Using ancient DNA to examine genetic continuity at the Mesolithic - Neolithic transition in Portugal

Two main mechanisms for the introduction of agriculture at the transition from the Mesolithic to the Neolithic in Portugal have been proposed: indigenous adoption and colonisation. Distinguishing between these mechanisms can be regarded as a question of genetic continuity or discontinuity at the transition. A genetic comparison of late Mesolithic and early Neolithic populations at the transition using ancient DNA is described here. Mitochondrial DNA (mtDNA) was extracted from human remains collected in several Mesolithic sites of the Sado estuary and from Neolithic cave sites. Phylogenetic analysis, based on the mitochondrial hypervariable region 1 (HVSI), and comparison with DNA from modern European populations was performed. The absence of mtDNA haplogroup J in the ancient Portuguese Neolithic sample suggests that this population was not derived directly from Near Eastern farmers. The Mesolithic and Neolithic groups show genetic discontinuity implying colonisation at the Neolithic transition in Portugal. A University of Oxford B Universidade de Lisboa at essentially the same moment as their appearance across the western Mediterranean (Zilhao 2001). The Neolithic is characterised by inland settlements, forming enclaves based on an agricultural economy, geographically separated from the hunter-gatherer settlements based in the coastal and estuarine regions. The earliest farming sites show evidence for a complete agricultural package, including domesticates, pottery and lithic technology which distinguishes these groups from the Mesolithic hunter-gatherers of the same region. Radiocarbon dating of sites throughout Portugal shows that the Neolithic and Mesolithic groups co-existed for 500 years (Zilhao 1993, Zilhao 2000). Figure 1 shows the changing settlement patterns of southern Portugal at the Neolithic transition. Stable isotope analysis of carbon and nitrogen in bone of human remains from Mesolithic and Neolithic burials in Portugal has shown that the Mesolithic groups had a diet comprising 50% marine foods, while the Neolithic farming groups had an entirely terrestrial diet (Lubell et al. 1994). This difference in diet demonstrates that the two different settlement patterns observed, that is, the shell middens and the agricultural enclaves, do not represent seasonal movements of a single population, but rather two distinct populations. Information from skeletal morphology, however, has been contradictory. Different studies have claimed evidence for population replacement based on skeletal differences across the transition (Fox 1996), or alternatively evidence for continuity of morphological traits (Jackes et al. 1997). The evidence from skeletal morphology has thus not been able to resolve whether the observed cultural, economic and dietary discontinuities reflect a genetic discontinuity. Given these circumstances, a direct study of the genetics of these populaHelen Chandler, Bryan Sykesa and Joao Zilhao 782 Figure 1. Geographic distribution of late Mesolithic ( ) and early Neolithic ( ) sites in south-central Portugal between 6000 and 4657 cal BC. Figure 2. A stylised simplified European mtDNA median network. The major haplogroups are designated by capital letters. tions is clearly desirable, and ancient DNA analysis on human remains from both Mesolithic and Neolithic sites in Portugal was thus performed. Modern European population genetics Certain properties of the human mitochondrial genome mean that the majority of modern population genetics studies compare the diversity within a section of the mitochondrial DNA (mtDNA) known as the hypervariable region I (HVSI). The mtDNA is separate from the nuclear DNA and is strictly maternally inherited. Thus, recombination, which occurs in chromosomal DNA, does not occur in mtDNA. In addition, mtDNA is present at approximately 1000 copies per cell and has a high mutation rate. The large number of studies carried out using mtDNA in modern Europeans means that a large comparative data set exists. The relationship between mtDNA sequence types can be represented on a median network diagram (Bandelt et al. 1999, Bandelt et al. 1995). Studies of modern Europeans have led to the development of a European mtDNA network that shows the presence of several different clusters of sequence types, known as clades or haplogroups. These have been given letter designations (Macaulay et al. 1999, Richards et al. 1998). The major European haplogroups are summarised in figure 2. Based on the variability observed within each of the different clusters it is possible to calculate the length of time that each haplogroup has been present in Europe. The ages of the major clusters are shown in figure 3. The ages of the clades show that most Europeans have an ancestry in Europe going back to the last glacial maximum with some dating back to the initial colonisation of Europe approximately 45 000 years ago. Around 15–20% of Europeans however, have mtDNA belonging to the haplogroup J which has been present in Europe for only around 8000–10000 years and which is derived from populations in the Near East. The results of modern mtDNA work thus imply a moderate input of population from the Near East with the beginning of farming in Europe, with haplogroup J being the main marker for the farming migration (Richards et al. 2000).