Isozyme Banding Patterns in Two Stands of Cetraria arenaria Kärnef.

Abstract

The objective was to explore the range of isozymes detectable in two stands of Cetraria arenaria Kiirnef. established in stabilized sand dunes along the eastern shore of Lake Huron. Proteins in crude extracts were separated by isoelectricfocussing and tests were made for peroxidase, catalase, esterase, carbonic anhydrase, acid phosphatase, alkaline phosphatase, lactate dehydrogenase, malate dehydrogenase and glutamate dehydrogenase activity. Some gels were stained also with Coumassie Brilliant Blue, a general protein stain. Storage of acetoneextracted thallus pieces at -200C for as long as 10-12 months had little effect on the isozyme banding patterns. Most of the bands appeared to be common among samples collected in the same stand, but some notable within-stand differences were observed with respect to esterases and acid and alkaline phosphatases. Only minor quantitative differences in proteins were observed between the two stands. Isozyme information could greatly facilitate phylogenetic studies in lichens. This is particularly true in the case of those species that lack characteristic lichen products synthesized along the acetate-polymalonate pathway. To date, lichen proteins separated with electrophoresis or isoelectricfocussing have been detected mainly through the use of general protein stains (Fahselt & Jancey 1977; Fahselt 1980). As taxonomic characters, bands detected in this way are at least as valid as any morphological trait for which the underlying biochemical explanation is unknown. More precision is possible, however, if comparisons 007-2745/83/129-134$0.75/0 This content downloaded from 157.55.39.100 on Thu, 25 Aug 2016 04:37:56 UTC All use subject to http://about.jstor.org/terms 130 THE BRYOLOGIST [Volume 86 are based upon proteins whose particular activities have been identified. Estimates of similarities among samples are more reliable if considerations can be narrowed to a subset of bands related by function rather than making comparisons within a larger, more heterogeneous group of proteins. Prior to the initiation of our studies, no extensive isozyme analyses had been previously undertaken in lichens. The degree of difficulty that would be encountered, therefore, in retaining enzyme activity through the lengthy extraction procedures that are necessary was unknown. Further, there has been no information available concerning the extent to which enzymes might vary within one taxonomic species, or even one stand. Our objective was to investigate the range of isozymes detectable in two local stands of Cetraria arenaria Kirnef. This is a species of dry o]ofn areas which was selected for our work because the thallus is relatively large and because its growth form permits only minimal contamination by substrate matter and other cryptogams. Additionally, though an uncommon species, in southern Ontario it grows abundantly in a few locations. Cetraria arenaria is not known to produce phenolic secondary compounds such as are typical of many lichens. MATERIALS AND METHODS Samples of C. arenaria were collected from open dunes with sparce herbaceous (mainly graminoid) cover on the southeastern shore of Lake Huron (43'13'N, 81'58'W). Voucher specimens were deposited in the University of Western Ontario herbarium. All collections were made on four occasions during May and June, 1981, from two stands (I and II) separated from each other by a distance of 1.2 km. The two sites were equidistant from the beach and similar botanically as well as geomorphologically. The topography of Site I sloped gradually toward the northwest (maximum slope approximately 8%) and Site II was essentially flat; there were no glaring heterogeneities of habitat at either location. Sampling points were selected at random in each site; within 15 cm from each of sixteen points enough intertwined thalli were collected to loosely fill a 0.23 kg (half-pound size) paper bag. The lichens were cleaned of leaves or other macroscopic debris within 24 to 48 hr and the terminal 1.5 cm of all branches were removed for analysis. The excised portions of material collected at one sampling location were combined and then extracted three times (for 30 min each time) with excess acetone before being stored at -200C. Preliminary studies showed that isozyme patterns of material stored at -200C were the same as those in thalli that had been analyzed immediately after acetone extraction without freezing. Therefore, only frozen material was analyzed in this work. Samples were first analyzed within 2 to 8 weeks and later after a period of 10 to 12 months. Disruption of cells was accomplished by twice grinding 0.5 g portions of acetone-extracted lichen thalli in a mortar and pestle with liquid nitrogen. The resulting lichen powder was mixed thoroughly with 14 ml cold 0.06 M phosphate buffer pH 6.6 and then ground for 5 min at about 0?C in a glass tissue grinder with a Teflon pestle powered by a Black and Decker variable speed 8/, drill. Centrifugation of the mixture at 12,100 g for 35 min (4?C) was sufficient to precipitate most of the cellular remains, however, low density (floating) debris was removed by subsequently filtering the supernatant at 4?C through sintered glass funnels with coarse porosity. Precipitation of proteins with ammonium sulfate, dialysis of redissolved proteins to remove salts and isoelectrofocussing (IEF) were carried out in basically the same manner as described earlier (Fahselt 1980). In the present study, extracts were maintained at temperatures no higher than 5 or 60C throughout; during isoelectrofocussing a Haake Refrigerated Bath and Circulator D3-G (Haake Inc., Saddle Brook, NJ), held the gel temperature at approximately 4-60C. Separations described were made on 5% polyacrylamide gels with a pH range of 4-6. For best results the power for a 1-mm thick, 25-cm wide gel was maintained at 1.8-2.0 W for the first 30 minutes and at 10 W for the remainder of the 3 to 31/2 hour run. The extract from each 0.5 g sample was divided into three equal parts and was sufficient for three simultaneous IEF separations. Measurements of pH across the gel were taken with an Ingold surface electrode (Ingold Electrodes Inc., Lexington, Massachusetts) at the end of each run, and the gel was refocussed for 10 minutes afterwards. Tests were made to establish the presence of peroxidase (PER), alkaline phosphatase (ALP), acid phosphatase (ACP), esterase (EST), carbonic anhydrase (CAN), lactate dehydrogenase (LDH), malate dehydrogenase (MHD) and glutamate dehydrogenase (GDH) activity (Brewer and Sing 1970). In the test for peroxidase, tetramethyl benzidine was substituted for the more carcinogenic benzidine used by Brewer and Sing. Their method of detection of acid phosThis content downloaded from 157.55.39.100 on Thu, 25 Aug 2016 04:37:56 UTC All use subject to http://about.jstor.org/terms 1983] FAHSELT & HAGEMAN: ISOZYME BANDING PATTERNS 131