Development of microsatellite markers for the polyploid <i>Damnacanthus indicus–D. major</i> species complex (Rubiaceae)

<i>Damnacanthus indicus</i> C. F. Gaertn. and <i>D. major</i> Siebold & Zucc. are taxonomically complex species with extensive morphological variation and polyploidy. To provide molecular tools for future taxonomic and population genetic studies of the polyploid <i>D. indicus–D. major</i> species complex, we assessed ploidy levels based on nuclear DNA content using flow cytometry and developed 25 polymorphic microsatellite markers. Flow cytometric analysis showed an approximately twofold difference in nuclear DNA content between diploid and tetraploid individuals, consistent with their reported chromosome numbers. Genotyping of 60 individuals representing diploid <i>D. indicus</i>, tetraploid <i>D. indicus</i>, and tetraploid <i>D. major</i> revealed substantial polymorphism, with expected (<i>H</i>_E) and observed (<i>H</i>_O) heterozygosity ranging from 0–0.812 and 0–0.917, 0.348–0.815 and 0.250–0.962, and 0–0.757 and 0–0.987, respectively. The developed markers will serve as valuable tools for future taxonomic reexaminations and for investigating the evolutionary processes that have shaped the <i>D. indicus–D. major</i> species complex.