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The species of the genus Calligonum L. (Polygonaceae) that appear in Tunisian deserts play an important role in maintaining local ecosystems and supply important natural resources in these regions. In this study, 31 individuals belonging to the 3 hypothetical species of the genus Calligonum (Calligonum arich Le Houer., Calligonum azel Maire, and Calligonum comosum L’Her.) were collected from 12 populations in 6 localities of the Tunisian desert and examined to assess the relationship between species. Phylogenetic analyses using 1 nuclear (ITS) and 2 plastid regions (trnL-trnF and rbcL) and genome size assessments are used in this study to evaluate the relationships between these hypothetical species. C-value results suggest the existence of 3 different species, which is also supported by phylogenetic tree topology. The paraphyletic pattern of C. comosum and the plausible origin of C. arich from C. azel suggest the existence of several mechanisms of isolation and speciation in the Sahara Desert for this genus. Additional studies are necessary to evaluate the population size, demographic tendency, and conservation status of these desert species and their genetic relationships with other congeners.