A DNA analysis of a 6,200-year-old massacre reveals that this ancient act of organized violence was an instance of indiscriminate killing, according to research published Wednesday that conducted the largest genetic analysis of an ancient massacre to date.

Potočani, Croatia, is home to the Eneolithic, or Copper Age, mass burial site discovered in 2007. Approximately two meters in diameter and one meter deep, the mass burial site contains the skeletal remains of 41 individuals of various ages.

Studies of other ancient massacres have found instances where the killing was the result of a battle's aftermath, the killing of a family or kinship group or killing because of age or sex. But none of those indicators are present in the genetic analysis conducted in this study, published March 10 in PLOS One.

The study defines a massacre as an intentional murder conducted by a group upon people who were not prepared for battle. Massacres can be directed at subsets of the population, such as an age group or specific sex. What makes a killing indiscriminate, the researchers said, is that it is not targeted at any particular group of individuals.

The findings of this study are significant, the researchers said, as they reveal that organized violence in the form of indiscriminate killing is not just a "feature of historic and modern times" but also a feature of prehistoric times.

Radiocarbon dates from the skeletal remains, in connection with an analysis of several pottery fragments found in the burial, indicate that the massacred people were of the Eneolithic Lasinja culture — a culture widely dispersed across the stretches of Croatia, western Hungary, northern Bosnia and eastern Austria. 

Eleven of the massacred victims were found with perimortem cranial injuries — wounds present at or near the time of death — located on the side or the back of the head. It is unclear what types of weapons were used to inflict the injuries. 

According to Mario Novak, an author of the study and a member of the Centre for Applied Bioanthropology at the Institute for Anthropological Research in Zagreb, Croatia, the injuries resembled a firearm-style execution to the back of the head.

"They really looked like they were made with modern weapons, but these people didn't have firearms or gunpowder," he said. "So these injuries were sustained by different types of weapons. Some kind of club or stone axes, or even some kind of metal object."

Given the archaeological context and the fact that the injuries were effectuated with different weapons, the study postulates that the massacre was a "single episode of execution."

To ascertain the reason for the execution, the researchers combined a forensic bioanthropological study of the injuries with genetic data of the victims. Based on a genome-wide ancient DNA analysis of 38 of the 41 victims, the researchers ruled out three of the usually suspected scenarios entirely. 

First, the massacre was not a targeted killing based on sex since the burial site had approximately equal numbers of males and females among the deceased — 20 and 21, respectively. 

"You can tell the sex of a skeleton based on differences between male and female skeletons," Novak said. "The problem with children is that you cannot tell, especially with smaller children, the sex based on its skeleton. So, we had to molecularly confirm the sex of the individuals." 

Likewise, if the massacre was the result of a battle, "Adult males are usually killed and younger females are usually taken into captivity," Novak told The Academic Times. But this would also have caused a disproportionate number of men to be found at the burial site. Thus, the massacre was neither the outcome of a battle nor an event targeting a specific sex. 

Second, because the ancestry of the victims was homogenous, the researchers ruled out the theory that the massacre occurred because migrants had arrived in the region. 

The genetic ancestry of the victims was mostly comprised of Anatolian, or modern-day Turkey, Neolithic ancestry that represents the first agricultural migrants who moved to Europe about 8,000 to 9,000 years ago. Their ancestry also contained a small proportion of genetic ancestry belonging to Western European hunter-gatherers. 

The findings matched the researchers' modeling of Potočani individuals "as a mixture of predominantly Anatolian Neolithic ancestry with [approximately] 9% Western European hunter-gatherer ancestry."

"Their genetic ancestry was basically the same," Novak said. "This tells us these people buried in this mass burial were the locals. They were not migrants from somewhere else — they were living in this area."

Lastly, the massacre was not a targeted killing of a family or kinship group. Based on a study of the autosomal chromosomes of the victims, the executed victims were not members of the same family. 

Approximately 70% of the analyzed victims did not have close kin among the deceased, suggesting that the massacre targeted a smaller subset of people from different kinships within the larger community. 

The reason for the indiscriminate killing, however, remains "the million-dollar question," Novak said.

One possible explanation could be that climatic change exacerbated by a significant increase in population led to a fight over the resources. But, "In this specific case, we really cannot tell," Novak said. "This period of time and this region is one big question mark. We don't know much about these people."

The researchers also said that an "important direction for work will be to study additional massacre sites to determine the prevalence of this pattern of ancient violence."

The study "Genome-wide analysis of nearly all the victims of a 6200 year old massacre," published March 10 in PLOS One, was authored by Mario Novak, Centre for Applied Bioanthropology, Institute for Anthropological Research; Iñigo Olalde, Institute of Evolutionary Biology, CSIC – University Pompeu Fabra and Harvard Medical School; Harald Ringbauer, Harvard Medical School and Harvard University; Nadin Roland Harvard Medical School and Broad Institute of Harvard and MIT; James Ahern, University of Wyoming; Jacqueline Balen, Archaeological Museum in Zagreb; Ivor Janković, Centre for Applied Bioanthropology, Institute for Anthropological Research and University of Wyoming; Hrvoje Potrebica, University of Zagreb; Ron Pinhasi, University of Vienna and Harvard Medical School; and David Reich, Harvard Medical School, Harvard University, Broad Institute of Harvard and MIT, and University of Vienna.