Phylogenetic relationships in the genus Nicotianawere investigated using parsimony analyses of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (nrDNA) and compared to previously published results from the plastid matKgene. In addition, origins of amphidiploid taxa in Nicotianawere investigated using the techniques of genomic in situ hybridization (GISH). All taxa regardless of ploidy produced clean single copies of the ITS region, even though some taxa are natural hybrids and others are artificial hybrids (allopolyploids). The patterns of relationships in Nicotianaare largely congruent with previous evolutionary ideas of Goodspeed, who studied their morphology and cytology, but some important differences are apparent. None of the subgenera of Nicotianais monophyletic, and although most of the currently recognized sections are coherent, others are clearly polyphyletic. Relying solely upon ITS nrDNA and matKanalysis to reveal phylogenetic patterns in a complex genus such as Nicotianais insufficient, and it is clear that conventional analysis of single data sets, such as ITS, is likely to be misleading in at least some respects about evolutionary history. ITS sequences of artificial as well as natural and well-documented amphidiploids are similar to one of their two parents, usually the maternal (but not always) and not in any sense themselves "hybrid". Knowing how ITS evolves in artificial amphidiploids gives insight into what the ITS analysis might reveal about naturally occurring amphidiploids for which little of their origin is known, and it is in this perspective that analysis of ITS sequences is highly informative.

Key words: amphidiploids, GISH, hybridization, ITS, matK, Nicotiana