Hybridization between different species is an accident of nature that may in some instances have a very important outcome: the origin of new species. Hybrids are typically unable to reproduce through ordinary sexual means because they contain incompatible chromosomes that do not pair during meiosis, the reduction division that produces the haploid cells that ultimately produce eggs and sperms. However, a second accident, chromosome doubling (polyploidy) may restore fertility by providing each chromosome with an exact duplicate with which it can pair. These two processes, hybridization and polyploidy, have combined in this way to produce a vast number of new plant species, termed allopolyploids. Allopolyploids include some of our most important crops, among them wheat, cotton, coffee, peanut, and tobacco. Nonetheless, surprisingly little is known about the precise cellular mechanisms that operate to produce allopolyploids, or about the circumstances that prevail in nature when successful allopolyploids become established. The present proposal is designed to bolster our knowledge of the origin of allopolyploids through detailed studies of natural hybrids between species of Dryopteris, a genus of ferns common in eastern North America. Dryopteris is an ideal model system for the following reasons: (1) as a fern, the products of meiosis in Dryopteris are spores that can be grown directly into plants rather than the pollen grains or embryo sacs of flowering plants which are much less amenable to further study; (2) Dryopteris plants are large and produce numerous spores, offering an enhanced opportunity to observe relatively rare events; (3) numerous hybrids are produce in nature, some of them very frequently, allowing for the variation between processes that pertain to individual kinds of hybrids to be evaluated. This study will consist of two phases, a laboratory phase and a field phase. In the laboratory, the production of spores with doubled chromosomes will be followed. Such spores are believed to be distinguishable as large spherical ("giant") spores that stand out from the remainder of the shriveled spores ordinarily produced by the hybrid. The genetic constitution and growth potential of these "giant" spores will be evaluated by growing them into juvenile ferns (gametophytes) and examining these using molecular and microscopic techniques. Attempts will also be made to produce adult ferns (sporophytes) from the gametophytes by both sexual and asexual means. In the field, clusters of hybrids will be studied to determine whether they are actually reproducing through polyploid spores, i.e. whether the hybrids are all first generation, or include some later generations. Molecular fingerprinting techniques will be used that can distinguish between these two possibilities. The results from these combined field and laboratory studies will provide important new information on the means by which hybrids can give rise to allopolyploid species.
