In 1978, populations of Isoëtes were discovered in the Little Tennessee and Hiwassee Rivers of southeastern Tennessee. These populations were reported as disjunct populations of I. macrospora Durieu, a decaploid species (2n=110) of boreal eastern North America. Examination of Isoëtes plants recently collected from the Hiwassee River reveals that these specimens are an undescribed species. Chromosome number and megaspore size and texture distinguish the Hiwassee River plants from I. macrospora. Chromosome counts from root tip squashes of Hiwassee specimens are 2n=88, the first octoploid quillwort reported for North America. Megaspores of Hiwassee River plants are large, ranging in size from 615-950 µm in diameter with an average diameter size of 760 µm. They have a bold rugate-reticulate texture and lack a distinctly ornamented girdle beneath the equatorial ridge. In contrast, megaspores of I. macrospora are smaller, ranging in size from 550-750 µm in diameter with an average of 650 µm. They have a thin cristate-reticulate texture and a prominent papillate to smooth girdle. Comparisons of nuclear ribosomal ITS nucleotide sequences indicate that the Hiwassee River specimens are similar to I. engelmannii A. Braun and I. valida (Engelm.) Clute. In the ITS region, I. engelmannii and I. valida differ from each other by eleven nucleotide substitutions and two 1 bp indels. At seven of these ITS nucleotide sites, the Hiwassee River specimens are like I. engelmannii and at the other four sites they are like I. valida. The Hiwassee River plants share one indel with I. engelmannii and the other indel with I. valida. Isoëtes engelmanii and I. valida, both basic diploids (2n=22), and their allotetraploid, I. appalachiana D.F. Brunton & D.M. Britton (2n=44), are sympatric within the area of the Hiwassee River octoploid.

Key words: Isoetaceae, Isoetes, octoploid, Tennessee