FIDDL: depth-matched negative controls distinguish genuine interspecific introgression from competitive-mapping artifact

Interspecific introgression is routinely detected by competitively mapping reads to a concatenated multi-species reference and calling regions where a non-focal species recruits coverage. Using strains that cannot contain the ancestry being detected, we show this design generates substantial false-positive signal through two mechanisms with opposite phylogenetic-distance signatures. Standard nuclear assemblies omit the mitochondrion and 2-micron plasmid, leaving high-copy cytoplasmic reads without a legitimate target; completing the reference preferentially removes signal from the most divergent donor. Genuine cross-species sequence conservation inflates the most closely related donor. Masking chromosome ends removes its subtelomeric part but plateaus at a non-zero floor, and the interior residual traces to conserved single-copy genes where a short read carries under one base of discriminating information. The floor grows with sequencing depth (1.19% of callable positions at 50×, 2.02% at 147×, 3.85% at 393× in a pure strain), is not mitigated by long reads, and appears at sub-diploid dosage – three properties widely read as evidence of authenticity. Because the discriminating information is below single-read resolution, no read-level filter separates artifact from introgression; we show three that fail. What works is locus-level: a consensus-phylogenetic test (29/29 specificity on confirmed artifact) and an allele-fraction donor-match test, complementary and validated in both directions on independent published introgression. We package the comparative controls as FIDDL ( False Introgression Detection via Depth-matched controls and Loci-recurrence ), an open-source tool, withdraw two of our own analysis-ready calls, and show re-analysis of published wild isolates reduces low-confidence introgression by ∼53% while leaving high-confidence signal intact.