The genus Nymphaea L. (water lily) is the most diverse genus in the family Nymphaeaceae, with more than 50 species worldwide, including 11 species distributed in Africa. The complex and variable morphology of Nymphaea makes it extremely difficult to accurately identify species based on morphological characteristics alone. DNA barcoding has the potential to identify species accurately. In this study, 158 Nymphaea populations from seven African countries were collected for species identification by ITS, trnT-trnF and rpl16. Additionally, the three candidate DNA barcodes were evaluated for genetic distance and barcoding gap. Based on the comprehensive analysis of sequence similarity, genetic distance method and phylogenetic tree, a total of 137 populations of seven Nymphaea species from African were well-identified, including N. lotus, N. petersiana, N. zenkeri, N. nouchali var. caerulea, N. micrantha and N. guineensis. ITS has more obvious advantages over trnT-trnF, rpl16 and trnT-trnF+rpl16 in the intraspecific and interspecific variation differences and barcoding gap and can identify most species. trnT-trnF and rpl16 can identify some species that cannot be identified by ITS. The results showed that it is more appropriate to apply the combination of ITS and trnT-trnF (or rpl16) as the DNA barcoding of Nymphaea. Additionally, this study further enriches the DNA barcoding database of Nymphaea and provides a reference basis for studying taxonomy, phylogenetics and evolutionary origin of Nymphaea.

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