An integrative identification approach reveals that subspecies hybridization in Bombus terrestris predates commercial use and varies with greenhouse density

Laboratory assessment of environmental stressors on wild bumblebees typically rely on workers from laboratory colonies founded by wild-collected queens. In Spain, where Bombus terrestris lusitanicus and Bombus terrestris terrestris coexist, accurately identifying the subspecies of wild individuals used in these experiments remains challenging. Since the 1990s, commercial colonies of B. t. terrestris have been widely used in Spain for greenhouse pollination, and previous studies have documented the escape and naturalisation of individuals in areas where B. t. lusitanicus is native. The present study integrates three methodologies-morphological traits, mitochondrial 16S rRNA gene, and microsatellites-to achieve accurate colony-level identification, enabling the differentiation of the two subspecies and their hybrids. Laboratory colonies were established with wild queens from Almería (southeastern Spain; high greenhouse density) and Madrid (central Spain; minimal greenhouse infrastructure), allowing the analysis of both queens and offspring. This approach allowed to determine colony subspecies composition for studies involving the use of wild individuals. Overall, our findings show widespread hybridization between both subspecies, with higher levels observed in areas of high greenhouse density, which may be associated with commercial use. Furthermore, the inclusion of commercial and historical specimens showed that introgression among wild bumblebees predated commercial colony use, though hybridization is currently more pronounced.