In this study, seven populations of A. flaccida were collected throughout their distribution in the grassy ecosystems of the Atlantic Forest biome in southern Brazil and were named according to the county of origin (Table 1). For all populations, vouchers were made and identified using the key for the genus Achyrocline (Less.) DC. in Brazil proposed by Deble . The number of plants sampled from each population and the characterization of the sampling sites according to original  and actual ecosystem  are shown in Table 1. From each population, leaves of plants that were spaced at least 20 m from one another were collected. The collected leaves were stored in silica gel until DNA extraction. DNA extraction was performed following the protocol of Doyle and Doyle . The concentration and quality of each DNA sample was checked on a 0.8% agarose gel using standard concentrations of the λ phage DNA (50, 100, and 200 ng).