Abstract
Horizontal gene transfer is a fundamental driver of prokaryotic evolution, facilitating the acquisition of novel traits and adaptation to new environments. Despite its importance, methods for inferring horizontal gene transfer are rarely systematically compared, leaving a gap in our understanding of their relative strengths and limitations. Validating horizontal gene transfer inference methods is challenging due to the absence of a genomic fossil record that could confirm historical transfer events. Without an empirical gold standard, new inference methods are typically validated using simulated data; however, these simulations may not accurately capture biological complexity and often embed the same assumptions used in the inference methods themselves. Here, we leverage the tendency of horizontal gene transfer events to involve multiple neighboring genes to assess the accuracy of diverse horizontal gene transfer inference methods. We show that methods analyzing gene family presence/absence patterns across species trees consistently outperform approaches based on gene tree-species tree reconciliation. Our findings challenge the prevailing assumption that explicit phylogenetic reconciliation methods are superior to simpler implicit methods. By providing a comprehensive benchmark, we offer practical recommendations for selecting appropriate methods and indicate avenues for future methodological advancements.