Sampling of the Brandenburg transect was carried out in cooperation with E. Heitlinger of the Humboldt University Berlin. Primary steps of ddPCR optimising were done in collaboration with D. Tautz during the stay of Z.H. at the Max-Planck Institute for Evolutionary Biology (Plön, Germany), with the help of A. Yanchukov and N. Thomsen. We also acknowledge CF Genomics CEITEC, Masaryk University (Brno, Czech Republic), for their support while generating all individual CN estimates presented in this paper. In total, we analysed 252 male mice live-trapped in two distinct transect regions across the HMHZ, the first one stretching from north-eastern Bavaria (Germany) to western Bohemia (Czech Republic) where Eastern Y chromosomes introgress across the zone deep into the Western territory (hereafter ‘Invasion transect’; N = 180) and the other situated in north-eastern Brandenburg (Germany) with no or negligible introgression (hereafter ‘Control transect’; N = 72) (Figure 1). Most of the captured males were dissected immediately in a field laboratory, whereas others were dissected in External Research Facility Studenec, IVB. One testis and the spleen were either placed in ethanol (when only CNV was being estimated) or snap-frozen in liquid nitrogen immediately after dissection and then stored at – 80 °C until use (when both CNV and expression were measured). Total DNA and RNA were extracted using AllPrep DNA/RNA Mini Kit (Qiagen GmbH, Hilden, Germany). DNeasy® 96 Tissue Kit (Qiagen) and Invisorb® Spin DNA Extraction Kit (Stratec Biomedical AG, Birkenfeld, Germany) were used for DNA extraction from ethanol-preserved samples (CNV analysis only). All the procedures followed the manufacturers’ instructions.

A remarkable gene copy number (CN) arms race system has recently been described in laboratory mice, where Slx;Slxl1 and Sly genes compete over transmission by altering fertilisation success of X and Y chromosome-bearing sperm, respectively. Here, we focus on this system in nature, where natural selection can counter CN/gene product escalation. Our model is house mouse subspecies hybridising in Europe. In some regions, Y chromosomes of the Eastern subspecies have introgressed onto Western genomic backgrounds, accompanied by sex ratio distortion in favour of males, consistent with the inbred-lines suggested mechanism: overabundance of SLY protein expressed by invading Y chromosomes. We take Slx as representative of the X side of this arms race and measure Slx|Sly CN and expression across an ‘Invasion’ transect where Ys introgress and a ‘Control’ transect with negligible introgression. Since we found similar Slx|Sly ratios in both transects, SLY overabundance is unlikely to explain the introgression. However, Slx CN is relatively low in the introgression area, suggesting Slx is less able to combat Sly effects here. Further, deterministic changes in Slx;Sly expression proportions versus CN proportions suggest standing variation for trans regulation of Slx|Sly is being co-opted in nature where their arms race reduces population fitness.