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Premise of the Study Microsatellite markers were developed for Polypodium appalachianum (Polypodiaceae) to facilitate investigation of species boundaries between P. appalachianum and its putative hybrid, P. virginianum, and potentially among other members of the Miocene‐age P. vulgare species complex. Methods and Results Forty‐eight primer pairs were designed from Illumina data and screened for successful amplification. Sixteen pairs were genotyped and evaluated for variability within and among three populations in North Carolina, Vermont, and New Hampshire. Twelve of these primer pairs were reliable and polymorphic, exhibiting one to 10 alleles per locus. Cross‐species amplification experiments were conducted for P. virginianum and four additional close relatives from the P. vulgare complex in order to maximize information about likely utility within a phylogenetic context. Conclusions These microsatellite markers will be useful in population genetics and species boundaries studies of P. appalachianum and P. virginianum, and likely in other species within the P. vulgare complex.

The typically lithophilic Polypodium appalachianum was discovered as a canopy epiphyte 35 to 40 m above ground on a horizontal branch of a champion-size Liriodendron tulipifera in the Great Smoky Mountains National Park. Occurring along with this first documentation of P. appalachianum from the tree canopy was an assemblage of normally terrestrial mosses, an unusual assortment of collembola (springtails), and a flightless proturan insect species previously known only from soil and litter. The distinctive features of this canopy habitat may duplicate some ecological conditions usually found only at ground level, establishing the opportunity for translocating an entire community and providing biologists with new insights on the origin of some epiphytes.