Ten rules for identifying horizontal gene transfers in deep plant phylogenies.

Horizontal gene transfer (HGT) is one of the most intriguing aspects of genome evolution, challenging classical views of vertical inheritance. Growing genome availability across the tree of life offers opportunities to discover HGT, yet recent reanalyses suggest many reported cases may not withstand scrutiny. Detecting ancient transfers is especially difficult, as phylogenetic signal erodes over time and analytical choices strongly shape conclusions. Here we propose 10 practical rules for evaluating HGT in deep plant phylogenies, organised around three themes: genomic foundation, rigorous analytical framework, and biological interpretation. A reliable genomic foundation requires excluding contamination, verifying genomic integration, and avoiding biased taxon sampling. Rigorous phylogenetics is essential, as deep phylogenies are sensitive to alignment parameters, substitution model, rooting strategy, and branch support. We illustrate this with three glycosyl hydrolase families: for GH9 and GH32, analytical choices can eliminate apparent HGT signals entirely, while GH18 shows why complex, multi-domain families resist confident inference. Finally, HGT signatures demand cautious, biologically plausible interpretation. Recurrent gene duplication, loss, and domain shuffling commonly produce HGT-like patterns and should always be considered as alternatives. Our rules offer a pragmatic checklist helping authors and reviewers distinguish robust HGT from artefacts, fostering more reliable evidence on HGT in plants.