Taxonomy and Species Identification

Isozyme analysis was first applied to fungal taxonomy during the 1960s (Clare 1963; Hall 1967; Meyer et al. 1964; Peberdy and Turner 1968). Since then it has been applied at many taxonomic levels from typing individual strains to delimitation and identification of species. The best enzymes for taxonomic purposes are those that are monomorphic within but different among taxa. A caveat is that isozymes cannot be used to infer phylogenetic relationships. This is because bands with the same migration rate on a gel in fact may not be identical. Furthermore, it is not possible to infer which allele is ancestral or to estimate the number of mutations that cause the isozymes to migrate differently; alleles of similar size may be more different evolutionarily than those with larger migration distances on a gel. Therefore, clustering algorithms such as neighbor joining (Saitou and Nei 1987) that allow for unequal rates of evolution on branches are not appropriate for isozyme data. Instead, isozyme data are analyzed usually by calculating a simple distance coefficient and drawing clusters with the Unweighted Pair Group Method with Arithmetic mean (UPGMA) (Michener and Sokal 1957). These analyses can be performed with several computer programs such as NTSYSpc (Rohlf 1998), POPGENE (http://www.ualberta.ca/ -fyeh/index.htm), PHYLIP (http://evolution.genetics. washington.edu/phylip.html), or PAUP* (http://paup.csit. fsu.edu/index.html). Cluster analyses always should be accompanied by bootstrap analysis or some alternative method of indicating the level of statistical support for particular groupings. Unfortunately, bootstrap analysis cannot be performed with NTSYS, but it is available with some of the other computer programs as well as the program WinBoot (Yap and Nelson 1996).

Specific applications of isozyme analysis to taxonomic questions include identifying strains of Trichoderma harzia-num (Zamir and Chet 1985), varieties of Verticicladiella wageneri (Otrosina and Cobb 1987), or anastomosis groups within Rhizoctonia (Damaj et al. 1993). The technique also can be used to identify fungal cultures to species (Six and Paine 1997). Isozyme analyses have confirmed a high level of genetic differentiation among host-associated varieties of Leptographium wageneri (Zambino and Harrington 1989) and have revealed previously unknown genetic subdivision within various species of Phytophthora (Mchau and Coffey 1994; 1995). In the rust genus Puccinia, isozyme variation can distinguish among species and also among formae speciales on different hosts (Burdon and Marshall 1981).

The most common use of isozyme analysis in fungal taxonomy is to divide isolates into species and to test how well biochemical species identification corresponds to classical taxonomy. Usually, species groups identified by isozyme analysis correspond quite closely with those identified morphologically (Hsiau and Harrington 1997; Oudemans and Coffey 1991a,b; St. Leger et al. 1992; Surve-Iyer et al. 1995). However, sometimes two or more taxa are found to be the same genetically (Oudemans and Coffey 1991b; Yoon et al. 1990) and are combined into a single species. The opposite also is a common result of isozyme analysis: single species frequently can be divided into two or more species based on previously hidden genetic differentiation uncovered by isozyme analyses (Altomare et al. 1997; St. Leger et al. 1992).

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