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The recognition, identification, and differentiation of closely related plant species present significant and notorious challenges to taxonomists. The  Maddenia  group of  Prunus , which comprises four to seven species, is an example of a group in which species delimitation and phylogenetic reconstruction have been difficult, due to the lack of clear morphological distinctions, limited sampling, and low informativeness of molecular evidence. Thus, the precise number of species in the group and the relationships among them remain unclear. Here, we used genome skimming to generate the DNA sequence data for 22 samples, including 17  Maddenia  individuals and five outgroups in Amygdaloideae of Rosaceae, from which we assembled the plastome and 446 single-copy nuclear (SCN) genes for each sample. The phylogenetic relationships of the  Maddenia  group were then reconstructed using both concatenated and coalescent-based methods. We also identified eight highly variable regions and detected simple sequence repeats (SSRs) and repeat sequences in the  Maddenia  species plastomes. The phylogenetic analysis based on the complete plastomes strongly supported three main subclades in the  Maddenia  group of  Prunus , while five subclades were recognized based on the nuclear tree. The phylogenetic network analysis detected six hybridization events. Integrating the nuclear and morphological evidence, we proposed to recognize five species within the  Maddenia  group, i.e.,  Prunus fujianensis, P. himalayana, P. gongshanensis, P. hypoleuca , and  P. hypoxantha . Within this group, the first three species are well-supported, while the gene flow occurring throughout the  Maddenia  group seems to be especially frequent between  P. hypoleuca  and  P. hypoxantha , eroding the barrier between them. The phylogenetic trees based on eight concatenated hypervariable regions had a similar topology with the complete plastomes, showing their potential as molecular markers and effective barcodes for further phylogeographic studies on  Maddenia .

On the Species Delimitation of the Maddenia Group of Prunus (Rosaceae): Evidence From Plastome and Nuclear Sequences and Morphology