The most important managed pollinator, the honeybee (Apis mellifera L.), has been subject to a growing number of threats. In Western Europe one such threat is large-scale introductions of commercial strains (C-lineage ancestry), which is leading to introgressive hybridization and even the local extinction of native honeybee populations (M-lineage ancestry). Here, we developed reduced assays of highly informative SNPs from 176 whole genomes to estimate C-lineage introgression in the most diverse and evolutionarily complex subspecies in Europe, the Iberian honeybee (Apis mellifera iberiensis). We started by evaluating the effects of sample size and sampling a geographically restricted area on the number of highly informative SNPs. We demonstrated that a bias in the number of fixed SNPs (FST=1) is introduced when the sample size is small (N≤10) and when sampling only captures a small fraction of a population’s genetic diversity. These results underscore the importance of having a representative sample when developing reliable reduced SNP assays for organisms with complex genetic patterns. We used a training dataset to design four independent SNP assays selected from pairwise FST between the Iberian and C-lineage honeybees. The designed assays, which were validated in holdout and simulated hybrid datasets, proved to be highly accurate and can be readily used for monitoring populations not only in the native range of A. m. iberiensis in Iberia but also in the introduced range in the Balearic islands, Macaronesia, and South America, in a time- and cost-effective manner. While our approach used the Iberian honeybee as model system, it has a high value in a wide range of scenarios for the monitoring and conservation of potentially hybridized domestic and wildlife populations.

SNP dataset retrieved from 176 whole-genome sequences (117 A. m. iberiensis, 28 A. m. carnica and 31 A. m. ligustica)The dataset contains a total of 2,366,382 SNPs detected between 117 A. m. iberiensis, 28 A. m. carnica and 31 A. m. ligustica. SNP calling was performed using three different variant calling tools: GATK's, SAMtools’ mpileup 1.1 and PLATYPUS 0.8.1 Details for SNP processing and quality control is provided in the paper and its supplementary information.iber_car_lig.vcf.gz