Gerald J. Gastony

Starch Gel Electrophoresis of Ferns: A Compilation of Grinding Buffers, Gel and Electrode Buffers, and Staining Schedules

The homosporous pteridophytes have been largely uninvestigated by electrophoresis, despite the fact that they offer many exciting research possibilities (Soltis et al., 1980). The paucity of electrophoretic studies of ferns and fern allies may be due in large part to the high concentrations of condensed tannins that many species contain (Cooper-Driver, 1976 and pers. comm.). These compounds render enzymes inactive by binding with them following cellular disruption, thereby frustrating researchers who have attempted electrophoretic analysis utilizing standard methods of sample preparation. The method of sample preparation developed by Kelley and Adams (1977a, b) in their analysis of enzyme variation in Juniperus was an important procedural breakthrough in overcoming the difficulties that result from the liberation of large amounts of phenolic compounds during tissue preparation. Recently, a simplified version of that method was applied by Soltis et al. (1980) to fern leaf tissue, facilitating rapid preparation of active enzyme samples and thereby making electrophoretic analyses of large numbers of individuals more feasible. In an attempt to improve methods of analysis of fern enzymes in starch gel electrophoresis, we have experimented with modifications of the method of sample preparation outlined by Soltis et al. (1980). We also have examined several different methods of sample preparation such as those of Gottlieb (1981a), Mitton et al. (1979), and Werth et al. (1982), and have evaluated the relative merits of each with fern tissue. Finally, during the course of our electrophoretic investigations of ferns we found that standard gel and electrode buffers and staining schedules, such as those of Brewer (1970) and Shaw and Prasad (1970), often provided unsatisfactory results when applied to ferns. We have determined gel and electrode buffers, as well as staining schedules, that provide clear starch gel enzyme banding for 22 enzyme systems in ferns. Requests for advice resulting from the recent surge of interest in fern enzyme electrophoresis have prompted us to compile our procedural data so that other researchers can take advantage of our experimentation. We hope that these data will stimulate more extensive electrophoretic investigation of pteridophytes and other electrophoretically difficult taxa. Gottlieb (1981b) recently reviewed aspects of enzyme electrophoresis primarily in gymnosperms and angiosperms. His discussion is equally relevant to understanding the potential applications and limitations of electrophoretic evidence in pteridophytes. Since homosporous pteridophytes have high chromosome numbers, it is tempting to invoke polyploidy in interpreting their enzyme band patterns. It is well

Detecting Enzyme Variation in the Fern Genus Bommeria: An Analysis of Methodology

Although numerous flowering plant species have been subjected to electrophoretic enzyme analysis, very few such studies have involved pteridophytes. This is largely because standard methodology typically results in little or no electro- phoretically detectable enzymatic activity in pteridophytes. The presence of large amounts of phenolic compounds in leaves of most pteridophytes suggests that the electrophoretic difficulties heretofore encountered in this group result from com- plexing of enzymes by these compounds following cellular disruption. Use of a grinding buffer containing dimethylsulfoxide and several compounds inhibitory to phenolic complexing, in combination with a grinding procedure utilizing polyvinyl- pyrrolidone and liquid nitrogen, was found to overcome the difficulties of tissue preparation that result from high concentrations of phenolic compounds. Modifi- cations of this grinding buffer and grinding procedure often resulted in reduced clarity of enzyme bands, or even a total loss of enzyme activity. In Bommeria, prep- aration of leaf tissue in liquid nitrogen and use of sodium tetraborate, sodium as- corbate, sodium meta-bisulfite, and sodium diethyldithiocarbamate were found to be of less importance than use of polyvinylpyrrolidone in obtaining active enzyme samples. We hope that discussion of our procedural data and provision of gel and electrode buffer recipes and staining schedules will stimulate further electrophoretic investigation of pteridophytes.

Chromosome Numbers and Apomixis in the Fern Genus Bommeria (Gymnogrammaceae)

Species of the fern genus Bommeria occur from the southwestern United States, throughout much of Mexico and into Guatemala, Honduras, Nicaragua and Costa Rica. Acetocarmine squashes of spore mother cells, root tips, and gametophyte meristems have yielded chromosome counts for the five putative species: n - 30, 2n - 60 in B. hispida (Mett.) Underw., B. subpaleacea Maxon, B. ehrenbergiana (KI.) Fourn., and B. knoblochii Maxon; n - 2n = 90 in B. pedata (Sw.) Fourn. The first four of these species are sexual, producing 64 functional spores per sporangium and requiring the addition of free water to gametophyte cultures for sporophyte initiation. Bommeria pedata produces both well developed sporangia containing 32 functional spores and shrunken sporangia with aborted contents. Although gametophytes of B. pedata produce structurally complete archegonia and functional antheridia, culturing and sectioning data indicate that sporophytes arise apogamously near archegonia whose contents are necrotic. The generally greater geographic distribution of B. pedata relative to its sexual congeners may be taken to indicate the present success of this apogamous species. BOMMERIA IS PRESENTLY RECOGNIZED as a genus of five more or less xerically adapted species which are found from the southwestern United States, throughout much of Mexico and into Central America. The approximate ranges of the several species (figs. 1-5) have been determined from herbarium specimen data. Bommeria hispida (fig. 3) is documented from the southwestern United States (western Texas and southern New Mexico, Arizona, and California, though the last not plotted due to lack of precise locality data for the single known collection), Baja California Sur, and throughout much of northwestern Mexico. Bommeria knoblochii (fig. 4) is known only from the original collections of Knobloch in the mountains of western Chihuahua and through recent re-collections from the same localities by Bye. Bommeria ehrenbergiana (fig. 2) has most commonly been collected in the states of Hidalgo and Puebla but reaches as far north as Nuevo Leon. Bommeria subpaleacea (fig. 5) ranges north to the state of Hidalgo and has been collected as far south as Oaxaca. Bommeria pedata (fig. 1) is known from Baja California Sur and is widespread throughout the mountains of northwestern, central, and southern Mexico and through southeastern Guatemala, with rare collections from Honduras, Nicaragua and Costa Rica.

Chromosome Numbers in the Fern Genus Anogramma, II

In an earlier report on chromosome numbers in the genus Anogramma (Gastony & Baroutsis, 1975), three new counts were established and all known previous counts were summarized. At that time, a count of n=26 for A. leptophylla from Europe (Kurita, 1971) was overlooked. We wish now to acknowledge this count, to report new counts made for populations of A. guatemalensis and A. leptophylla, and to suggest an explanation for some of the variant counts previously reported for A. leptophylla. The general techniques for chromosome preparations were those previously discussed (Gastony & Baroutsis, 1975). To maximize chromosome staining, however, a propionic-iron-haemotoxylin stain (Henderson and Lu, 1968; Rigby, 1973) was applied to fixed mitotic cells of Anogramma gametophytes. The stained chromosomes were visually enhanced by use of phase microscopy in analysis and photographic work. To promote spreading and separation of mitotic cells during squashing, material was treated with one of two preparations: Glusulase (Endo Laboratories Inc., Garden City, NY), a commercially available enzyme mixture from the intestinal juice of the snail Helix pomatia, was applied full strength to gametophyte tissue for four hours (Faberge, 1945); Driselase (Kyowa Hakka Kogyo Co., Tokyo, Japan), a fungal-produced enzyme mixture, was applied as a 10% (w/v) aqueous solution according to the Glusulase schedule. Both preparations were equally satisfactory for softening cell walls. The potential of this enzyme technique in working with gametophyte chromosomes has been more fully discussed by Gastony (1977). Chromosome counts forA. guatemalensis, published here for the first time, are based on three unequivocal counts. The counts reported for A. leptophylla, however, are based only on the material illustrated in Figs. 3, 4, 8, and 9. Sources of spores cultured to provide living material of the taxa herein reported are: A. guatemalensis, Gastony 1037, Depto. Chimaltenango, Guatemala; A. leptophylla, 7 Oct 1972, Mickel, Edo. Oaxaca, Mexico; A. leptophylla, 20 Oct 1972, Esterhuysen, Cape Province, South Africa. Voucher specimens of the plants raised from spores are deposited at IND.