Amplified fragment length polymorphism (AFLP) data were generated for most of the; similar to 12 species and subspecies of endemic New Zealand Plantago (Plantaginaceae) to test species boundaries and interpret polyploid origins. Phylogenetic, network, principal-coordinates, structure, and quantitative analyses of the AFLP data were generally congruent and complementary regarding the main lineages, and the incongruences allowed some inferences of alloploidy and hybridization. Within the largely diploid group III, P. novae-zelandiae, P. lanigera, P. obconica, and P. aucklandica were genetically distinct. Within group II, there was little genetic differentiation between octoploids P. masoniae and P. triandra, and multiple alloploid origins were inferred for decaploid and dodecaploid cytotypes of P. unibracteata. Within group I, the 16-ploid P. sp. "Sylvester" is suggested to be an autoploid of octoploid P. raoulii, octoploid P. picta is clearly a genetically distinct entity, and species boundaries of octoploids P. raoulii, P. spathulata, and P. aff. spathulata are unclear. Additional genetic and molecular cytogenetic studies on New Zealand species and their close relatives from Australia and southern South America are needed to further investigate polyploid origins. AFLP is a useful tool for elucidating the origins and evolutionary history of closely related polyploid species in taxonomically challenging groups such as endemic New Zealand Plantago.
