1e-po1e body (SP3) on nuc1ear membrane of meiotic prophase nuc1eus. x 15,000. 96 Plate 32 97 outer 1ayers are e1ectron-dense, whi1e the midd1e 1ayer is e1ectron- transparent. The 1ast wa11 1ayer to be 1aid down in the ornamented exospore (about 200-550nm thick, secondary wa11 gggsu Merkus, 1974). In g1utara1dehyde-0504-fixed mature spores, this 1ayer is granu1ar, homo- genous in appearance, s1ight1y e1ectron dense, and thicker than the endo- spore. Ascospores are uninuc1eate with numerous. spherica1 g1obu1es (about 0.75um in diameter) on either side of the nuc1eus. E1ongate mito- chondria (up to 2.0um 1on9) and numerous ribosomes are conspicuous features of the sporop1asm. Condensed materia1 is deposited within the investing membrane on the endospore prior to epi- and exospore wa11 formation (P1ate 32, Figure 3). Lomasomes are common1y found on the sporop1asma- 1emma in maturing ascospores. At this stage of deve1opment, the investing membrane is irregu1ar in out1ine and c1ose1y associated with the spore in g1utara1dehyde-0504 fixed materia1. However, in KMnO4 fixed materia1, the investing membrane is anywhere from 0.4-2.5um from the deve1oping spore wa11 (P1ate 32, Figure 2). A spherica1 monog1obu1ar spind1e-po1e body (about 1um in diameter), is associated with the differentiated nuc1ear enve1ope at meiotic pro- phase (P1ate 32, Figure 4). This organe11e is composed of e1ectron-dense . granu1ar materia1 and 1ies in an indentation of the nuc1eus dista1 to the nuc1eo1us. G. Ascospore Germination When ascospores are discharged in mass onto di1ute JPA under constant f1uorescent i11umination at 24-25°c, up to 95% germination was discernib1e within 36h (P1ate 33, Figure 1). An increase in spore size (about 3 times increase in vo1ume) as a resu1t of water intake, is the 98 PLATE 33 F195. 1-5. Peziza gue1epjdotia. Scanning e1ectron and Tight microgrpahs showing ascospore germination. Fig. 1. Scannin e1ectron micrographs showing c1usters of germinating ascospores (about 95%). x 660. Fig. 2. Scanning e1ectron micrograph of genminating ascospore with sing1e po1ar germ-tube. Note ruptured ascospore wa11. x 2800. Fig. 3. Light micrograph showing 1atera1 germ-tube from ascospore. x 9M1 Fig. 4. Scanning e1ectron micrograph showing subpo1ar germ-tube. x 2500. Fig. 5. Scanning e1ectron micrograph showing sing1e po1ar germ-tube. Arrow indicates 1ongitudina1 fracture in ascospore wa11. x 2800. 99 Plate 33 100 first visib1e sign of germination. Concomitant with spore swe11ing is the gradua1 disappearance of the crown of g1obu1es on either side of the asco- spore nuc1eus. Prior to germ-tube emergence, numerous vacuo1es are dis- cernib1e throughout the cytop1asm. Germ-tube emergence genera11y occurs successive1y at the po1es (P1ate 33, Figure 2). Emergence of the germ- tubes may occur in a 1atera1 (P1ate 33, Figure 3) or subpoTar (P1ate 33, Figure 4) position. The ascospore wa11 is ruptured irregu1ar1y and some- times sp1its 1ongitudina11y (P1ate 33, Figure 5). 0ccasiona11y, emergence of the po1ar germ-tubes is simu1taneous (P1ate 34, Figure 1). Germ-tubes are soon de1imited from the ascospore (P1ate 34, Figures 3-4) but the hyphae is not conspicous1y constricted at its point of emergence from the spore. The ca11ose-pectic markings of some germinated spores are not discernib1e (P1ate 34, Figure 2). The mu1tinuc1eate germ-tubes genera11y become intricate1y branched after they grow to a 1ength of 20-30um (P1ate 34, Figure 4). Up to three germ-tubes have been observed on an ascospore (P1ate 34, Figure 3). H. Excipu1oid Stroma When grown on a rich medium such as YMA, Peziza qugJepidotia produces undifferentiated, ca11us-1ike, excipu1oid structures. These begin as hyphoid processes that are characterized by their brown co1or, thick annu1ar septa, ge1atinous materia1 which appears here as membranous materia1 on the myce1ium, and swo11en apices (P1ate 35, Figures 1-2). These processes anastomose and by cora11oid to irregu1ar dichotomous branching produce a prosenchymatous mass (P1ate 35, Figures 3-4). With further growth (P1ate 35, Figure 5), the g1obose and fi1amentous ce11s are compressed into a pseudoparenchymatous textura angu1aris (P1ate 35, 101 PLATE 34 Figs. 1-4. Peziza que1epidotia. Scanning e1ectron and 1ight micrographs of germinating ascospores. Fig. 1. Scanning e1ectron micrograph of ascospore with po1ar germ-tubes. x 2800. Fig. 2. Scanning e1ectron micrograph of ascospores with po1ar germ-tubes. Note that ornamentation on 1ower spores is not discernbi1e. x 1800. Fig. 3. Scanning e1ectron micrograph of ascospore with three germ-tubes. x 3500. Fig. 4. Light micrograph of branching germ-tube. x 875. 102 Plate 34 103 PLATE 35 Figs. 1-6. Peziza que1epidotia. Scanning e1ectron and 1ight micrographs Fig. Fig. Fig. Fig. Fig. Fig. of excipu1oid stroma. 1. Scanning e1ectron micrograph of excipu1oid stroma initiation. x 650. 2. Scanning e1ectron micrograph of excipu1oid stroma initiation. Note spherica1 swe11ing of apica1 ce11. x 1300. 3. Scanning e1ectron micrograph of hypha1 anastomosing of stroma initia1. x 780. 4. Light micrograph of excipu1oid stroma initia1. x 210. 5. Scanning e1ectron micrograph of deve1oping excipu1oid stroma. x 105. 6. Light micrograph showing pseudoparenchymatous excipu1oid stroma. x 180. ..... w? 104 Plate 35 105 Figure 6). As these pu1vinate growths are typica11y conf1uent, as much as one-ha1f of the petri dish may be covered by a continuous sheath of this excipu1oid materia1 (P1ate 36, Figure 1). At this point, most of the fi1amentous ce11s (about 6um in diamter) have been transformed to chains of giant g1obose ce11s (about 20-30um in diameter, P1ate 36, Figures 2-4). Cryofractured and thin-sectioned materia1 revea1 that most of these ce11$ are high1y vacuo1ated. Fibrous materia1 is discernib1e in the vacuoTes of ce11s that have been freeze-fractured. Irregu1ar pu1vinate excipu1ar growths (up to 11 mm wide x 10 mm ta11 in P1ate 36, Figure 5) deve1op within two weeks. These undergo the same co1or changes as the maturing ecta1 excipu1a of apothecia. A1though g1obose ce11s are continue1y formed within the matrix, they disappear as they are incor- porated into the textura angu1aris (P1ate 36, Figure 6). At maturity, these excipu1oid growths are hemispherica1 with the centra1, basa1 region ho11ow (P1ate 37, Figure 1). The tissue is uniform1y textura'angu1aris (P1ate 37, Figures 2-5) with thick ce11 wa11s and sma11 chains of beaded and fibrous materia1 discernib1e in some of the vacuo1es. Septa1 pores, which measure up to 2.5um, have a conspicuous rim of encrusted materia1 (note arrow, P1ate 37, Figure 5). Intrace11u1ar connections and mem- branous spherica1 bodies 1-3um in diameter are c1ear1y discernib1e. The inner surface of the hemispserica1 , excipu1ar growth is c1othed with fi1amentous hyphae (P1ate 37. Figure 6). 106 PLATE 36 Figs. 1-6. Peziza gue1epidotia. Scanning e1ectron and 1ight microgramn Fig. Fig. Fig. Fig. Fig. Fig. of maturing excipu10id'stroma. 1. Coa1escing pu1vinate excipu1oid stroma. x 40. 2. Pu1vinate excipu1oid stroma. x 40. 3. Scanning e1ectron micrograph of freeze-fractured excipu1oid stroma showing g1obose ce11s and connecting hyphae. x 495. 4. Light micrograph of pseudoparenchymatous, undifferentiated excipu1oid stroma. x 155. 5. Photograph of mature convo1uted excipu1oid stroma. x 1.4. 6. Light micrograph of mature excipu1oid stroma. x 120. I. 26'.“ a 5' ”’5 ‘ w :-' 0’; ' n. I Plate 36 108 PLATE 37 Figs. 1-6. Peziza que1epidotia. Scanning e1ectron micrographs of Fig. Fig. Fig. Fig. Fig. Fig- cryofractured excipuTOid stroma. 1. Cryofractured excipu1oid stroma. x 17. 2. Magnified portion of Fig. 1 showing angu1ar ce11s of stroma. x105 3. Magnified portion of Fig. 2. Showing textUra angu1aris. x 416. 4. Cryofractured excipu1oid stroma showing textura angu1aris. Note 1arge vacuo1es. x 1110. 5. Cryofractured excipu1oid stroma showing angu1ar ce11s. Arrow indicates septa1 pore with encrusted materia1. x 1110. 6. Inner surface of excipu1oid stroma in Fig. 1. x 500. 109 Plate 37 110 DISCUSSION A. Imperfect States The ch1amydospores (sgg§u_Kendrick, 1971) and botryose so1itary conidia of Peziza que1epidotia i11ustrate the p1eomorphic (sgg§g_Hennebert, 1971) nature of the asexua1 phase. A1though the ch1amydospores of Peziza_ que1epidotia are variab1e in size and shape, they are a1ways thick-wa11ed and either termina1 or interca1ary. They are broad1y attached to the vacuo1ated parent conidiogenous ce11s and are de1imited by a singTe cross- wa11. A1though on1y a few species of Peziza_are known to produce ch1amydo- spores in cu1ture (Brefer, 1891; Paden 1967, 1972; Berthet, 1964a) detai1ed cu1tura1 observations on other 222152.5PP- may revea1 their common occur- rence (Paden, 1972). The ch1amydospores of Peziza que1epidotia are mor- pho1ogica11y simi1ar to those produced by E, ostracoderma (Paden, 1972) and P, brunneoatra (Paden, 1967). There is on1y one pub1ished account of ch1amydospore germination in Peziza. About 20% of the ch1amydospores of E, brunneoarta germinate by one or two germ-tubes within 48h on potato- carrot agar at 22°C (Paden, 1967). Paden aISo noted that a germ tube was occasiona11y produced at the ch1amydospore base. Aside from basa1 emer- ,gence, ch1amydospore germination in P, gue1epidotia is simi1ar. In E, gue1epidotia, ch1amydospores are occasiona11y produced termina11y or intercaTary on the germ-tubes of ascospores. Simi1ar observations were made by Whitney and Parmeter (1964) for Trichophaea where ascospores germinating on poor media frequent1y produced ch1amydospores. 111 Corre1ative time-1apse photomicrography and scanning e1ectron micro- scopic observations on the botryose soTitary b1astospores of Peziza que1epidotia revea1 ho1ob1astic, determinate retrogressive conidiogenous ce11s, sing1e conidia, and mu1tip1e synchronous conidiogenous 1oci (termino1ogy of Kendrick, 1971). This wou1d p1ace it in Hughes' (1953) conidiogenetic section 1B together with the other known imperfect states of Pezjga_(i.e. 0edocepha1um and Chrome1osporium). If c1assified according to Pirozynski (1971), E, que1epidotia and Chrome1osporium wou1d be in group E (conidiogenous ce11s born on branched, differentiated supporting hyphae). 0edocepha1um wou1d be c1assified in Pirozynski's conidiogenetic section B (sing1e conidiogenous ce11 on a 1atera1 hypha). The conidio- phores of P, que1epidotia are a1most a1ways regu1ar1y dichotomous and composed of severa1 conidiogenous ce11s supported by a steri1e sta1k of one or more ce11s. In some of the dimunitive conidiophores of E, que1epidotia, a11 of the ce11s may be conidiogenetic. These conidio- phores wou1d be c1assified in Pirozynski' group C. 0ccasiona11y, the conidiophore is reduced to but a sing1e conidium per conidiophore. These wou1d be p1aced in Pirozynski's group C. That the imperfect state of Peziza que1epidotia can be c1assified in three of Pirozynski's groups based of conidiogenous ce11 types i11ustrates the variabi1ity of this imperfect state and the artificia1 nature of this conidiophore c1assifi- cation scheme. According to Barron (1968), 0edocepha1um, Chrome1osporium wou1d be p1aced in the Botryob1astosporae. This series is characterized by the simu1taneous production of conidia on sw011en conidiogenous ce11s re- ferred to an ampu11ae. As the conidiogenous ce11s of Peziza que1epidotia are not conspicuous1y swo11en, Barron's series definition wou1d have to 112 be expanded to accompany E, que1epidotia. According to Prof. Hennebert, the imperfect state of E, gueTepidotia probab1y be1ongs to an undescribed genus (Korf, 1973a). In recent years, there have been a number of detai1ed 1ight and TEM' ;_ studies on Hughes‘ 1953) conidiogenetic section 18. Co1e (1969, 1973b) and Kendrick et a1 (1969) studied the botryose b1astospores of Gonatobotryum apicu1atum and found the fo11owing sequence of events: conidiophore e10nga- tion, ampu11a formation, short pause before denticaT formation, simu1taneous primary b1astospore deve1opment, asynchronous deve1opment of an acropeta1 chain of secondary b1astospores from each primary b1astospore, and apica1 percurrent pro1iferation of the conidiogenous ce11 which then produces a new ferti1e apex. The u1trastructura1 detai1s of conidium ontogeny of the botryose so1itary b1astospores of 0edocepha1um (Cook, 1974) and Chrome1o- SQorium (Hughes and Bisa1putra, 1970) are simi1ar in a number of respects: both are determinate, ampu11ae swe11 to maximum size before dentic1es appear, dentic1es reach maximum 1ength before the conidia are b1own-out, the conidiogenous ce11 wa11 is not ruptured during dentic1e and spore ontogeny (i.e. the ampu11a wa11 remains continuous and expands to form the integu- ment of the new conidium), nuc1ei enter the conidium 1ate in deve1opment, and a septum fbrms in the dentic1e de1imiting the near1y mature conidia. In Chrome1osporium (Hughes and Bisa1putra, 1970), a doub1e-wa11ed septum . with a midd1e 1ame11a deve1ops by the centripita1 invagination of the inner wa11 of the conidiophore. The midd1e 1ame11a functions as an abcission 1ayer. This thin 1ayer appears to disso1ve a11owing the two ha1ves of the septum to separate. At conidium maturity in Chrome195porium, the ampu11a is virtua11y devoid of:cytop1asm. This is not the case in 0edocepha1um roseum where Cook (1974) found the ampu11a fi11ed with 113 cytop1asm, food vacuo1es and a few organe11es. A1though the conidiogenous ce11s in E. que1epidotia are genera11y devoid of cytop1asm, some may remain in the conidiogenous ce11s. Both Cook (1974) and Hughes and Bisa1putra (1970) found vescicu1ar e1ements in the ampu11ae, dentic1e and conidia. Cook (1972, 1974) suggests that the 1arger vesic1es contain wa11-bui1ding materia1s and the sma11er vesic1es contain wa11-softening enzymes. Conidium ontogeny in Peziza que1epidotia is simi1ar to 0edocepha1um and Chrome1osporium in that: it is determinate, dentic1es deve10p to their maximum 1ength before the ba110ning of their tips, the ampu11a wa11 does not appear to be ruptured during dentic1e and conidium deve1opment, and septa deve1op in the dentic1e sta1k de1imiting the near1y mature conidia. Transmission e1ectron microscopic observations wi11 be needed to determine the re1ationship of conidiogenous ce11-dentic1e-conidium. As two to three septa are formed in the dentic1es of Peziza que1epidotia, this de- 1imitation process appears to be more comp1icated than in 0edocepha1um and Chrome105porium where on1y "one" septum is formed. As in 0edocepha1um (Cook, 1972), the conidiophore apex (i.e. 3-4.5um) of E, que1epidotia is devoid of conspicuous vacuo1es when viewed with differentia1 interference phase-contrast i11umination. Cook (1972) found Spitzenkorpers in the conidiophore apex and hypha1 tips in 0edocepha1um. The cytop1asmic organ- ization in the conidiophore apex in P, qge1gpidotia wi11 need to be examined in the transmission e1ectron microscope and compared with 0edocepha1um. Likewise, a transmission e1ectron microscopic study of conidium ontogeny in Peziza is needed to fu11y characterized conidio- genesis and to unequivoca11y determine its 1ocation in hyphomycete c1ass— ification schemes. 114 Conidium ontogeny in Peziza qge1epjdoita, as measured from the first signs of dentic1e formation through conidium maturation, takes about 390 min in the cu1ture time-1apse chamber. Comparative times were recorded in petri dishes. Co1e (1969) noted that "the times quoted in the photo- micrographic sequences obtained in the cu1ture chambers may not be the precise rate under other conditions." He did suggest that the proportion of the tota1 time required for each stage wi11 remain about the same. Simi1ar resu1ts were obtained for Peziza que1epidotia. Times quoted for the conidium ontogeny in Hughes' conidiogenetic section 18 are as fo11ows: 0edocepha1um roseum (115 min, Cook, 1972), Chrome1osporium (120-240 min, Hughes and Bisa1putra, 1970), and'GOnatobotryum apicuTatum (85 min for primary b1astospore formation, Co1e, 1969). The time required from dentic1e appearance through maximum e1ongation is about 20% of the tota1 time required for conidium ontogeny in fig que1epidotia, 0edocepha1um roseum, and GonatObotryum apicu1atum. A1though time-1apse and scanning u1trastructura1 observations on conidium ontogeny are very much in vogue (Co1e and Ramirez-Mitche11, 1974; Co1e and A1drich, 1971; 001e, 1973a, 1973b, 1974; Harvey, 1974), prior to this investigation there have been no attempts to take materia1 viewed in time-1apse cu1ture chambers and view these identica1 conidiophores in the e1ectron microscope. Not on1y does the time-Tapse cu1ture chamber used in this study meet Co1e's (1969) requirements but it a1so has the fo110wing advantages: 1) inexpensive and commercia11y avai1ab1e, 2) reso1ution better due to higher refractive index of 1iquid medium, 3) no prob1em with condensation or dessication, 4) most of the conidiophores cou1d be photo- graphed, 4) thin enough for differentia1 phase-contrast i11umination (=higher reso1ution), 4) po1arized 1ight reduces heat, 6) gas exchange is 115 not a prob1em because an air bubb1e can be 1eft in the chamber to one side of the agar square, and 7) the agar square bearing the conidiophores can be removed in any stage of deve1opment and easi1y prepared without damage for the transmission or scanning e1ectron microscope. A1though the imper- fect state of Peziza gge1epjdotia has been the on1y conidiophore studied in this cu1ture chamber, this time-1apse microchamber technique may find wide app1ication in myco1ogica1 1aboratories. If conidiogenetic hyphae in petri dishes are a11owed to just begin to differentiate before being p1aced in the microcu1ture chamber, this reduces contamination and the time in the cu1ture chamber from severa1 days to hours. Likewise, if conidiogenetic hyphae (i.e. committed to deve1opment) are p1aced in the microchamber, these hyphae shou1d be more 1ike1y to continue to deve1op up to maturity than norma1 vegetative hyphae. Conidium germination in Pe2iza que1epidotia is simi1ar to the pre- vious1y pub1ished reports for 0edocepha1um (Brefe1d, 1891; Vui11emin, 1886; Dodge, 1937; Webster et a1, 1964; Berthet, 1964a) and Chrome1osporium (Wo1f, 1958; Fergus, 1971). As in chroneiosoorium, the conidia of Peziza have the capacity to germinate on di1ute media. The first sign of _ germination is an increase in conidium diameter as a consequence of water intake. This contradicts Hawker's (1966) hypothesis that spores that can germinate in water undergo 1itt1e or no swe11ing before germ-tube emergence. Genera11y, germ-tubes initia11y rupture the parent ce11 wa11 dista1 to the dentic1e. If more than one germ-tube is formed, these are successive and inserted 1atera11y near the dentic1e. The jn_§itu_germination of conidia, as reported here for Peziza que1epidotia, appears to be common in 0edocepha1um (Brefe1d, 1891; Berthet, 1964a; Gamundi and Rana11i, 1964; Cook, 1974). Gamundi and Rana11i (1964) observed that the conidia that 116 had germinated in situ cou1d give rise to more conidiophores. The most detai1ed observations to date on in situ conidium germination are those of Dixon (1971) on 0edocepha1um roseum. Her scanning e1ectron microscopic observations revea1ed that the ampu11ae were co11apsed. Cytop1asm may provide some structura1 support in the conidiogenous ce11s of 0edocepha1um and Peziza. The high1y vacuo1ated conidiogenous ce11s appear to be more susceptab1e to co11apse. In nature this “co11apse” as a resu1t of dessication may provide enough disturbance to dis1odge the conidia and aid in conidium dispersa1. As was noted for 0edocepha1um (Dixon, 1971), in_situ conidium germination in'Peziza was unsynchronized. Transmission e1ectron microscopic observations on conidium germination in 0edocepha1um roseum by Dixon (1971) revea1ed a break between the outer wa11 of the conidium and the germ-tube. As no new wa11 1ayers appeared to have been formed at germination, this wou1d p1ace 0edocepha1um in type I of Bartnicki-Garcia's c1assification of spore_germination. Recent work by Gu11 and Trinci (1971) demonstrates that Bartnicki-Garcia's c1assifi- cation (1969) is probab1y of no taxonomic significance. Transmission e1ectron microscopic study of conidium germination wi11 be necessary to determine the re1ationship of germ-tube to the conidium wa11s in Eegizg_ que1epidotia. The conidum germ-tube of Peziza gye1epidotia and other conidia (Dixon, 1971; Gu11 and Trinci, 1971, and Sea1e, 1973) are covered with muc1iaginous materia1. This materia1 probab1y protects the incipent _ germ-tubes from dessication. As a resu1t, the germinating conidia may adhere to one another. This adhesion has sometimes been misinterpreted as hypha1 fusions (Dixon, 1971; and Sea1e, 1973). A1though there were no discernib1e changes in the conidium surface architecture during 117 germination in E, que1epidotia, Sea1e (1973) reported changes in the macroeonidia‘ of Neurospora crassa. As his preparative methods were crude (i.e. air drying), it is difficuTt to draw any conc1usions from his work. 8. Apothecium Ontogeny The corre1ative 1ight microscopic and scanning u1trastructura1 ontogeny study on the stipitate-piTeate apothecia (he1ve11oid sensu Eckb1ad, 1968) of Peziza que1epidotia revea1ed that deve1opment is paragymnohymenia1 (termino1ogy of Brumme1en, 1967) and parthenogenetic. A1though species of Eggiga_are genera11y thought to be somatogamous (Haupt, 1953; Kimbrough, 1970; Gwynee-Vaughan, 1922), there have been no detai1ed studies to support this contention. This is primarTy due to the inabi1ity of previous investigators to obtain ferti1e ascomata of Peziza_in pure cu1ture (Arx, 1970). AS‘Peziza'qge1epidotia readi1y fruits in pure cu1ture (0'Donne11 and Beneke, 1973), it was possib1e to observe a11 phases of apothecium ontogeny. In this species, ascogonia1 coi1$ were not discernib1e in seria11y, thick-sectioned (i.e. 1.5-2.0um) apothecia over 1mm ta11. As the few investigations on Eezjga_used materia1 co11ected in nature (i.e. much 1arger than 1mm) and paraffin sectioning (inferior to p1astic), this may in part exp1ain the somatogamous reports for Peziza, In view of this fact, the common1y he1d be1ief that somatogamy is more common among the he1ve11oid fami1ies (Eckba1d, 1968; Kimbrough, 1970) shou1d be reconsidered. A reso1ution of this prob1em wi11 not be forthcoming unti1 detai1 studies on p1astic embedded apothecia are made on the ear1y stages co11ected in nature or ferti1e ascomata obtained in cu1ture. A1though variation in the morpho1ogy and deve1opment of apothecia 118 has been the subject of numerous 1ight microsc0pic studies (Kimbrough, 1970), there have been re1ative1y few morphogenetic studies of the perfect stages of ascomycetous fungi conducted with the scanning e1ectron micro- scope (E11iott and Cor1ett, 1972; Sea1e, 1973; Locci, 1972; B1anchard, 1972; 0'Donne11 and Hooper, 1974; 0'Donne11, Fie1ds, and H00per, 1974). The paucity of research on myce1ia1 stages with the scanning e1ectron microscope is due in part to the high water content of these stages and the fragi1e nature of the various funga1 structures (Hawker, 1971). How- ever, with the advent of the critica1 point drying technique, most fragi1e funga1 structures can be viewed in the scanning e1ectron microscope with minima1 shrinkage, distortion and co11apse of the hyphae and other struc— tures (Hayat and Zerkin, 1973). Of the studies 1isted above, on1y those by 0'Donne11 and associates emp1oyed the method of critica1 point drying. The natura1 re1ationships of the intact structures are preserved by this method. The much higher reso1ving power and greater depth of focus of the scanning e1ectron micrOscope makes it possib1e to reso1ve detai1s which cannot be seen in the 1ight microscope. In some cases, scanning e1ectron micrographs_give more re1iab1e and better reproducab1e resu1ts than drawings or 1ight micrographs. A case in point wou1d be the intricate structures of the initia1 stages of apothecium ontogeny. With the techniques detai1ed above, it is possib1e to reso1ve and accurate1y record the natura1 re1ationships of the ascogonia1 coi1s and investing hyphae. Since the surface features of a11 stages of apothecium ontogeny can now be viewed in the scanning e1ectron microscope, the corre1ation of these e1ectron micrographs with 1ight microscopic observations on who1e mounts and p1astic-embedded, thick-sectioned apothecia shou1d provide for a comp1ete structura1 and functiona1 image of apothecium morphogenesis. In addition, 119 Tongitudina11y cryofractured apothecia viewed in the scanning e1ectron microscope further c1arify the "three-dimensiona1" re1ationship of the excipu1a, ascogonia1 coi1, ascogenous hyphae, subhymenium, and hymenium. The sa1ient aspects of apothecium deve1opment in Peziza que1epidotia are simi1ar in P, gue1epidotia to those described in most Peziza1es (Brumme1en, 1967). However, an ascogonium arises as a 1atera1 thickened branch near mature or maturing Botrytis-1ike conidiophores (§§n§u_Hennebert, 1973). That the ascogonia1 primordia are invariab1y produced in c1ose proximity to these conidiophores suggests that the coi1s may be produced in response to hormone(s) produced by the conidia or conidiophores. How— ever, fusions between the ascogonia1 coi1 and conidia or any other structure have never been observed. Even though the conidiophores become ensheathed and integrated into the apothecia, it shou1d be noted that E, que1epidotia is homotha11ic and compatabi1ity types are therefore absent. As was noted by Bistis (1957) on Ascob01us stercorarius, the asco— , gonium of Peziza gueTepidotia is determinate in growth. Previous studies have demonstrated the investing hyphae arise from any vegetative hyphae in the vicinity of the ascogonium (Bistis, 1956; Paden and Stan1ake, 1973; Brumme1en, 1967; Gwynne-Vaughan and Wi11iamson, 1932). In Peziza, in- vesting hyphae arise from the ascogonia1 sta1k, from norma1 vegetative hyphae in the vicinity of the coi1 and the sta1k ce11s of the conidiohore. - According to Bistis (1956), the directiona1 growth of the investing hyphae is probab1y a chemotrophic response to hormone(s) produced by the asco- . gonium. A1though some genera typica11y deve1op from severa1 ascogonia (Moore, 1963; Kish, 1974; Jain and Morgan-Jones, 1973; Dangeard, 1907), the fruit-bodies of P, que1epidotia norma11y deve1op from a sing1e asco- gonium. A1though the prosenchymatous investing hyphae soon become 120 pseudoparenchymatous, the ecta1 and enta1 excipu1a are not differentiated unti1 the mesohymenia1 phase. This differs from species of Saccobo1us and Ascobo1us (Brumme1en, 1967) where differentiation between the f1esh and excipu1um occurs at a very ear1y phase of deve1opment. A unique feature of paragymnohymenia1 deve1opment in E, que1epidotia is the e1eva- tion of the ascogonia1 coi1 as the stipe deve1ops. The coi1 is positioned between the enta1 excipu1um and subhymenium up to the ear1y mesohymenia1 phase. At subsequent phases, the coi1 is positioned between the midregion of the stipe and the subhymenium. As is characteristic formany genera (Kimbrough, 1970), the septa in the coi15 break down a11owing free nuc1ear migrations between adjacent ce11s. On one occasion, a giant ascogonia1 ce11 was observed in the subhymenium of a midmesohymenia1 phase apothecium. This ce11, which probab1y resu1ted from the expansion of coi1 ce115 that had Tost their septa, was e1evated about 0.6mm off the substrate, A1ter- native1y, this ce11 may have resu1ted from the expansion of an ascogenous hypha at the 1eve1 of the subhymenium (i.e. a secondary coi1). However, primary and secondary coi1s have never been observed in a sing1e apothecium. Deve1opment of the paraphyses, which grow monopodia11y and dista11y,‘ conform to the genera1 Peziza1es pattern (Brumme1en, 1967). These appear before the ascogenous hyphae and are derived from the excipu1ar ce11s and from hyphae of the ascogonia1 sta1k. Simi1ar observations were made on Mycoarctium ci1iatum (Jain and Morgan-Jones, 1973) where basa1 ascogonia1 ce11s produce binuc1eate paraphyses. (A1though the paraphyses of Peziza gue1epidotia are frequent1y binuc1eate, they may a1so be tri- or quadri- nuc1eate. As in Neottie11a (Rossen and Westergaard, 1966), the nuc1ei in the paraphyses are e1ongated. Prior to the sympodia1 pro1iferation of ascogenous hyphae from one or more "prive1iged ce11s", there is a 121 concomitant en1argement of ascogonia and disso1utionment of some of the septa a11owing nuc1ear migrations between adjacent ce11s. This behaviour is characteristic for other parthenogenetic Peziza1es (Schweizer, 1923; Cutting, 1909; Ram1ow, 1915; Fraser, 1913). As with most members of the Peziza1es, the ascogenous hyphae are dikaryotic. In Mycoarctium two types of ascogenous hyphae were reported (Jain and Morgan-Jones, 1973) as fo11ows: primary consisting of typica11y binuc1eate, dikaryotic narrow ce11s giving rise to secondary hyphae consisting of 1arge mu1tinuc1eate, dikaryotic ce11s. The ascogenous hyphae of Egzjzg_differs, however, in that binuc1eate, dikaryotic narrow ce11s (primary ascogenous hyphae sensu Jain and Morgan- Jones, 1973) pro1iferate from the coi1 and give rise to 1arge, mu1tinuc1eate, dikaryotic ce11s from which croziers arise (see Figure 6 in Appendix). Crozier deve1opment is of the p1eurorhynque type (sgn§g_Chadefaud, 1943). As is characteristic for other opercu1ate Discomycetes (Corner, 1929b; Brumme1en, 1967), ascus maturation in‘Eegig§_proceeds centrifuga11y with the o1dest asci forming at the center of the apothecium. At the 1ate- mesohymenia1 phase, opercu1ate asci protrude 25-35% of their 1ength above the 1eve1 of the paraphyses. As discharged, protruding asci have never been observed, it is assumed that these asci are immature and have moment- ari1y outgrown the paraphyses and cortex. As is common in He1ve11a and other genera with stipitate fruit-bodies (Corner, 1929b), differentiation of the stipe in P, gue1epidotia takes p1ace before any termina1 expansion. The stipe of Peziza, which exhibits a strong positive phototrophic response, is c1othed on the upper ha1f with conco1orous (i.e. ye11ow-brown) squamu1es composed of inf1ated subg1obose to irregu1ar ce11s and dark-brown sca1es composed of cemented hair-1ike e1ements. The hyphoid processes arise from ecta1 excipu1ar ce11s (Korf, 1973a). A1though Korf (1973a) pointed 122 out the gross simi1arity between P_. que1epidotia and A1euria asterigma (Egziza sensu Korf, 1973a; Boudier, 1885), and noted differences in the squamu1es and sca1es between these species, he did not make reference to Vui11emin's (1886) report on A1euria asterigma. Vui11emin noted that this coprophi1e produced ascogonia as "un sco1ecite enrou1e en tire- bouchon" and an 0edocepha1um imperfect state. A1though P. (maepidotia produces simi Tar ascogonia, it has never been reported growing on dung and does not produce an 0edocepha1um imperfect state. C. Cryofractured Apothecia The microanatomy of the enta1 and ecta1 excipu1a of Peziza quelepidotia, as revea1ed by median 1ongitudina11y sectioned ascocarps, is characteristic for the genus (Eckb1ad, 1968; Korf, 1973a, 1973b; Rifai, 1968). The re- so'l ution of tissue types in cryofractured apothecia viewed in the scanning eTectron microscope is superior to other methods present1y emp1oyed. A1- though the microanatomy of the apothecium is of considerab1e taxonomic Va'lue on generic to 'fami1iar 1eve1$ (Eckb1ad, 1968), the microanatomy in "1051;. genera of the Peziza1es is sti11 in need of intensive study (Korf, 1972a). The cryofracture technique shou1d provide an exce11ent means for reso1ving the tissue types in apothecia and the natura1 re1ationship of the ascogonia1 coi1, ascogneous hyphae, asci and paraphyses. This 1ast Point is of some importance since Jain and Morgan-Jones (1973) recent1y reported asci and paraphyses have a comnon origin in Mycoarctium ci1iatum. D- Vegetative Nuc1ear Condition The mu1tinuc1eate condition of the excipu1ar ce11s and paraphyses 1" fieziza que1epidotia is characteristic for most opercu1ate Discomycetes 123 (Eckb1ad, 1968). Very sma11 p1aque-shaped spind1e-po1e bodies (approximate1y 0.6-0.9um) were found at the po1es during mitosis in vegetative ce11s. Simi1ar findings were reported for Ascobo1us stercorarius (Zick1er 1970, 1971) where typica1 intranuc1ear somatic mitosis was observed. Zick1er a1so observed that mitosis was extreme1y rapid (approximate1y 6-7 min). In Peziza qggJepidotia, a gTobu1ar, amorphous e1ectron-dense spind1e- po1e body (ca1ottes extranuc1eaires sen§u_2ick1er, 1973) was observed in an indentation of the meiotic prophase nuc1eus. Simi1ar observations were obtained for Podospora and Sordaria (Zick1er, 1973) where two or more "cathtes extra-nuc1eaires" (=spind1e-po1e bodies) were situated on the nuc1ear membrane. These spind1e-po1e bodies continue to persist after the formation of the p1aque-shaped spind1e-po1e body (Zick1er, 1973). Schrantz (1970a) reported simi1ar "ca1ottes po1aires" (=spind1e-po1e bodies) on the ascogenous hyphae in the fOrm of severa1 sma11 fragments. The spind1e-po1e body of Xy1aria po1ymorpha at prophase I appears to differ in that it is not entire1y contiguous with the nuc1ear membrane and it is not found in an indentation of the nuc1ear membrane (Schrantz, 1970a; Beckett and Crawford, 1970). The g1obu1ar spind1e-po1e body of P, que1epidotia is simi1ar to those reported for Coprinus (Thie1ke, 1974; Raju and Lu, 1973). The behaviour and possib1e function of these spind1e-po1e bodies during ascosporogenesis in P, que1epidotia has previous1y been discussed (Appendix, 0"Donne11 et a1, 1974). E. Septa of Paraphyses A1though the ascomycetous septa are genera11y referred to as being simp1e septate, there are numerous reports of 1aminar (diaphragme pora1 sensu Schrantz, 1970) and striate structures associated with the pore 124 rim of ascogenous hyphae (Zick1er, 1973; Futardo, 1971; Schrantz, 1970b; Pepin, 1971), ordinary vegetative hyphae (Brenner and Carro11, 1968), conidiophores (Dixon, 1971; Hughes, 1971), asci (CarroT1, 1967; Schrantz, 1970b; Pepin, 1971) and paraphyses (Schrantz 1964, 1970b; Bracker, 1967; Pepin, 1971). The striate structures, which are three-dimensiona11y a series of concentric cy1inders (Bracker, 1967; Brenner and Carro11, 1968; Cook, 1971), are a consistent feature of the paraphyses in Pegiza_ gueTepidotia. A1though Schrantz (1970b)and Pepin (1971) reported cup- shaped, osmiophi1ic structures associated with the basa1 septa1 pores of paraphyses in Peziza spp., the occurrence of "hand1e-bar shaped" structures in association with septa1 pores as reported here for P, gge1epidotia, has never been reported. Additiona1 e1ectron microscopic observations are needed on paraphyses to determine whether these striate and 1aminar structures are structura11y distinct or on1y different configurations of simi1ar structures. A1though the e1aborate pore structures have been suggested as re1ated to septum formation (Cook, 1971) and subce11u1ar sieves during ascosporogenesis (Carro11, 1967), the function and composition of these septa1 structures remains to be determined. F. Ascosporogenesis and Ascospores Ascosporogenesis in Peziza gue1epidotia conforms to the fami1iar ascomycetous pattern of free ce11 formation (Bracker, 1967; Merkus, 1973, 1974). As was noted for severa1 species in the Pyronemataceae (Merkus, 1974), primary and secondary ascopore wa11s are formed successive1y. The endospore (primary wa11) is a thick e1ectron-transparent 1ayer contiguous with the p1asma1emma. Additiona1 transmission e1ectron microscopic observations on E, que1epidotia wi11 have to be conducted to determine 125 whether the 1ayered epispore wa11 is derived from the endospore (i.e. primary wa11 materia1) as suggested by Merkus (1974), or from secondary wa11 materia1. The exospore, or ornamented secondary wa11, is somewhat . granu1ar in appearance in Peziza, This ornamented 1ayer appears to resu1t from the condensation of e1ectron-dense materia1 on the epispore wa11 (Merkus, 1974). Simi1ar resu1ts were obtained by Schrantz (1970b) in E, Biggie, The exospore of this species, however, is e1ectron opaque. A crown of guttu1es is discernib1e on either side of the ascospore nuc1eus in mature ascospores of Peziza que1epidotia. This differs from an ear1ier report (Korf, 1973a) c1aiming that mature ascospores in this species are devoid of guttu1es. These po1ar guttu1es, which presumab1y function as endogenous reserves, are osmiophi1ic and simi1ar in appear- ance to Woronin bodies. In cryofractured ascospores, these gutta1es behave as 1ipodia1 materia1.* The primary and secondary wa115'(§en§u_ Merkus, 1974) can a1so be reso1ved in freeze-fractured ascospores. A1- though the cyanophi1ic exospore of‘Peziza que1epidotia has been reported as 1ight1y marked (Korf, 1973a), scanning e1ectron microscopic observations revea1 that the ca11ose-pectic ornamentations of the exospore form a comp1ete reticu1um. As ascospore ornamentation has been of immense importance in the taxonomy of Peziza_and the Peziza1es (Le Ga1, 1941, 1947; Eckba1d, 1968), the scanning e1ectron microscope shou1d prove to be a va1uab1e took in future work at the species 1eve1 in the group (Dissing, 1972; McKnight and Batra, 1974; E11iott and Kaufert, 1974). G. Ascospore Germination A1though ascospore germination in the Peziza1es has been the Subject of numerous 1ight microscopic investigations in recent years (Berthet, 126 1964b; Paden, 1974, 1975), there are no pub1ished reports of the u1tra- structura1 aspects of germ-tube emergence in the opercu1ate Discomycetes. The on1y Ascomycetes that have been investigated in detai1 are those that germinate through preformed germ-pores (Lowry and Sussman, 1968; Me1éndez- Howe11 and Cai11eux, 1969; Me1éndez—Howe11, 1970, 1972; Cai11eux and Me1éndez-Howe11, 1970). In Peziza que1epidotia, there are no germ-pores and germ-tube emergence is achieved by rupturing the ascospore wa11. In addition to mechanica1 rupturing, hydro1ytic/or proteoTytic enzymes are thought to p1ay a ro1e (Smith and Berry, 1974). A1though some members of the Sarcoscyphaceae (Pfister, 1973; Paden, 1973, 1975) and Pezizaceae (Berthet, 1964a) produce ho1ob1astic conidia direct1y on the ascospore . germ-tubes (simi1ar to microcyc1ic conidiations), this has never been observed in Peziza que1epidotia. The reported changes in the surface architecture for SaccharomyCes during germination (Rousseau et a1, 1972) are probab1y artifactua1 due to poor technique (i.e. air drying). Germ- inating spores are more susceptab1e to distortion as a resu1t of water imbibition and mean decrease in wa11 thickness (Gu11 and Trinci, 1971). That the surface features of the exospore during germination in P, gue1epidotia are partia11y obscurbed may be due to muci1age, f1attening of the exospore due to spore swe11ing. /or enzymatic degradation of the exospore. Transmission e1ectron microscopic observations are needed to determine the re1ationship of the germ-tube to the parent ascospore wa11. H. Excipu1oid Stroma In an ear1ier pub1ication (0'Donne11 and Beneke, 1973), Peziza gueTepidotia was reported to have produced sc1erotia-1ike structures on enriched media (i.e. yeast-ma1t agar). A detai1ed study of the ontogeny 127 of these vegetative masses revea1ed that the initia1 aggregation of hyphae is simi1ar to the three different methods of sc1erotium formation described by Townsend and Wi11etts (1954). The hyphae are cemented together by a muci1agenous matrix that appears as membranous materia1 on the ce11s at a11 stages when viewed in the scanning e1ectron microscope. This muci1agenous materia1 is secreted from the vegetative hyphae (Moore, 1965). At maturity, these pu1vinate to cerebriform, pseudoparenchymatous structures are f1eshy and sometimes form a continuous cover over one-ha1f of a petri dish cu1ture. As these structures are not hard and not differentiated into we11 defined zones, they most c1ose1y resemb1e the 1oose or Rhizoctonia-type of sc1erotium (Sensu Townsend and Wi11etts, 1954). However, on1y a sma11 centra1 part of the Rhizoctonia-type sc1erotium is pseudoparenchymatous whereas a11 of PeZiza is at maturity. A1though Townsend and Wi11etts (1954) suggest that the Rhizoctonia-type is resistant to drought, it is not 1ike1y that the pseudoparenchymatous structures of ‘ Peziza function in a simi1ar capacity. In addition, the surface features of the pseudoparenchymatous structures of Peziza viewed in the scanning e1ectron microscope are c1oser to Botrytis cinerea (Wi11etts, 1969). There- fore, Wi11etts (1969) suggestion that the surface features of sc1erotia may be of taxonimic importance may have to be reconsidered. A1though some OpercuTate Discomycetes produce true sc1erotia (Moore, 1962; Moreau and Moreau, 1956; Whitney and Parmeter, 1964), the pseudoparenchymatous structures of Peziza_do not fit the structura1 and functiona1 definition of a true sc1erotium (Townsend and Wi11etts, 1954; Ainsworth, 1961). The structures of Peziza gue1epidotia are stroma-1ike and are mophoTogica11y identica1 to the ecta1 excipu1um in the apothecium. However, as these stromatic masses do not produce spores, they do not 128 fit Ainsworth's (1961) strict definition of a stroma. I suggest that these indeterminate" vegetative masses in E, que1epidotia be referred to as excipu10id stroma because of the strong resemb1ance to the ecta1 excipu1um. A1though Dixon (1971) noted pu1vinate "comp1ex structures" (probab1y exci- pu1oid stroma) in artificia1 cu1ture of 0edocepha1um roseum, she did not describe the morpho1ogica1 features of these structures. 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All nuc1ear divisions appeared to be intranuclear. Plaque-shaped spindle-pole bodies were discernible during the syn- chronous mitotic division in the crozier, the reductional division in the ascus and the mitotic division preceding sporogenesis. The intranuc1ear reductional division was of the ‘Neurospora type’ with no time interval between karyogamy and the onset of meiosis. This type of meiosis is further characterized by the presence of highly contracted homologues at synapsis and the absence of leptotene. A com- parison of the behaviour of spindle-pole bodies in this species with other members of the Euascomycetidae is presented. INTRODUCTION In recent years, ascosporogenesis in the Euascomycetidae has been investigated exten- sively at the light-microscopic level (Singleton, 1953; Carr & Olive, 1958; Doguet, 1960; Lu, 1967; Zickler, 1967; Beckett & Wilson, 1968) and ultrastructural level (Reeves, 1967; Carroll, 1969; Wells, 1970; Zickler, 1970; Beckett & Crawford, 1970). We made a light microscopical investigation of the behaviour of the nuclei and spindle-pole bodies (Aist & Williams, 1972) during ascosporogenesis in the Discomycete Peziza quelepidotia Korf & O’Donnell. METHODS Cultures of Peziza quelepidotia were grown in Petri dishes on a medium of Jiffy-7 pellet infusion agar (prepared by homogenizing three Jiffy-7 pellets per litre plus 20 g agar) at 25 i 1 °C with continuous white light supplied by fluorescent tubes. Apothecia in various stages of development were harvested and cut longitudinally into thin pieces. The ferti1e slices were immediately fixed in modified BAC fixative containing 9 parts ethanol, 6 parts propionic acid and 2 parts 10 ‘Z, aqueous chromic acid (Lu & Raju, 1970) for 24 h at room ‘ temperature (25 i 1 °C) and stored at o to 4 °C until examined cytologically. The hymenial pieces were hydrolysed in N-HCI in a 65 °C water bath for 20 min. The acid was replaced with water and the tissue left at room temperature for at least 3 days. The tissue was further hydrolysed for 45 min in N-HC] at 65 °C and the acid was decanted and replaced with 50 % propionic acid. This solution was placed in a 65 °C water bath for 5 min and then imme- diately cooled in an ice bath. 304 141 K. L. O’DONNELL, W. TAT AND E. S. BENEKE “b ' O .-. ’ \3" . / 121 ‘D 142 Ascosporogenesis in Peziza quelepidotia 305 The stain employed was propionic iron haematoxylin (Lu, 1967; Lu & Raju, 1970). A small piece of hymenium was placed on a glass slide in a drop of their solution ‘B’ and covered with another glass slide. The asci were spread out by pounding the top glass slide and then the slides were separated. A drop of solution ‘A’ was added to solution ‘B’ and the asci were further squashed under a coverslip. The slides were briefly heated over an alcohol lamp and then ringed with dental sticky wax. All slides were examined and photographed with phase-contrast illumination on a Zeiss Photomicroscope II fitted with a Planapo 100/1-3 objective and an achromatic—aplanatic phase condenser (numerical aperture = 1-4). Photographs were taken with a built-in 35 mm camera on Kodak Panatomic-X film exposed at ASA 25 and developed in either Kodak D-76 (1: 1) or Microdol-X (1 :3). All prints were made on Kodak Kodabromide F-5 paper. RESULTS Ontogeny of a typical Dangeardian crozier (Fig. 1) was initiated by the migration of two nuclei into a terminal ascogenous hypha. These nuclei underwent a synchronous mitotic division (Figs. 2 to 5), at which time plaque-like spindle-pole bodies (SPBs; Aist & Williams, 1972) were discernible. During this division, there was a marked decrease in the nucleolar size. The binuc1eate ascus mother cell was then delimited by septation (Fig. 6). As this penultimate cell elongated (Figs. 7 t0 9) there was a concomitant proliferation of the ulti- mate cell. The antipenultimate cell nucleus then migrated into the ultimate cell where these nuclei underwent a synchronous mitotic division (Fig. 10). Karyogamy took place shortly after the binuc1eate ascus mother cell (penultimate) began to extend (Figs. 10 to 12). At karyogamy the nucleoli were always situated at opposite ends of the respective nuclei (Fig. 11). In the late zygotene to early pachytene nucleus (Fig. 13) the nucleoli had fused, forming a large crescent-shaped nucleolus. Relational coiling of some homologues was discernible at full pachytene (Fig. 14). At early diplotene, the chromo- somes became more extended and the homologous chromosomes began to separate (Fig. 15). Two lightly staining amorphous bodies were discernible on the nuclear membrane at this stage and may have represented replicated SPBs or nuclear blebs. As the homologues continued to separate, the meiotic nucleus entered the diffuse diplotene stage (Fig. 16). There was no apparent decrease in the size of the nucleolus at this stage. Inability to find diakinesis suggests that this stage was relatively short in this fungus. At metaphase I, the bivalents lined up forming a distinct equatorial plate (Fig. 17). The presence of a distinct meiotic metaphase plate is an uncommon feature in the fungi (Olive, 1965). The intranuc1ear Unlabelled arrows are used to locate the spindle-pole bodies. Lengths of scale bars are approximate. Fig. 1. Nuclear migration into Dangeardian crozier (left cell). Fig. 2. Synchronous mitotic metaphase in crozier. Fig. 3. Mitotic metaphase in crozier. Fig. 4. Mitotic telophase in crozier. Fig. 5. Interphase following synchronous mitosis in crozier. Fig. 6. Binucleate ascus mother cell (AMC) subtended by ultimate (U) and antipenultimate (AN) cells. Fig. 7. Elongation of binuc1eate ascus mother cell (AMC). Fig. 8. Binucleate ascus mother cell (AMC) subtended by extending ultimate cell (U). Fig. 9. Binucleate ascus mother cell (AMC) subtended by binuc1eate crozier (C). 143 306 K. L. O’DONNELL, w. TAI AND E. s. BENEKE \ .. o .3 AMC ‘ ‘ ‘ I ~ M \f“ 2N... ' I . O J .\ -3 ~ - 1'. _' " f. I ’fihmg. ‘ Ritmr mo.".' ‘3 1__1 10 l_J 11 f ‘ w 12 . . 14 16 .l. '1 311m . 311m \ 17 18 Unlabelled arrows are used to locate the spindle-pole bodies. Lengths of scale bars are approximate. Fig. 10. Synchronous mitotic metaphase in crozier subtending a young diploid ascus. Fig. 11. Karyogamy in a young ascus. Arrows locate the nucleoli (Nu). fig. 12. Binucleate ascus mother cell (AMC) and young diploid ascus (2N) subtended by ultimate 0e (U). Fig. 13. Late zygotene to early pachytene nucleus with a crescent-shaped nucleolus (Nu). Fig. 14. Full pachytene with relational coiling of some homologues. Fig. 15. Diplotene with two amorphous bodies (SPBs or nuclear blebs ?) contiguous with the nuclear membrane. Fig. 16. Diffuse diplotene with extended homologues and two amorphous bodies (SPBs or nuclear blebs ?) contiguous with the nuclear membrane. Fig. 17. Metaphase I with intranuc1ear spindles and plaque-like SPBs at the poles. Fig. 18. Metaphase I with spindles perpendicular to the long axes of the ascus. 144 Ascosporogenesis in Peziza quelepidotia 307 ‘3‘ 3 “m 3 pm ’ 9 gt 5 um - 3 11m A 4 11m I—J 4 iim 6 pm 24 g1 25 Unlabelled arrows are used to locate the spindle-pole bodies. Lengths of scale bars are approximate. F'g. 19. Metaphase I with 14 to 15 bivalents. ' . 20. Anaphase I with continuous spindle fibres. F' 21. Telophase I with regrouped chromosomes at the poles. . 22. Interphase I with a plaque-like SP3 on each nuc1eus. ' . 23. Prometaphase II with replicated SPBs. ' . 24. Prometaphase II with SPBs migrating to the poles. ’g. 25. Metaphase II with polar SPBs and chromosomes on distinct equatorial plates. ._.'_H. gene 313:" mono: El 145 308 K.L. o’DONNELL, w. TAI AND E. s. BENEKE i'l 1}. ,_ , f (yum . 26 L__1_ 27 1___1 § 28 w~ 4 11m l‘l ‘JJ .— 1 Unlabelled arrows are used to locate the spindle-pole bodies. Lengths of scale bars are approximate. Fig. 26. Anaphase (A) and telophase II ('1') showing asynchronous anaphase movements in adjacent nuclei. Fig. 27. Telophase II with linearly arranged nuc1ei. Fig. 28. Interphase [I with SPB visible on one nucleus. Fig. 29. Prophase III with replicated SPBs. Fig. 30. Mitotic metaphase III with plaque-like SPBs at the spindle poles. Fig. 31. Mitotic anaphase III showing three of the four spindles. 146 A scosporogenesis in Pezi:a quelepidotia 309 4 0"V 6 [1111 ,1- ‘1 ....._.. ... 1'1" ’ (‘3 . . $ ,\ ' :7 611111 a L___.| 33 0 .g c \ 511m g4 36 ’ a" an» fi- . "~ o- . . 1‘13; _5 )1111 - 2 nm ,L.-—’ 35 |———1 37 it. It: Unlabelled arrows are used to locate the spindle-pole bodies. Lengths of scale bars are approximate. Fig. 32. Mitotic telophase III with SPBs visible on some of the nuclei. Fig. 33. Interphase III (the beaked nuclear stage) with greatly enlarged crescent-shaped SPBs. Fig. 34. Spore delimitation showing a SPB near one end of a developing ascospore. Fig. 35. Spore delimitation showing retraction of a SPB from the developing spore wall. Fig. 36. Various stages of spore delimitation showing the SPB near one end of the spore initials. Note that two dark-staining bodies (SPB ?) appear on the nuclear membrane of nuclei that have retracted from the spore wall. Fig. 37. Spore initia1 with SPB near one end of developing spore wall. 310 K. L. 0’DONNELL, W. TAI AND E. S. BENEKE 148 Ascosporogenesis in Peziza quelepidotia 311 spindle at metaphase I was typically found to be parallel to the long axis of the ascus but occasionally a perpendicular arrangement was seen (Fig. 18). In favourable squashes of metaphase I(Fig. 19) and anaphase I asci, there appeared to be 14 to 15 bivalents and 28 to 30 chromosomes, respectively. This haploid chromosome number is high for the genus Peziza (Olive, 1962). Chromosome separation was asynchronous at anaphase I and the spindle fibres persisted until late anaphase. At late anaphase, the nuclear membrane appeared to break down and a semi-persistent nucleolus (Pickett-Heaps, 1970) was eliminated. Spindle-pole bodies were not discernible at anaphase I. At telophase I, the spindle was no longer visible and the chromosomes were arranged in two groups (Fig. 21). A rod-shaped SPB was discernible on each interphase I nuc1eus (Fig. 22). Replication of these plaques took place before prometaphase II (Fig. 23). The plaques then separated (Fig. 24) and migrated to the metaphase II poles (Fig. 25). Metaphase II spindles were typically parallel to the long axis of the ascus; however, one (Fig. 26) or both spindles were sometimes perpendicular. Asynchronous separation of the chromosomes was common at anaphase I to 111. Asynchronous anaphase movements of adjacent nuclei within an ascus are also common. The nuclei at telophase II (Fig. 27) were typically well spaced and linear in the ascus. The SPB on each interphase II nucleus (Fig. 28) replicated (Fig. 29) and then the daughters moved to the poles (Fig. 30). Although the intranuc1ear spindles at metaphase III were characteristically perpendicular to the long axis of the ascus, they were sometimes found oriented in any direction. Spindle-pole bodies, which were not discernible at anaphase III (Fig. 31), were clearly visible on telophase III nuclei (Fig. 32). At interphase III, the beaked nuclear stage (Fig. 33), a SPB was situated on each nucleus and appeared to be contiguous with the nuclear membrane. The SPB of each interphase III nucleus appeared to be situated near the ascus wall. The nucleolus in each interphase III nucleus was located distal to the SPBs. The crescent-shaped SPB of each nucleus appeared to be contiguous with one end of the developing ascospore wall (Figs. 34, 36, 37). As the spore matured, the SPB was retracted from the spore wall (Figs. 35 to 36) and appeared as two dark-staining bodies on the nuclear membrane of each spore nucleus (Fig. 36). The SPBs were not discernible from before secondary wall deposition (Fig. 38) through to spore maturation. The mature ascus (Fig. 39) normally contained eight uninucleate, haploid spores with a number of globules situated on either side of each ascospore nucleus. On rare occasions, binuc1eate (Figs. 40, 41) and trinucleate (Fig. 42) ascospores were observed. Asci with polymorphic, aberrant ascospores (Fig. 43) were also sometimes observed. Unlabelled arrows are used to locate the spindle-pole bodies. Lengths of scale bars are approximate. Fig. 38. Ascus with eight uninucleate spores prior to secondary wall deposition. Fig. 39. Mature ascus with eight uninucleate spores with a number of dark-staining globules situated on either side of each unstained spore nucleus. Mounted in Melzer’s IKI (1 :1). Fig. 40. Ascus with two binuc1eate and four uninucleate ascospores. Fig. 41. Ascus with one aberrant, binuc1eate, allantoid spore and several aborted spores. Fig. 42. Ascus with one trinucleate and five uninucleate ascospores. Fig. 43. Ascus with polymorphic, aberrant ascospores. 149 312 K. L. o’DONNELL, w. TAI AND 13. s. BENEKE DISCUSSION In Peziza quelepidotia, SPBs were discernible during all nuclear divisions. Very small SPBs (approx. 06 ,um) were found at the poles of the synchronous mitosis in the crozier. Similar findings were reported for Pyronema domesticum (Hung & Wells, 1971) where the spindle was composed of ‘ at least 10 microtubules’. Spindle-pole bodies were not discernible at telophase in the crozier up to pachytene. At diplotene, two amorphous bodies (1 to 2 am in length) were contiguous with the nuclear membrane and may have been progenitors of the plaque—like SPB. Alternatively, they may have been nuclear blebs (see Fig. 13 in Beckett & Crawford, 1970) since Wells (1970) reports that the SPB is not discernible with the light microscope in Ascobolus stercorarius at prophase. In X ylosphaera polymorpha (Schrantz, 1970; Beckett & Crawford, 1970), the SPB appears in the electron microscope as an electron-dense body near the fusion nucleus. Beckett & Crawford (1970) indicate that this body ‘divides’ at diplotene and migrates to the poles where it forms plaque-like SPBs. U1trastructura1 evidence will be needed in order to determine whether this is so in P. quelepidotia. As in the Ascomycetes, replication of the SPBs takes place in the Basidiomycete Coprinus lagopus (Lu & Raju, 1970; Raju & Lu, 1970) at the diffuse diplotene stage. The replicated SPBs in Coprinus do not separate until diakinesis (Raju & Lu, 1973). At metaphase I, the plaque-like SPBs were approximately 09 ,um long in Peziza quelepi- dotia and were found on each side of a distinct metaphase plate. Lu (1967) interprets the perpendicular orientation of the SPB at metaphase I as a possible transitory stage. There is some credence to this interpretation in P. quelepidotia since spindles perpendicular to the long axes of the ascus in anaphase and telophase I nuclei have never been observed. The intranuc1ear position of the meiotic apparatus is only discernible in overstained asci. Inability to discern SPBs at anaphase I may have been due to chemical changes within the plaque (i.e. depolymerization) or more probably competition with the chromatin and the nucleolus for the stain. In a closely related species (Ascobolus stercorarius), the ultra- structural integrity of the plaques at anaphase I has been demonstrated (Zickler, 1970). Replication of the plaque-like SPBs took place at prophase II and III in P. quelepidotia. Similar findings have been reported for Gelasinospora (Lu, 1967) and Ascobolus (Wells, 1970). An apparent increase in the plaque size was discernible in Peziza from metaphase I (approx. 09 am) to interphase I (approx. 16 pm). In addition, the plaques were seen to increase in size at metaphase III (approx. 18 ,um) and interphase III (approx. 28 pm). The SPBs almost tripled in size from metaphase I to interphase III which suggests ‘the synthesis of some SPB material’ (Raju & Lu, 1973). The squash technique presents one problem in that measurements are only approximate because of the enlargement of the SPBs, nucleoli and chromosomes during flattening. The Ascobolus-type plaque (sensu Zickler, 1970) at interphase III in Peziza quelepidotia appeared to be contiguous with the plasma membrane of each spore initial. An ultra- structural analysis of spore delimitation in X ylosphaera polymorpha (Beckett & Crawford, 1970) reveals that the astral-ray complex, and not the plaque, was contiguous with the ascus vesicle. Although astral rays have never been observed in the light microscope at any state of division in P. quelepidotia, it is very probable that they were present during spore delimita- tion. The fate of the SPB—astral-ray complex in the Euascomycetidae after spore delimita- tion seems to be variable. In P. que1epidotia and Sordariafimicola (Doguet, 1960) the plaques appear to be retracted from the plasma membrane of the ascospores and are later discernible on the nuclear membrane of the spores. However, this observation of the behaviour of the 150 Ascosporogenesis in Pesto quelepidotia 313 SPB in the spore of P. quelepidotia has not been confirmed in the electron microscope. A situation similar to that in Peziza may exist in Pustu1aria cupularis (Schrantz, 1970) and Ascobolus stercorarius (Wells, 1970) where a plaque-like SPB is discernible on the young ascospore nucleus. A different situation is found in Podospora anserina (Zickler, 1970) and X ylosplzaera polymorpha (Beckett & Crawford, 1970) where the SPB becomes dissociated from the nuclear membrane. In X. polymorpha the SPB may replicate in the spore (Beckett & Crawford, 1970), as an additional SPB is discernible on the nuclear membrane. Spindle- pole bodies may possess some degree of autonomy, since Heslot (1958) demonstrated that enucleate spores contain plaques in mutants Of Sordaria macrospora. In addition to the structural changes that take place in the SPBs during ascosporogenesis within a species (Lu, 1967; Beckett & Wilson, 1968; Beckett & Crawford, 1970; Zickler, 1970; Wells, 1970), there are reports of structural differences in the SPBs between species. Zickler (1970) and Wells (1970) found a rod-shaped SPB in Ascobolus stercorarius. In addition, rod-shaped SPBs have been reported in a number of other Euascomycetidae (Schrantz, 1970; Westergaard & von Wettstein, 1970; Hung & Wells, 1971). In several species of Penicillium, Laane (1970a, b) reported a bipartite centriole (spindle-pole body). Bipolar SPBs were observed by Girbardt (1971) in Neurospora, Chaetomium and Asper- gillus. The structure of these bipolar SPBs is similar to that reported for some of the Hetero- basidiomycetidae (Wells, 1971 ; McCully & Robinow, 1972 a, b) and Homobasidiomycetidae (Girbardt, 1968, 1971; McLaughlin, 1971; Raju & Lu, 1973). Raju & Lu interpret the diglobular SPBs in the Homobasidiomycetidae as representing ‘the duplicated form of the monoglobular state’. Distinct plaque-like SPBs have evolved in the Protozoa (Acanthamoeba: Bowers & Korn, 1968; Thalassophysidae: Hollande, Cachon & Cachon, 1969) and centric diatoms (Litho- desmium: Manton, Kowallik & von Stosch, 1969). There is circumstantial evidence that the SPBs provide the cell with discrete loci that control the distribution of microtubules (Pickett- Heaps, 1969; Tilney, 1971). It is also possible that the SPBs are reservoirs of microtubule proteins that are eventually organized into microtubules by some other structure (Aist & Williams, 1972). In addition, there is evidence to suggest that the SPBs may play an active role in nuclear migrations in fungi (Girbardt, 1968; Aist & Williams, 1972), possibly in positioning the nuclei before sporogenesis. However, further ultrastructural and comple- mentary cytochemical studies need to be conducted on taxonomically diverse protists before functions can be attached to the multifarious SPBs and homologies be drawn. REFERENCES AIST, J. 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