Collinear genes and karyotype evolution resolve the phylogenetic uncertainty of biebersteiniaceae within sapindales

The phylogenetic placement of the herbaceous family Biebersteiniaceae, a monotypic lineage with an exceptionally low chromosome number (2n = 10), has remained contentious within the order Sapindales, particularly regarding the relationships among Biebersteiniaceae, Sapindaceae, and Nitrariaceae. To address this, we generated a chromosome-level genome assembly for Biebersteinia heterostemon. Phylogenetic analyses based on single-copy nuclear genes and plastome sequences yielded strongly conflicting topologies. In contrast, a species tree reconstructed from collinear genes supported a third, well-defined topology: Biebersteiniaceae is sister to a subclade comprising Anacardiaceae, Meliaceae, and Rutaceae that further is sister to the subclade comprising only Sapindaceae. We further reconstructed the ancestral karyotype of Sapindales and traced karyotype evolution across the order. Reconstruction of ancestral karyotypes revealed that Anacardiaceae, Meliaceae, and Rutaceae share a chromosomal fusion supporting their monophyly. Biebersteiniaceae shares an additional fusion with this subclade, supporting its sister relationship. Furthermore, Biebersteiniaceae, Anacardiaceae, Meliaceae, Rutaceae, and Sapindaceae share another fusion that is absent from Nitrariaceae, suggesting that Nitrariaceae represents the earliest-diverging lineage among these families. Our study demonstrates that integrated phylogenomics based on collinear genes and karyotype evolution can resolve long-standing deep phylogenetic conflicts where traditional analyses fail, providing a robust framework for understanding trait evolution.