The Ultrastructure of Ascospore Delimitation in Saccobolus Kerverni

The mechanics of spore delimitation within the ascus have long been a topic of controversy among ascomycete cytologists. Lateral fusion of recurving astral rays around each nucleus following the mi-totic, third division in the ascus (6), progressive differentiation of spore membranes in a granular layer of cytoplasm around each nucleus (5), fusion of epiplasmic vacuoles (8), and invagination of a peripheral membrane system in the ascus (3) all have been cited in attempts to describe this process under the light microscope. Carroll (2) and Reeves (12) provide more complete discussion of the process. Early observations of the ultrastructure of developing ascospores revealed only that following meiosis each nucleus somehow is surrounded by at least one membrane, outside of which the spore wall develops (7). Later studies have demonstrated that each spore is delimited by two membranes , an inner plasma membrane and an outer investing membrane, between which the spore wall is deposited (1, 4, 9-11, 12, 13). Recently, Reeves (12) has shown that in Pyronema domestica the spore membranes originate in an open-ended peripheral cylinder of double m e m b r a n e which initially surrounds all eight nuclei in the ascus. As the m e m b r a n e cylinder constricts between the seriate nuclei, it pinches out a row of ascospores. T h e present study was carried out to investigate the origin of the spore membranes in the disco-mycete, Saccobolus. Petri plates containing a Difeo corn-meal agar culture medium (Difco Laboratories, Detroit, Mich.) supplemented with yeast and malt extracts were in-oculated with the fungus and incubated at 25°C with a 12 hr, light-dark cycle. When a mycelial mat had covered about half of each plate, the cultures were subjected to cold shock at 4°C for 3-5 days and then were returned to room temperature. At the periphery of the old mycelium, where the new growth began, a dense ring of apothecia formed within which the asci developed in near synchrony. Small blocks of agar with developing apothecia on their upper surfaces were cut from the plates, trimmed of excess agar, and placed in 2% KMnO4 for 2 hr (15 min in vacuo) at 4°C; they were then washed 6-8 times in distilled water and soaked for 4-6 hr in a 0.5% solution of uranyl acetate. The tissue was dehydrated in a graded alcohol series and embedded in an epoxy resin. Sections were …