Scanning ultrastructural ontogeny of eugymnohymenial apothecia in the operculate Discomycetes Ascodesrnis nigricans and A.

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1 Scanning ultrastructural ontogeny of eugymnohymenial apothecia in the operculate Discomycetes Ascodesrnis nigricans and A. sphaerospora K. L. O'DONNELL, G. R. HOOPER, AND W. G. FIELDS^ Dep(~rtmc>nt of Botorzy crnd Platzt Pntl~ology and Center for Electron Optics, Michigan St~te University, East Lorzsitzg, Michigcrn Received July 21, 1975 O'DONNELL, K. L., G. R. HOOPER, and W. G. FIELDS Scanning ultrastructural ontogeny of eugyrnnohyrnenial apothecia in the operculate Discornycetes Ascodesmis nigricans and A. spl~aerospora. Can. J. Bot. 54: The developmental ontogeny of eugymnohyrnenial apothecia in Ascodesrnis nigricans van Tieghern and A. spl~aerospora Obrist, hornothallic operculate Discornycetes (Pezizales, Pyronemataceae), was investigated from initiation of the T-shaped garnetangial initials through spore discharge. Branching of these T-shaped side branches gave rise to garnetangia that paired and coiled helically. Paraphyses proliferated from the base of the rosette of garnetangia and enveloped the coils, which appeared to undergo plasrnogarny at their apices. Although a loose peridiurn of paraphyses was formed, a true excipulurn was absent. Displacement of the opercula on the positively phototropic asci was towards the more strongly illuminated side. Infroduction Ascodesmis nigricans and A. sphaerospora are coprophilic, homothallic, opercblate Discomycetes that lack a true excipulum. Since the gametangia and hymenium are fully exposed, apothecium development in this genus, which is gymnocarpic (Corner 1930) or eugymnohyrnenial (van Brummelen 1967), has been the subject of numerous light microscopic investigations (van Tieghein 1876; Zukal 1886; Dangeard 1903, 1907; Claussen 1905; Swingle 1934; Usher 1960; Obrist 1961). Since the intricate structures and natural relationships of the initial and subsequent stages of apothecium ontogeny are sometimes difficult to resolve with the light microscope, this investigation was initiated to obtain the maximum information concerning the surface architecture of the initial stages of apothecium ontogeny in Ascodesrnis nigricans van Tieghem and A. sphaerospora Obrist through spore discharge. 'Michigan Agricultural Experiment Station Journal Article Number ZDeceased, November 30, 1975, in East Lansing, Michigan. Material and Methods Cultures of Ascodesmis rzigricans (IF0 9199) and A. ~pho~rosporo (isolated in Texas by W. G. Fields) were grown on modified Leonian's medium (Obrist 1961) under continuous white light supplied by fluorescent tubes at "C. Preparative methods for the scanning electron microscope were identical with those detailed in a previous investigation (O'Donnell and Hooper 1974) with the exception that the isoamyl acetate series was omitted during the critical-point drying procedure. Observations To obtain a complete struct~iral analysis of apothecium ontogeny, all scanning electron micrographs were correlated with whole mounts and thick-sectioned, plastic-embedded apothecia. The descriptive terminology of van Brummelen (1967) for hymenial development was used in this investigation. Since the morphogenetic events of apothecium ontogeny in Ascodesmis nigricans and A. sphaerospora are essentially identical, the following represents a combined general account of their development. Archihyrnenial Phase Stout lateral knobs are the first sign of apothecium formation (Fig. 1, arrow). These hyphae FIGS Scanning electron micrographs of apothecium ontogeny in Ascodeai7is. Fig. 1. A. sphaerospora. Arrow indicates stout lateral knob. Note cluster of T-shaped gametangial initials (archihymenial phase). x Fig. 2. A. sphaerospora. Archihymenial-phase apotheciurn showing pairing of gametangial initials. x Fig. 3. A. nigricarrs. Helically coiled gametangia in archihymenial phase. x Fig. 4. A. nigricans. Arrows indicate proliferation of paraphyses from base of helically paired gametangia (prohymenial phase). x 1860.

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3 574 CAN. J. B01 become delimited at their base by septa and then branch dichotomously. The result is a cluster of T-shaped gametangial initials. These initials elongate; the pairing is initiated between hyphae of the same terminal dichotomy or from adjacent branches (Figs. 2-5). Subsequent to the concomitant elongation and pairing of the gametangial initials, the hyphal pairs enlarge up to 3.5 pm in diameter, coil helically (Figs. 3-9, and function as gametangia (i.e., ascogonia and antheridia). Gametangia appear septate (Fig. 5, arrows locate septa). Protuberances appear on some hyphal elements but not consistently. Prohymenial Phase Numerous paraphyses proliferate from the base of the gametangia (Fig. 4, arrow) and begin to envelope the coils which appear to have undergone plasmogamy at their apices (Fig. 6, arrow). The septate paraphyses continue to proliferate around the cluster of gametangia (Fig. 7) and eventually form a loose cover over the ascogenous hyphae (Fig. 8). On rare occasions, some of the paraphyses branch. A true ectal excipulum, however, is completely absent in this genus. Mesohymenial Phase Within the next 24 h, ascogenous hyphae give rise to club-shaped asci (Fig. 9). As the asci mature, they become constricted below into a short stipe (Fig. 10). Membranous material is discernible on the asci and paraphyses (Figs ). Telohymenial Phase At maturity, the broadly clavate asci protrude above the level of the paraphyses (Fig. 1 I). A mature apothecium, which is about 0.2 mm in diameter, consists of only a fascicle of asci and paraphyses supported by a few pseudoparenchymatous cells. As a result of the successive nature of ascus maturation, immature to mature asci are discernible in apothecia in this phase. As the asci mature, an operculum or lid develops as a circumscissile zone of weakness at each ascus apex (Fig. 12). At spore discharge, the ascospores are forcibly ejected from the positively VOL. 54, 1976 phototropic asci. In Ascodesmis sphaerospora, ascospore ornamentation consists of reticulations that form spines at their junctions (Fig. 13). The isolate of A. nigricans used in this study produces ascospores with spines and short ridges. Posthymenial Phase The opercula, which are about km in diameter, generally remain attached to the collapsed, dehisced asci. Displacement of the opercula is towards the source of illumination (Fig. 14). Discussion The morphogenetic events associated with fruiting-body formation in Ascodesmis nigricans and A. sphaerospora reported here are essentially similar to the light microscopic studies reported previously for A. sphaerospora (Usher 1960; Swingle 1934) and A. microscopia (Dangeard 1907; Claussen 1905). In all of these species, several gametangia are involved in the ontogeny of a single apothecium. Although several workers (Claussen 1905; Dodge 1914; Swingle 1934; Gwynne-Vaughan and Barnes 1937) have reported that the gametangia (i.e., antheridia and ascogonia) arise from separate T-shaped rosettes, this study and others (Dangeard 1907; Usher 1960) demonstrate that paired gametangia may arise from the same terminal dichotomy. That antheridia (Gaumann and Dodge 1928) and ascogonia (Usher 1960) are incorrectly reported to be non-septate is probably due to the late phase at which the gametangia become septate (van Brummelen 1967). Although Usher (1960) reported that the antheridia are larger than the ascogonia in A. sphaerospora, this was not confirmed in this study. The tuberculate protuberances that we observed on the apothecial initials of A. sphaerospora (Figs. 1, 5, 6) are interpreted as artifacts since these were not a constant feature. In most studies, the paraphyses of Ascodesmis are reported as unbranched (Obrist 1961 ; Eckblad 1968); however, Usher (1960) and this investigation clearly show that the paraphyses may Fig. 5. A. sphaerospora. Cluster of helically coiled septate gametangia (archihymenial phase). Arrows indicate septa in gametangia. x Fig. 6. A. sphaerospora. Prohymenial-phase apothecium showing proliferation of paraphyses from base of gametangia. Arrows may indicate plasmogamy. x Fig. 7. A. spliaerospora. Prohymenial-phase ascocarp showing envelopment of gametangia by paraphyses. x Fig. 8. A. sphaerospora. Lateral view of prohymenial-phase apothecium showing pseudoexcipulum of paraphyses. Arrow indicates branching paraphysis. x 1460.

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5 576 CAN. J. BOT. VOL. 54, 1976 Fig. 9. A. nigricnris. Fascicle of paraphyses and asci in midmesohymenial phase. x Fig. 10. A. rrigricn?~~. Midmesohymenial-phase apothecium showing asci with constricted bases. Note membranous material on asci and paraphyses. x Fig. 11. A. spl~oerosporn. Mature eugymnohymenial apothecium in telohymenial phase showing fascicle of clavate asci and filiform paraphyses. Note membranous material on asci. x 600. Fig. 12. A. sp\rnerosporo. Ascus apex showing large operculum. x Fig. 13. A. sphnerosporn. Dehisced ascus with reticulate ascospore. x Fig. 14 A. spl~nerosporn. Senescent telohymenial-phase apothecium showing collapsed, dehisced asci with opercula attached. x 900.

6 O'DONNELL ET AL. 577 occasionally branch. Gaumann and Dodge (1928) incorrectly indicated that the paraphyses disappear at maturity, but this may be due to the fact that they become completely translucent and their contents disappear (Usher 1960). The translucency of the senescent paraphyses may be due to the secretion of gelatinous material from their protoplasts (Moore 1965; Korf 1973), which appears in the scanning electron microscope as membranous material on the paraphyses and asci. The appearance of gelatinous or membranous material on hyphal elements in the scanning electron microscope is not an uncommon occurrence (Elliott and Corlett 1972; Colotelo 1974; O'Donnell and Hooper 1974; O'Donnell et al. 1974). The operculum in Ascodesmis is defined by an annular indentation similar to that reported for Ascobolus stercorarius (Wells 1972). As reported for other Pezizales at spore discharge (Buller 1934; Ingold 1966), the opercula in Ascodesrnis are displaced towards the more strongly illuminated side. The biological significance of this unilateral displacement, which is a coroliary of the positively phototropic ascus, has been amply discussed elsewhere (Buller 1934; Usher 1960; Ingold 1966). The superficial ontogenetic similarity of Ascodesmis with members of the Gymnoascaceae resulted in speculation that the two taxa are directly related phylogenetically (Gaumann and Dodge 1928; Usher 1960). However, recent taxonomic studies of Ascodesmis by Le Gal (1949) and Obrist (1961) suggest a high evolutionary level (i.e., simplicity as a result of reduction) based on spore ornamentation, coprophilic habit, and operculate asci. As a result, Korf (1973) erected the subfamily Ascodesmidiodeae in the Pyronemataceae (Pezizales) which accomodates Ascodesmis. Acknowledgments We express our sincere appreciation to Arthur 0. Ackerson, the scanning electron microscopy laboratory supervisor, for his expert instruction in the use of the SEM and preparatory equipment. The senior author thanks Dr. E. S. Beneke for his support. BRUMMELEN, J., VAN A world monograph of the genera Ascobolrrs and Saccobolrrs (Ascomycetes, Pezizales). Persoonia(Supp1.). 1: BULLER, A. H. R Researches on fungi. Vol. VI. Longmans, Green and Co., London. CLAUSSEN, P Zur Entwickelungsgeschichte der Ascomyceten Bo~idiera. Bot. Zeit. 63: COLOTELO, N A scanning electron microscope study of developing sclerotia of Sclero~irlia sc[erotiorum. Can. J. Bot. 52: CORNER, E. J. H Studies in the morphology of Discomycetes. IV. The evolution of the ascocarp. Trans. Br. Mycol. Soc. 15: DANGEARD, P. A Sur le genre Ascodesmis. Le Botaniste, 9: L'origine du pkrithkce chez les Ascomycktes. Le Botaniste, 10: DODGE, B The morphological relationshipsofthe Florideae and the Ascomycetes. Bull. Torrey Bot. Club, 41: ECKBLAD, F The genera of the operculate discomycetes. A re-evaluation of their taxonomy, phylogeny and nomenclature. Nytt Mag. Bot. (Oslo), 15: ELLIOTT, M. E., and M. CORLETT Light microscope and scanning electron microscope observations of Ciborin accrinn. Can. J. Bot. 50: GAUMANN, E. A,, and C. W. DODGE Comparative morphology of fungi. McGraw-Hill BookCo., Inc., New York. GWYNNE-VAUGHAN, H. C. I., and B. BARNES The structure and development of the fungi. 2nd ed. Cambridge University Press, Cambridge. INGOLD, C. T Aspects of spore liberation: violent discharge. 111 The fungus spore. Editc~d by M. F. Madelin. Butterworths, London. pp KORF. R. P Discomycetes and Tuberales. 111 The fungi: an advanced treatise. Vol. IVA. A taxonomic review with keys: Ascomycetes and Fungi Imperfecti. Edited by G. C. Ainsworth. F. K. Sparrow, and A. S. Sussman. Academic Press. New York. pp LE GAL, M Deux Discomycetes ma1 connus: Ascodes~nis nigriccrns van Tieghem et A.scodc~.srr~is microscopic,n (Crouan) Le Gal, non Seaver. Rev. Mycol. 14: MOORE. E. J Fungal gel tissue ontogenesis. Am. J. Bot. 52: OBRIST. W The genus Ascodesmis. Can. J. Bot. 39: O'DONNELL, K. L.. W. G. FIELDS, and G. R. HOOPER Scanning ultrastructural ontogeny of cleistohymenial apothecia in the operculate Discomycete Ascobolris fiirfiirciceris. Can. J. Bot. 52: O'DONNELL, K. L.. and G. R. HOOPER Scanning ultrastructural ontogeny of paragymnohymenial apothecia in the operculate Discomycete Pc,ziztr c/rrcdepidotia. Can. J. Bot. 52: SWINGLE. D. B Fertilization in Ascodesmis rligriccrrrs van Teigh. Am. J. Bot. 21: TIEGHEM, P., VAN Sur le developpementdu fruit des Ascodc~srnis, genre nouveau de I'ordre des Ascomycetes. Bull. Soc. Fr. 23: USHER, M. L A comparative study of the developmental stage of two coprophilic fungi, Amnrrronsclts verrrrcosrrs and Ascodesrnis sp. nov. M.Sc. Thesis, University of Toronto, Toronto, Ontario. WELLS, K Light and electron microscopic studiesof Ascobolrrs stc~rc~ornriri.~. 11. Ascus and ascospore ontogeny. Univ. Calif. Publ. Bot. 62: ZUKAL, H Mycologische Untersuchungen. Denkschr. Kais. Adad. Wiss. Wien. 51: