The Appalachian Asplenium complex consists of six fertile species: three diploids and their three allotetraploid derivatives. This reticulate evolutionary pattern, originally proposed on the basis of morphological and cytological evidence, was later corroborated by analyses of flavonoid composition. Presented here are data from starch gel electrophoresis of 11 enzyme systems, coded by 15 interpretable loci, which are highly consistent with the proposed relationships in the Appalachian Asplenium complex. The diploids are strongly differentiated (genetic distance (D) = 0.67 to 1.30); each diploid possesses unique alleles at several loci. Each allotetraploid expresses the heterozygous, or less often homozygous, enzyme phenotypes expected for combinations of parental alleles at most loci. Exceptions are the expression of a novel PGI-2 allele and the loss of expression of parental IDH alleles in both allotetraploids. The importance of reticulate evolution in plants is well documented (Lewis 1980). This type of evolution involves hybridization of differentiated genomes resulting in high levels of heterozygosity. The application of the isozyme technique seems particularly relevant to gain additional insights into the evolution of polyploid complexes. This technique detects allelic variants (allozymes) at equivalent gene loci, thus facilitating analysis of genetic variation within and between species. Allopolyploids may be expected to exhibit heterozygous phenotypes at loci for which the presumed parental taxa possess electrophoretically different alleles. Indeed, early isozyme studies of plants often addressed the identification of diploid progenitors of allopolyploid crops (Cherry et al. 1972; Garber 1974; Reddy and Garber 1971; Sheen 1972). More recently Roose and Gottlieb (1976) demonstrated the utility of allozymes in confirming relationships and assessing heterozygosity in Tragopogon. However, given the preponderance of such complexes in nature and the general lack of allozyme studies on them, the technique is at present underexploited. Reticulate evolution is well known among most genera of temperate ferns (Walker 1979). The use of allozyme analysis to address problems of systematics and evolution in ferns has awaited development of techniques that allow extractions of active and electrophoretically resolvable enzymes from tissues noted for their high levels of phenolic compounds. Such techniques are now available (Soltis et al. 1980, 1983; Werth et al. 1982) and are in use in several laboratories. The evolutionary relationships among Asplenium species endemic to eastern North America were clarified by Wagner (1954) in a classic study of their morphology and meiotic chromosome behavior. Wagner showed that three diploid species, A. platyneuron, A. montanum, and A. rhizophyllum, gave rise to three allotetraploid derivatives, A. ebenoides (platyneuron x rhizophyllum), A. pinnatifidum (montanum x rhizophyllum), and A. bradleyi (montanum x platyneuron) (fig. 1). Studies on cytology (see Walker 1979 for review) and flavonoids (Smith and Levin 1963; Smith and Harborne 1971; Harborne et al. 1973) of the six fertile species as well as a number of sterile hybrids provided data consistent with the concept of relationships as originally hypothesized by Wagner (1954). The present paper reports allozymic evidence that further corroborates Wagner's hypotheses, and demonstrates the potential, as well as some of the limitations, of allozyme data to elucidate parentages of allopolyploid taxa. MATERIALS AND METHODS Field collections from two or three populations of each fertile taxon as well as one sterile allodiploid A. ebenoides were obtained from isolated localities (table 1). Up to sixty individuals of each species were collected per population. However, collection of A. ebenoides from the unique tetraploid population in Alabama was limited to three juvenile sporophytes.