The ultrastructure of Leptogium cyanescens Ach. K6rb. and its cultured phycobiont, Nostoc commune Vauch., were examined using scanning electron microscopy and transmission electron microscopy. Scanning electron microscopy showed that the isidia break at the point of contact with the thallus, suggesting a possible dehiscence mechanism. Transmission electron microscopy showed that isidia are surrounded by a cortical layer of fungal cells; internally, isidia contain algal and fungal cells interdispersed in an amorphous matrix. The lichen gelatine was found to consist of four layers on both the upper surface and the lower surface of the lichen thallus. Cells of the inner portion of the thallus were found to be dispersed in an amorphous matrix which is continuous with layer four of the gelatine. The ultrastructure of lichenized and cultured Nostoc commune was compared. A tubular array previously unreported was found in cells of the phycobiont. Cultured cells contain numerous paracrystalline structures and a membrane-bounded inclusion. Leptogium cyanescens is a gelatinous lichen in the Collemataceae in which Nostoc commune Vauch. is the blue-green phycobiont. Collemataceae is placed in the Lecanorales (Hale, 1967). Both algal and fungal cells of members of the Collemataceae are embedded in an algal gelatine (Degelius, 1954). Scanning electron microscopy of lichen thalli has been limited to observing cortical hyphae (Hawksworth, 1969; Hale, 1973, 1975), crystals of lichen substances (Reznik et al., 1968; Peveling, 1970; Fahselt et al., 1973) and rhizines (Reznik et al., 1968). No detailed morphological study of lichen isidia has been made although several authors (Dobson & Hawksworth, 1976; Hale, 1975) have looked at lichen isidia to help solve systematic problems in various lichen groups. Previous investigations of lichens with blue-green phycobionts (Ahmadjian, 1967; Drew & Smith, 1967; Peat, 1968; Peveling, 1969; Roskin, 1970; Paran et al., 1971; Jacobs & Ahmadjian, 1973; and Peveling, 1973) have found that the only difference between blue-green phycobionts and their cultured forms is the abundance of osmiophilic globules in the phycobiont. Peveling (1969) found the number of plastoglobules were doubled in degenerating cells. Jacobs and Ahmadjian (1973) found plastoglobuli to be absent in cells of the phycobiont of Hydrothyria venosa, a freshwater lichen, collected during the summer and present in cells of the phycobiont collected in the fall. Recently Bousfield and Peat (1976) observed "microtubule-like" structures in ' Cells were kindly identified by Dr. Francis Drouet, Philadelphia Academy of Sciences, Philadelphia, Pennsylvania. 2 Department of Botany, Rutgers University, New Brunswick, New Jersey 08903. 3 Department of Biological Sciences, Herbert H. Lehman College of CUNY Bedford Park Boulevard West, Bronx, New York 10468. This content downloaded from 157.55.39.55 on Tue, 23 Aug 2016 04:41:27 UTC All use subject to http://about.jstor.org/terms 446 THE BRYOLOGIST [Volume 80 the phycobiont of the lichen Collema tenax; they have cultured the phycobiont and intend to compare its morphology to that of its lichenized counterpart. The mycobiont of Leptogium cyanescens is an Ascomycete which exhibits the usual inclusions found in fungal cells (Bracker, 1967). In addition, concentric bodies previously observed in numerous lichenized fungi (Brown & Wilson, 1968; Peat, 1968; Peveling, 1969; Griffiths & Greenwood, 1972) and in non-lichenized fungi (Griffiths & Greenwood, 1972; Grannett, 1974) have also been reported. The study of lichens, such as Leptogium cyanescens, with bluegreen phycobionts are especially interesting since these lichens are composed of a prokaryotic organism (blue-green alga) and a eukaryotic organism (fungus) living together symbiotically. Thus, this investigation was undertaken to examine in detail the morphology of Leptogium cyanescens and its cultured phycobiont in the hope of lending a better understanding on how the symbiotic relationship is maintained in lichens. MATERIALS AND METHODS Leptogium cyanescens was collected from Shenandoah National Park and southwestern Louisiana, where it was found growing on rocks and trunks of deciduous trees. Portions of lichen thalli were cleared from debris and fixed in the following ways. Scanning Electron Microscopy (SEM).-Tissue was fixed in 3% glutaraldehyde in phosphate buffer at pH 7.2 at 4 C for one h. The tissue was then washed in several changes of phosphate buffer and dehydrated in a graded series of ethanol and freon TF. Tissue was then critical-point dried in a Bomar SPC-900 critical point drier, mounted on copper stubs with double stick tape and coated with gold in a Hummer 1 (Technics). Tissue obtained from herbarium packets was cut into small pieces and mounted without fixation onto copper stubs as described above. Tissue was examined with a JSM-U3 scanning electron microscope operating at 25 kv. Transmission Electron Microscopy (TEM).-Portions of the lichen thallus were prepared according to the following: 1) small pieces of freshly collected lichen thalli were suspended in 1% OsO4 in Michaelis buffer at pH 6.3 for three h at room temperature according to the method of Pankratz and Bowen (1963) and 2) small pieces of freshly collected lichen thalli were suspended in 3% glutaraldehyde in phosphate buffer at pH 7.2 for one h at 4 C, washed in several changes of phosphate buffer and resuspended in 1% OsO4 according to the method of Pankratz and Bowen (1963). Tissue prepared in either way was then dehydrated in a graded series of ethanol and propylene oxide and embedded in Epon 812 according to a modification of the procedure of Luft (1961); Propylene-oxide:Epon 3:1, 30 min; 1:1, 48 h; 1:2, 48 h; pure epon, 24 h under incandescent lamp; 48 h in 45 C oven; 5-8 days in 65 C oven. The authors found this modification necessary to obtain proper infiltration of the epon into the lichen thallus. Algal cells isolated from the lichen thallus via serial dilutions of a lichen homogenate (mortar and pestle) were collected from colonies grown in Bolds medium (Deason & Bold, 1960), modified Fitzgerald medium (Fitzgerald et al., 1952) and Cyanophycean medium (Starr, 1964), pelleted by centrifugation and prepared for transmission electron microscopy in the manners described above. The sections were post stained with saturated uranyl acetate in methanol (Stempak & Ward, 1964) or lead salts (Reynolds, 1963) either singly or in combination. Cells were examined in a Hitachi HU 11E electron microscope operating at 75 kv.