The history of our species is marked by numerous events that have shaped the current lifestyle and diversity of different human populations. Among the known points of inflection is the emergence of our species in Africa about 200,000 years ago, the departure of some modern humans from Africa around 60,000 years ago, their coexistence with Neanderthals and Denisovans – sex included – and their arrival in America about 15,000 years ago.
But nothing else has transformed our way of life more radically than the Neolithic revolution (a concept coined by archaeologist V. Gordon Childe in 1923). We are used to seeing documentaries where hunter-gatherers are shown in environments such as rain forests or deserts that seem alien to us; however, only about 8,000 years ago, during what we know as Mesolithic period, all the inhabitants of Europe were also hunter-gatherers, and had a way of life that disappeared with the arrival of the Neolithic.
The transition to the Neolithic age started with the so-called Fertile Crescent (a region shaped like a crescent moon extending from the valley of the Nile to the Tigris and Euphrates rivers) around 11,000 years ago, shortly after the last ice age. Aided by an increasingly warmer climate, humans developed farming and cattle raising and abandoned the nomadic livelihood that had accompanied them for millions of years. That allowed the first Neolithic societies to establish permanent settlements, have safer food sources and increase the effective population, which subsequently led to the emergence of the first great civilizations. The nature and population dynamics of this transition have been a matter of archaeological debate for decades. That is, did the idea of agriculture spread from its origin point and forced hunter-gatherer populations to abandon their ancestral way of life? Or was there rather a migration of farmers from the Middle East who replaced the indigenous inhabitants of Europe?
Genetics can answer these questions, because it is evident that the demographic processes of the past must have modelled the variation in current populations. But we need to keep in mind that these populations are the result of multiple overlapping evolutionary phenomena that took place during the Palaeolithic, Mesolithic, Neolithic and beyond. This is where palaeogenetics comes in, allowing us to analyse DNA obtained directly from human remains from different periods and obtain evolutionary information.
The radical change in the way of life to an economy of food production led to adaptations that are reflected in our genome. Several factors intervened in this phenomenon. Diet, for instance, which was far less protein-heavy and, in some cases, was based almost exclusively on cereal carbohydrates. Eventually new food resources appeared, such as dairy products, associated with raising livestock such as cows, goats or sheep. This change in diet probably led to genetic changes related to metabolism; the persistence in adulthood of the enzyme necessary for digesting lactose is just one example.
In addition, pets transmitted to humans, in a process known as zoonosis, a number of infectious diseases that probably include influenza, tuberculosis, measles, smallpox, whooping cough and mumps, many of which we still suffer. As populations became larger and more sedentary, pathogens propagated faster and epidemics became more frequent, forcing the immune system to adapt (in fact, the current populations are descendants of humans who survived these diseases). In short, the new way of life entailed changes in multiple aspects that we should be able to trace in genomes from periods prior to the Neolithic.
For all these reasons, it was very interesting a priori to analyse a European Mesolithic genome. In 2013, the only European prehistoric genome available to us was that of Ötzi, called the Iceman. It is the body of a man from the Copper Age (dated 5,300 years ago) discovered in 1991 in the Tyrolean Alps, 3,210 meters high, spectacularly preserved in ice. Because the cold helps preserve the DNA, it is logical for it to be the first recovered genome. But Ötzi is a Late Neolithic individual and therefore he cannot provide information about Europeans before the Neolithic.
«Palaeogenetics allows us to analyse DNA obtained directly from human remains from different periods and obtain evolutionary information»
|Julio Manuel Vidal Encinas/Institute of Evolutionary Biology (UPF-CSIC)|
In October 2006, several speleologists explored a small cavity in the Cantabrian Mountains at about 1,500 metres high, near the Leonese municipalities of La Braña-Arintero. After penetrating thirty feet down a narrow tunnel and overcoming a vertical well, they found two skeletons of Mesolithic hunters.
«Pets transmitted to humans, in a process known as zoonosis, a number of infectious diseases that we still suffer»
The genome of La Braña
In October 2006, several speleologists explored a small cavity in the Cantabrian Mountains, about 1,500 metres high, near the Leonese municipalities of La Braña-Arintero. After penetrating thirty feet down a narrow tunnel and overcoming a vertical well, they found a nearly complete skeleton in foetal position on a ledge. A few meters away, at the bottom of a well, there was another skeleton. Both were adult men. The dissemination of the news in the local media brought the regional government of Castilla y León to organize the complex extraction of the two skeletons, which were labelled as La Braña 1 and 2. The operation was led by archaeologist Julio Manuel Vidal Encinas.
One detail caught his attention: a stalagmite that had grown over some of the bones indicated they could be quite old. When the remains of the second man were removed, they found numerous atrophic perforated deer canines. Such teeth are typical Mesolithic hunters ornamentation, who wore them sewn to their clothes. The subsequent carbon-14 dating gave dates close to 7,000 years old, confirming this attribution.
At that time, new platforms for massive parallel sequencing (also known as second generation technologies) were still being implemented, and with a classical approach based on polymerase chain reaction (PCR) it would have only been possible to recover small fragments of these individuals’ mitochondrial DNA (a small cytoplasmic genome responsible for providing energy to cells). We already had a dozen Mesolithic sequences of central and northern Europe that showed a remarkable genetic uniformity: all belonged to the U4 or U5 mitochondrial lineages. Among the latter, most had the same sequence. This indicated that, with high probability, Mesolithic Europeans were very uniform from the genetic point of view. This notion is consistent with the fact that they established highly mobile populations over a large geographic area.
In 2013 we started testing different samples of the La Braña 1 individual in order to sequence it completely, and we managed to locate a genomic library generated from the dental roots of the third right upper molar, which had a DNA content close to 50%. That means that for every hundred sequences that we generated with the Illumina company platform, about half were human (the rest, as often happens in all ancient samples, were mostly bacterial sequences). This high efficiency is quite unusual in samples of a similar age (and even more recent ones) and it can only be explained by the exceptional conditions of the site, regarding height, thermal stability and low temperature, which have helped preserve the DNA. After doing a whole sequencing reaction in the Denmark Sequencing Centre, we managed to recover the genome with 3.4× coverage. That means that each of the 3,200 million nucleotides that make up our genome was represented on average in three to four different sequences. It is a low but sufficient coverage to carry out different types of genomic analysis.
Comparison of the La Braña 1 genome with partial genomic data of Neolithic and current European individuals allowed us to confirm that the hunters who inhabited Europe before the arrival of the Neolithic did not show genetic affinities with Neolithic farmers. The León individual was, curiously enough, related with current populations of Scandinavia such as the Swedes and the Finns. That would be the consequence of a process of Neolithic expansion that replaced local populations in southern Europe, where the climate was more favourable to agriculture, but partly assimilated them in colder latitudes. In Scandinavia, farmers and hunters lived together for several millennia, which resulted in encounters between them.
But what interested us the most was discovering the genetic changes that could result from the Mesolithic-Neolithic transition. We started looking at a list of genes that had been characterised as the product of recent natural selection in current Europeans. Selected genetic variants drag genetic context around them, creating areas of low genetic diversity that can be recognized by studying the current variation. These genes showed very frequency or even fixed variations (i.e., present in every individual) in Europeans, in contrast to other human populations. Our intention was to see if La Braña 1 had the ancestral alleles (i.e., identical to those of African populations) or the derivatives alleles (shared with current European) in these genes.
The La Braña 1 individual showed, surprisingly, variants derived from a number of immune system genes that had previously been associated with resistance to pathogens and their transmission via zoonosis. Clearly, many of the immunological events that had shaped the genome of current Europeans were prior to the Neolithic. That also meant that the adaptive changes produced by the transmission of pathogens from domestic animals should be among those genes in which La Braña carried ancestral alleles. An alternative but less likely possibility is that Neolithic pathogens entered Europe before the farmers themselves, and also decimated hunter populations, which were much smaller. A similar phenomenon took place in America, where Amerindian communities were decimated by smallpox and other diseases brought by Europeans even before meeting any of them.
|Julio Manuel Vidal Encinas/Institute of Evolutionary Biology (UPF-CSIC)|
La Braña 1 remains. The effectiveness of sequencing, quite unusual in ancient samples of a similar age (and even more recent), can only be explained by the exceptional conditions of the site, regarding height, thermal stability and low temperature, which have helped preserve the DNA.
«The results of the analysis in this first Mesolithic genome offer an idea of the potential of palaeogenomics»
Stages in the reconstruction of the portrait of a Mesolithic hunter-gatherer European. It is not possible to know the exact shade of skin tone; it should clearly be darker than the skin of current Europeans, but perhaps not as dark as sub-Saharan Africans. In any case, this is a unique phenotype that no longer exists in current European populations.
Blue eyes and dark skin: a unique phenotype
Unexpectedly, among the genes in La Braña presenting the ancestral gene variant, the two with that had an essential role in the light pigmentation of Europeans were found (SLC45A2 and SLC24A5). Derived variants that lead to lighter skin tones are present in virtually all current Europeans. We decided to extend the list to other pigmentation genes involved in hair colour and, to a lesser extent, in the skin and discovered that this Mesolithic individual still presented African variants in some genes such as MC1R, TYR and KITLG. In all likelihood, and against common belief so far, the clear pigmentation did not exist or was not widespread during the Mesolithic era. But there were more surprises to come: we also discovered that La Braña had the genetic variants in genes HERC2/OCA2, which are responsible for blue eyes in modern humans. That is, our guy had brown skin and bright eyes in a genomic context that was otherwise unequivocally European (actually, closer to Scandinavians than to any other current population). It is not possible to know the exact shade of skin tone; it should clearly be darker than the skin of current Europeans, but perhaps not as dark as sub-Saharan Africans. In any case, this is a unique phenotype that no longer exists in current European populations.
The results of the analysis in this first Mesolithic genome will stand for confirmation upon sequencing more samples in the future, but right now they offer an idea of the potential of palaeogenomics to rebuild migration and adaptive processes of human populations. The study of more ancient genomes, in different spaces and times, is the beginning of a new and exciting vision of European prehistory that will close more than a century of archaeological and anthropological debates.
«The study of more ancient genomes, in different spaces and times, is the beginning of a new and exciting vision of European prehistory»
Olalde, I. et al., 2014. «Derived Immune and Ancestral Pigmentation Alleles in a 7,000-Year-Old Mesolithic European». Nature, 507: 225-228.