mtDNA Analysis of Ancient Croatian Skeletal Remains

This project is a collaboration between the Forensic Science program here at Penn State, and the University Center for Forensic Science at the University of Split in Croatia.

Skeletal remains were recovered by scientists from the University of Spilt from a Late Middle Ages gravesite in Šopot-Benkovac, Croatia, dating back to somewhere between the 13th to the 16th centuries, with potential roots that extend back to the Early Middle Ages.

Map of Croatia

An exploratory excavation of this site occurred in 1929.  No remains were removed at that time.  However, a stone fragment of Duke Branimir was discovered, referring to him as the Duke and Lord of Croats.  Branimir was the ruler of Dalmatian Croatia from 879 to 892, and was recognized by Pope John VIII as the Duke of Croats.  During Branimir’s reign, Croatia maintained its independence from both Frankish and Byzantine rule.  As a result, when the system of honors was instituted in the Republic of Croatia after its independence in 1991, the Order of Duke Branimir was established as one of the highest state honors for merit in establishing, maintaining and promoting an independent and sovereign Republic of Croatia.

Archaeological excavations in 1985 and 1988 resulted in the recovery of skeletal remains associated with approximately 75 individual graves.  Pagan burial rituals were discovered in an area of the gravesite where most of the individuals were buried according to Christian traditions, suggesting that certain individuals within the Šopot-Benkovac site were associated with transient population groups.  Croatia has a rich history, including recent scientific data that suggests more than three quarters of contemporary Croatian men are descendants of Old Europeans who arrived before and after the Last Glacial Maximum, more than 20,000 years ago ( Primorac et al, CMJ 2011).  The remaining portions of the male population are linked to descendants of peoples arriving in this part of Europe through a southeastern route in the last 10,000 years, mostly during the neolithization process.  Therefore, Croatia is seen as having an increasingly important role as a gateway of population migration into Europe.

The goals of our work on the skeletal samples from the Šopot-Benkovac gravesite include an assessment of the ancestral origin of the remains, with the hopes of better understanding how they fit into the history of population migration through the central coastal region of Croatia.  Mitochondrial DNA sequence of the skeletal remains is being generated in our laboratory using Sanger and deep sequencing approaches (454 LifeSciences GS Junior instrument).  An example of our work in this area on pristine samples has been published (Holland et al CMJ 2011), and we have shown in the laboratory that results can be obtained from challenging sample types; e.g., hair shafts.  Examples of multiple individuals buried within the same grave were also documented at the Šopot-Benkovac gravesite.  Therefore, potential kinships will be assessed.  Finally, we hope to tap into low-level mtDNA heteroplasmy present in the skeletal samples and assess how this information can be used to increase the discrimination potential of mtDNA analysis (The mtDNA heteroplasmy research page).