Allozyme and basidiospore contributions to phylogenetic studies of Agaricus species

A review of literature on the classification of the genus Agaricus showed a great deal of confusion exists regarding relationship between species. Traditional methods used in mycological systematics have been inadequate to resolve many problems found when classifying this genus. Thus recourse to new techniques are required. As a result, methods were evaluated for the study of the allozymes of Agaricus species. Methods investigated included use of polyacrylamide gel electrophoresis and horizontal starch gel electrophoresis, composition of extraction buffers, quantity of mycelium needed for extraction, electrophoretic buffer systems, and staining systems. The method arrived at enabled successful resolution of 10 mycelial enzymes encoded by twelve polymorphic putative loci, Aat-1, Acp, βGlu, Gk-1, Gk-2, Gpt, Ha-1, Lap-1, Mdh-1, Mdh-2, Mpi, and Pgm-2. An evaluation of allozymes from mycelium and basidioma parts revealed that additional allozyme information was available when more than one Agaricus tissue was tested. Mycelium was chosen because it could be produced under standardised and controlled culture conditions. From a comparison of shaken and stationary liquid culture, shaken culture was chosen because it allowed production of more mycelium at a faster rate. Of the conditions tested for enzyme preservation, storage at -80ºC, of mycelium and extracted mycelia supernatant was successful for at least three months, and storage of freeze dried mycelium was successful for two years at that temperature. Depending upon species, the levels of mycelial enzyme activity may be influenced by culture method and period of incubation, and different electromorphs may be revealed by changing culture conditions. It was concluded that for allozyme analysis, a single standardised method should be used to grow, extract and store mycelium for the strains representative of the species investigated. Experiments also showed that for uniformity, mycelia should be of a similar stage of physiological development. It was considered that by decreasing differences due to cultural and electrophoretic methods, mycelial allozyme analyses and phylogenetic investigations based on such analyses will be improved. The twelve putative loci, from eleven Agaricus species were successfully used as a source of data for exploring cladistic relationships of Agaricus species based upon Phylogenetic Analysis Using Parsimony (PAUP). Leucoagaricus leucothites and Agrocybe parasitica were used as outgroups for analysis. The results supported the contention that the traditional Section Bitorques, and the Section Agaricus, Group Campestris were each monophyletic. The monophyly of these traditional Sections was also supported. Using computer image analysis basidiospore characters were measured with a greater speed and objectivity than has been possible using the light microscope. This method allowed the analysis of new variables, including area, and circularity, as well as length, breadth and elongation. PAUP was used to analyse the results obtained from the same eleven Agaricus species, again using Leucoagaricus leucothites and Agrocybe parasitica as outgroups. These analyses supported the phylogenetic usefulness of Agaricus basidiospores, and emphasised the need for the use of additional characters to improve the classification of this genus