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The genetic variation within and among seven Tunisian natural Hypericum humifusum L. populations belonging to three bioclimatic zones (sub-humid, upper semi-arid, and lower semi-arid) was assessed using random amplified polymorphic DNA markers. Eight selected primers produced a total of 166 bands, of which 153 were polymorphic. The genetic diversity within a population, based on Shannon’s index and percentage of polymorphic loci, was relatively high. The level of variation among populations did not differ significantly. However, the variation among populations grouped according to their bioclimates was significant.

A high differentiation and a low gene flow were observed at all spatial scales among all populations. The upper semiarid populations exhibited the highest differentiation. The relationship between genetic and geographic distances was not significant indicating that structuring occurred due to founding events. The UPGMA analysis based on Nei & Li’s coefficients showed that individuals from each population clustered together. The cluster analysis based on genetic distances among populations did not show clear groupings relevant to geographical distances or bioclimates.

The high differentiation among populations even through a small geographic range implies the collection of seeds from all populations to preserve, ex-situ, extant variation in the species. Populations from the upper semi-arid zone showing the highest genetic diversity should be first prospected.