Aplicación de la biología molecular en el estudio antropológico de las poblaciones humanas del pasado: análisis del ADN mitocondrial

In 1985 it was proved for the first time the possibility to recover DNA from mummified tissue; since then, the developments in the techniques of molecular biology that refer to the extraction and analysis of DNA of varying age and specially, the development of the Polymerase Chain Reaction (PCR) allowed to collect data from extinct populations in a straightforward way. Thus, the main purpose of this work was to obtain direct results, from archaeological human remains, on the genetic composition of a group of populations located across the Basque territory. We have analysed samples recovered from the sites of San Juan Ante Portam Latinam (Laguardia, Alava), dated 5,070 ± 150 - 5,020±140 BP; Longar (Viana, Nafarroa) dated 4,445±70-4,590±90 BP; Urratxa III (Orozko, Bizkaia) dated 3,405±70-3,475±80 BP; Pico Ramos (Muskiz, Bizkaia) dated 4,100±110-4,790±110 BP and Aizpea (Aribe, Nafarroa) dated 6,600±50 BP (Etxeberria & Vegas, 1988, Armendariz & Irigarai, 1995; Muñoz & Basaguren, 1997; Zapata, 1991; Cava, 1994). The data obtained constitute a sample of the genetic variability existing in the Neo-Eneolithic period. The comparison of the genetic data from the prehistoric populations to the already published data on present day population allowed us to contrast some of the hypotheses proposed on the evolution of the Basque population based on the latter approach. Most of the recent research on ancient DNA (aDNA) has been focused on the investigation of mitochondrial DNA (mtDNA), since there are thousands of copies of the mitochondrial genome per cell; this increases its survival and facilitates its recovery, in contrast for instance to the amplification of nuclear DNA, which results a much more arduous task if not impossible. Restriction enzymes, as opposed to sequencing, were chosen as the routine method for mtDNA analysis, since it constitutes a more efficient and feasible approach for these kind of studies at the population level.

The troublesome nature of the archeological samples, required a precise optimization of the techniques; thus, a significant part of our research has been dedicated to adjust and implement the techniques involved, as besides, this work constitutes the initiation of a new line of research in the U.P.V./E.H.U. One of the main characteristics of aDNA is the inverse relationship between the efficiency of amplification and the length of the expected amplification product, which can be explained as a function of the degradation of the input DNA (aDNA). Additionally, the nucleotides in the sequence may have been also modified as a result of the oxidative and hydrolitic reactions that take place in the tissue once the organism is dead. Thus, during the amplification, most of the damaged molecules are not going to be successfully replicated, as the activity of the Taq polymerase will be inhibited. As expected, we have been able to observe a high correlation between efficiency of amplification and age of the sample, being the prehistoric samples those rendering the lowest yield, as a consequence of the damage in their DNA. The greatest danger in aDNA work is the contamination of our aDNA samples with exogenous, contemporaneous DNA. This leads us to follow a series of esterility meassures in order to avoid contamination, such as the realization of contamination controls during extraction and amplification, and the duplicate analysis of the samples. This, obviously implies an extra effort and a greater investment of resources, but becomes necessary to guarantee the reliability of the results.

These are the first genetic data on prehistoric populations from the Basque Country, from which it is possible to rise some hypotheses on the origin of the human groups that settled down in our territory in prehistoric times. Thus, the distribution of the mtDNA haplogroups observed is similar to that described in a sample of modern Basques, with some exceptions: we have not detected haplogroup V in any of the prehistoric samples here analytzed. This promts to questioning a recently published hypothesis (Torroni et al. 1998) which supports an origin for this haplogroup in a region North of the Iberian Peninsula, about 10,000-15,000 years ago, and a subsequent expansion towards Europe.

In addition, we have been able to observe that the sample of Longar shows the greatest differentiation within the three prehistoric populations analyzed, which could be explained as a consequence of a different origin for this population. However, a differentiation due to genetic drift cannot be ruled out if we consider the possible reduced size of human populations at the time. This study, in allowing to relate genetic data with archaeological, morphometric, and linguistic data, constitutes a novel contribution to the study of the biological history of the human populations. The ongoing analysis of a greater number of prehistoric sites will eventually enable us to find an answer to these and other evolutionary questions