Searching for Molecular Markers to Differentiate Bombus terrestris (Linnaeus) Subspecies in the Iberian Peninsula

Bumblebees (genus Bombus Latreille) are pollinator insects of great ecological and economic importance, which commercial use for pollination has increased since the 80s. However, the introduction of foreign Bombus terrestris (Linnaeus) has resulted in a decline of native bumblebee populations in Japan, Chile and Argentina among others. To study the potential introgression of commercial B. terrestris into the Iberian endemic subspecies Bombus terrestris lusitanicus Kruger it is necessary to find a precise molecular marker that differentiates both subspecies. For this purpose, comparative analyses were carried out between B. t. lusitanicus and Bombus terrestris terrestris (Linnaeus) from Spain and from Belgium by sequencing the nuclear genes elongation factor 1-alpha and arginine kinase and the mitochondrial gene 16S ribosomal RNA, and genotyping with eleven microsatellite loci. No differentiation was observed at the nuclear level, but haplotypes found within the 16S sequence correlated with the morphological characterization of the subspecies. In a case study including individuals sampled before the establishment of bumblebee rearing companies and others from recent samplings, we detected hybrid individuals (those with non-matching morphological subspecies and 16S haplotype) more frequently in the south supporting the naturalization of commercial B. t. terrestris and introgression events between both subspecies. This marker should be used in Iberian populations with the aim to support management and conservation actions in endemic populations of B. t. lusitanicus.