Ancient mitochondrial DNA extraction from Bison bison long bones from Head-Smashed-In Buffalo Jump, UNESCO World Heritage site

• Reliable, cost-effective method for ancient DNA research on archaeological samples. • A shared haplotype, bHapB, persisted from ancient to modern bison. • Novel haplotypes in ancient HSIBJ bison are absent in contemporary populations. • Bison experienced a loss of genetic diversity due to the 19th-century bottleneck. Head-Smashed-In Buffalo Jump (HSIBJ) is a UNESCO World Heritage Site located on the southern end of the Porcupine Hills, near Fort Macleod, Alberta, Canada. Using a system of drive lanes, Indigenous groups drove herds of bison over the cliff edge for thousands of years. The well-stratified deposits at the base of the cliff offer a unique opportunity to investigate the genetic diversity of the American bison before European contact and their genetic bottleneck in the 19th century. We extracted ancient DNA from twenty-one bison long bones, amplified and sequenced the mitochondrial DNA control region. Comparisons between ancient and modern bison populations revealed novel haplotypes in the HSIBJ population, suggesting a loss of genetic diversity due to the bottleneck. Furthermore, we discovered a shared haplotype between the bison hunted at the site and modern populations, which may help elucidate the complex history of living herds.