Umar Imtiaz1,2, Khubaib Shakoor1,2, Xiangshe Wang1, Kede Liu2, Deguan Tan1* and Jiaming Zhang1,2*
1Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Science, China; 2College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
*Corresponding author: zhangjiaming@catasitbb.cn (JZ); tandeguan@catasitbb.cn (DT)
Duckweeds (Lemnaceae) are among the smallest and fastest-growing aquatic flowering plants and are increasingly recognized for their ecological, nutritional, and biotechnological importance. Despite growing global interest, information on duckweed diversity and genetic structure in South Asia remains limited. In this study, we conducted the first integrative assessment of duckweed diversity, distribution, and chloroplast genetic variation across Pakistan using combined morphological and molecular approaches. A total of 142 duckweed field samples were collected from freshwater habitats across Punjab, Sindh, Khyber Pakhtunkhwa, and Baluchistan, yielding 232 axenic strains. From these, 53 representative isolates were selected for molecular analysis based on species representation, geographic coverage, and DNA quality. Morphological identification revealed four dominant species: Lemna aequinoctialis, Spirodela polyrhiza, Wolffia borealis, and Wolffia globosa, with L. aequinoctialis being the most widespread. Genetic diversity was assessed using two chloroplast DNA markers, the rps16 intron and the atpF–atpH intergenic spacer. The rps16 marker exhibited higher polymorphism and greater haplotype resolution than atpF–atpH, particularly within L. aequinoctialis, which showed multiple haplotypes and moderate nucleotide diversity. Regional analyses indicated elevated genetic diversity in southeastern Pakistan, while northwestern populations, composed mainly of S. polyrhiza and W. globosa, exhibited fewer haplotypes but greater nucleotide divergence, suggesting geographic isolation. Phylogenetic analyses confirmed species identities and demonstrated the superior resolving power of rps16 for population-level differentiation. Overall, this study provides foundational insights into duckweed biodiversity and genetic structure in Pakistan and establishes a validated molecular framework for future ecological, evolutionary, and applied research on Lemnaceae in South Asia.