Mitochondrial haplogrouping of the ancient brown bears (Ursus arctos) in Bulgaria, revealed by the APLP method

In order to investigate the detailed zoogeographical history of brown bears (Ursus arctos) in Bulgaria and their relationships with populations in neighboring regions of Europe and Asia, the amplified product length polymorphism (APLP) method for the mitochondrial DNA (mtDNA) haplogrouping was applied to ancient bone remains. The results showed that haplogroups for 12 of 31 samples (38.7%) were successfully classified using the APLP method, although partial sequences of the mtDNA control region were determined with PCR product-direct sequencing for only 6 samples (19.4%). Even among 25 samples, of which nucleotide sequences could not be determined, the APLP method successfully classified haplogroups of 6 samples (6/25, 24.0%), indicating an advantage of the APLP method. In Bulgaria, although both the Balkan/Italian lineage of mtDNA (clade 1b) and the Eastern European lineage (clade 3a1) have been identified from modern bears as reported in other studies, all bear remains examined in the present study had APLP haplogroup W. The mtDNA phylogenetic analysis showed that the ancient Bulgarian brown bears had clade 1b. This indicates that clade 1b was originally distributed in Bulgaria, whereas those with clade 3a1 could have entered from Romania to Bulgaria. In addition, the APLP and phylogenetic analysis of recent skin samples from Turkey showed that they have mtDNA of the Middle Eastern/Turkish lineage (referred to clade 7a). Therefore, Bulgaria on the Balkan Peninsula might have been located between two distribution borders: one is between clades 1b and 3a1, and the other is between clades 1b and 7.