Hexastylis contracta and H. rhombiformis are imperiled herbaceous perennial plants endemic to southeastern North America. Hexastylis contracta exhibits a disjunct distribution, being found on the Cumberland Plateau of Tennessee and Kentucky and also 300 km east in the Blue Ridge Mountains of North Carolina. The distribution of H. rhombiformis is generally sympatric with H. contracta in North Carolina, with allopatric populations to the north and south in the southern Appalachian Mountains. This study involved an examination of morphology, micromorphology, Internal Transcribed Spacer (ITS) region of nuclear rDNA, and Randomly Amplified Polymorphic DNA (RAPDs) comparing H. contracta and H. rhombiformis. A morphological analysis using PCA revealed that H. rhombiformis is distinct from Hexastylis contracta. Examination of the internal calyx trichomes and reticulations using Scanning Electron Microscopy (SEM) showed that H. rhombiformis has high raised reticulations and trichomes with four to five basal cells, whereas H. contracta has very small inner calyx reticulations and trichomes with single basal cells. The ITS results indicated that H. contracta (sensu stricto) is generally monomorphic for the ITS region, with no variation between the two disjunct distributions, suggesting a fairly recent geographic separation of the species. Hexastylis rhombiformis exhibited variability in the ITS region, with two individuals similar to H. contracta, two individuals similar to H. arifolia var. ruthii, while four individuals possessed unique sequence patterns. The RAPD data showed differences between the two species, and also supported the hybrid designation for some individuals. These results corroborate the morphological analysis suggesting that some specimens identified as H. rhombiformis may be of hybrid origins. The combined data from morphological, micromorphological, and RAPD analyses support the separation of H. contracta and H. rhombiformis, and strongly suggest that H. rhombiformis should be recognized as a segregate species.

Key words: Hexastylis contracta, Hexastylis rhombiformis, ITS, morphology, RAPDs, species boundaries