Electrophoretic evidence for the origin of fern species by unreduced spores

Allopolyploid speciation is well documented in the ferns, but data from enzyme electrophoresis have only recently shown that certain sexual and agamo.sporous taxa are autopolyploids. Autopolyploidy may arise through fertilization involving gametophytes from unreduced spores, a mechanism previously proposed to account for the origin of allopolyploid Asplenium plenum. This report assesses the ability of unreduced spores to function in the origin of polyploid fern species by using enzyme electrophoresis to test their hypothesized role in the origin of A. plenum. Six isozymes of the enzymes PGI, PGM, TPI, 6PGD, and LAP are species-specific for the taxa proposed as parental under two competing hypotheses for the origin of this species. Electrophoretic data reject the more conventional hypothesis involving simple hybridization and agree perfectly with expectations under the more complex hypothesized origin via unreduced spores. The mechanism whereby unreduced spores have functioned in this case is no different from that by which they would function in the origin of autopolyploid taxa and may be more common in the origin of fern species than previously suspected. MOST DISCUSSIONS OF speciation in ferns have dealt either with geographic speciation resulting from gradual changes in gene frequencies associated with migration or with allopolyploid speciation. Tryon (1985, 1986) has particularly discussed the former process, and Wagner (1954) provided a classic example of the latter in the Appalachian Asplenium complex, the conclusions of which were electrophoretically confirmed by Werth, Guttman and Eshbaugh (1985). Recent enzyme electrophoretic studies, however, indicate that certain sexually and asexually reproducing fern taxa have arisen through autopolyploidy (Gastony, 1985; Windham and Haufler, 1985), leading to the present effort to seek the mechanism whereby autopolyploid taxa originate. This paper uses data from enzyme electrophoresis to test the hypothesis that unreduced spores have functioned in the allopolyploid origin of fern species and therefore may function in autopolyploid origins as well. This hypothesis is tested in an allopolyploid system because variant species-specific electrophoretic markers are unequivocally interpretable in such a system. Wagner and Whitmire (1957) were the first to demonstrate the apparent functioning of unreduced spores in the origin of an allopoly' Received for publication 26 December 1985; revision accepted 22 April 1986. Research supported by NSF grant BSR 82-06056. 1 thank Christopher J. Scelonge for careful work as laboratory technician, Warren H. Wagner, Jr. for providing plants of Asplenium cristatum, Thomas Darling, Jr. for directions to localities cited, and J. Frank Roessel for permission to collect Asplenium specimens on his property. ploid. They reported that about 25 gametophytes arose from a sowing of thousands of spores from the sterile allodiploid hybrid Asplenium ebenoides, and that fertilization among these gametophytes yielded fertile allotetraploids. This implied that a small percentage of unreduced diploid spores was produced by the sterile hybrid and that these were the source of the observed gametophytes. Morzenti (1962) elucidated the mechanism by which supposedly sterile hybrid ferns produce gametophytes. In addition to producing a vast majority of small, distorted, empty spores, the hybrids also formed up to 16 giant spherical spores per sporangium. In effect, some of the spore mother cells were simply tranisformed into large spores. She demonstrated that gametophytes derived from these large spores could produce active sperm but only non-functional archegonia and noted that although these gametophytes did not produce sporophytes, they might backcross with gametophytes of one of the parental taxa. The proposal that gametes from unreduced spores actually do function significantly in the reproductive biology of ferns can be tested by analyzing the derivation of the hybrid species Asplenium plenum. Two hypotheses for the origin of this species have been proposed. On the basis of morphology and cytology, Wagner (1963) reasonably proposed that A. plenum is the allotetraploid derivative of a hybrid between the sexual diploids A. abscissumn and A. cristatum (Fig. 1, 2). Morzenti (1967) alternatively proposed that the sporogenetic process she elucidated in 1962 might have_ enabled