Despite apparent ethological isolation based on specialized pollination systems, hybridization between day-flowering Zaluzianskya microsiphon and night-flowering Z. natalensis has been proposed due to intermediate individuals found in sympatric populations of these species. The extent of this putative hybridization was investigated using inter-simple sequence repeat (ISSR) markers and principal components analysis (PCA) of morphological traits. The species are genetically similar, but show some intra- and interspecific variation in band frequencies. Neighbor-joining analyses of the ISSR data demonstrated that although the species largely formed distinct groups, several individuals from the sympatric populations of each species and the "hybrids" clustered together rather than with members of their own species. These results are consistent with hybridization, although they could also indicate historical similarity. Nine loci were present only in individuals of Z. microsiphon, the "hybrids," and sometimes the sympatric individuals of Z natalensis. In contrast, only one locus showed the reverse pattern. This suggests unidirectional gene flow from Z microsiphon to Z. natalensis, which is also supported by population-level examinations of four loci. Ordination revealed separate phenotype clusters for each species, with hybrid individuals located in between but often closer to the Z natalensis cluster. One hypothesis is that hybrids are backcrossing with Z natalensis, leading to introgression of Z. microsiphon genetic material.
