Matched genome-background controls refine phylogenetically supported horizontal gene transfer candidates across Actinomycetota

Horizontal gene transfer (HGT) is a major source of bacterial genome innovation, yet genome-scale detection remains vulnerable to taxonomic sampling, nomenclatural instability, and close-lineage sequence similarity. Here, a staged similarity, phylogenetic, genomic-context, and matched-background framework was applied to 1,143 Actinomycetota proteomes. A DIAMOND reference database containing 5,486,888 proteins was used to prioritize cross-genus similarity anomalies, followed by maximum-likelihood phylogenies for an enriched validation panel. Of 264 tree-ready proteins, 77 formed strongly supported donor-associated topologies. Genomic inspection located all 77 proteins in their expected source assemblies and grouped them into 72 operational loci; 46 proteins initially showed coherent donor-like neighborhoods. However, comparison against 12,499 control proteins sampled away from candidate loci across the same source genomes revealed extensive genome-wide donor affinity. Thirty-five of the 77 strong phylogenetic candidates were classified as background-donor-dominated, whereas only 13 proteins remained locally donor-enriched relative to matched genomic background. These 13 proteins collapsed into 10 operational candidate events, including especially strong multi-protein signals in Calidifontibacter-Naumannella and Gordonibacter-Eggerthella comparisons. Retrospective screening-feature analysis further showed that donor-enrichment patterns strongly predicted strong trees, whereas final localization was associated with larger outside-versus-same-genus score contrasts and lower source-donor pair burden. Several additional candidates carried plasmid, short-contig, contig-end, mobile-element, or nomenclatural cautions. The results show that phylogenetic support and donor-like gene neighborhoods can still overstate localized HGT when the recipient genome as a whole has unusual affinity to the proposed donor group. Matched genome-background controls provide a practical additional safeguard for HGT inference in densely sampled and taxonomically unstable bacterial clades.