Resolving Phylogenetic Conflicts in Pandanales: The Dual Roles of Gene Flow and Whole-Genome Duplication

Tian Shi Jian He ^*

• Beijing Forestry University, Beijing, China

Accurate phylogenetic reconstruction is essential for understanding evolutionary relationships and biodiversity. Despite advances in molecular systematics, the relationships within Pandanales-encompassing Cyclanthaceae, Pandanaceae, Stemonaceae, Triuridaceae, and Velloziaceae-remain unresolved. Here, we analyzed transcriptomic and genomic data from 20 samples covering all five families, assembling 2,668 single-copy orthologous genes (SCOGs) and performing phylogenetic analyses using both coalescent-and concatenation-based methods, as well as plastid genome data. Although all three phylogenies were strongly supported, they exhibited significant topological incongruence. Based on our gene flow analysis using HyDe, we propose that the concatenation-based topology likely reflects the actual evolutionary history of Pandanales. Further investigation uncovered two notable ancient gene flow events-one between Velloziaceae and Triuridaceae, and another between Triuridaceae and the C-P clade (Cyclanthaceae + Pandanaceae)while coalescent simulations and QuIBL analyses indicated that gene flow, rather than incomplete lineage sorting, is the primary source of conflict at certain nodes. We also detected five whole-genome duplication (WGD) events in Pandanales, including two newly recognized here: two situated near the Cretaceous-Paleogene boundary (~66 Ma) on the stems of Stemonaceae and Pandanaceae, one on the stems of Triuridaceae during the mid-Paleogene, and two within Velloziaceae near the Paleogene-Neogene boundary (~24-26 Ma), potentially facilitating adaptation and diversification under changing environmental conditions. Together, our results highlight the dual roles of WGD and gene flow in shaping Pandanales evolution, resolve long-standing phylogenetic conflicts, and offer insights into the mechanisms driving plant diversification.