Horizontal gene transfer (HGT) is the transfer of genetic material between organisms that are not parent and offspring, and may even be from different species. Unlike traits passed from parent to offspring, HGT lets organisms gain new traits through contact or proximity with another individual. Many examples exist in nature, but the role of HGT in eukaryotes is still debated. In many cases, there is clear evidence that HGT has occurred, but few known mechanisms to explain how or why it happens. This project builds on a recent discovery of a clear HGT mechanism in fungi, a major group of eukaryotes, to answer key questions about how this process works. The work will improve understanding of fungi that affect human, plant, and environmental health. It may also support new advances in evolution and biotechnology, since gene transfer between eukaryotic species could help solve problems in plant breeding, medicine, and drug discovery. In addition, this project will help build the workforce by training early-career researchers, supporting team-based student learning, and expanding mentoring programs. Understanding how species exchange genetic information through HGT is critical for understanding the genetic bases of adaptation. Yet despite its importance for generating adaptive variation, HGT in eukaryotes has been near-impossible to study because the field lacks a system for hypothesis-driven experimentation. This project addresses this knowledge gap by capitalizing on the PI’s