A New Technique to Investigate Cell Layers of the Capsule Wall Using Frullania (Hepaticae) as a Case Study

Abstract

A new technique is reported for examining anatomical features of the different layers of the capsule wall in hepatics. This technique involves treating the capsule with a pectinase preparation to separate the layers, followed by clearing and staining to enhance detail of the cell wall architecture. The potential taxonomic value of capsule wall characters for some New Zealand Frullania species is discussed as a case study. Characters of particular significance include the morphology of cell wall thickenings in both the internal and epidermal layers. The possible chemical components present in the cell wall thickenings of the capsule wall layers are also discussed. Anatomical characters of both gametophyte and sporophyte are of considerable taxonomic importance in the study of mosses, liverworts, and other cryptogams (Zander 1983). Regarding hepatics, Schuster (1966) stated that there are many potentially significant taxonomic and phylogenetic sporophyte characters present in the capsule walls of Jungermanniae. In most hepatics, the capsule wall is composed of one to several cell layers, enclosing a mixture of spores and elaters, and when mature splits open into four equal valves (Scott 1985). In the liverwort genus Frullania Raddi, the capsule wall is composed of two layers, an epidermal and an internal layer (Kamimura 1961; Schuster 1992; Yuzawa 1991). Schuster (1966) drew particular attention to the anatomy of the capsule wall and emphasized the value of examining the pattern of cell wall thickenings in the different cell layers. Despite having drawn attention to this useful character many years earlier, Schuster (1992) in his revision of North American Frullania reported that the capsule wall was poorly studied. For example, there was only limited reference to this character in revisions of Frullania in Australasia (e.g., Hattori 1980; Hodgson 1949), Asia (e.g., Hattori 1972, 1981a), or the Pacific (e.g., Hattori 1981b). However, there are practical difficulties in examining the capsule wall, particularly when attempting a precise interpretation of the cell wall architecture of the different cell layers. Because the valves of the capsule are often small (e.g., 300 pxm long X 250 pxm wide in some smaller Frullania species) and are often heavily pigmented, special preparative and microtechnical procedures are required to examine them. In addition, the multilayered structure of the capsule gives background shadow effects when examined using the compound light microscope. These problems may account for the paucity of capsule wall studies in most revisions of Frullania. Similar difficulties have been reported for other hepatics. For instance, Bartholomew-Began (1991) revealed that the capsule of Steereomitrium minutum E. O. Camb. was identical in form and anatomy to that of Haplomitrium species, and that the apparent differences in the capsule wall anatomy were the result of misinterpretations. We have developed a new technique that largely overcomes the difficulties involved in interpreting characters associated with the different cell layers of the capsule wall. This technique involves partially digesting the capsule with an enzyme preparation to facilitate separation of the cell layers. The separated layers are then cleared and stained to enhance detail of the cell wall architecture. This paper describes the technique using Frullania as a case study, but the technique is likely to be equally useful in other hepatic groups. In addition, we assess the potential taxonomic value of capsule wall characters of some New Zealand Frullania species and discuss the possible chemical components present in the cell wall thickenings in the capsule cell lay-