RICERCA ITALIANA     

The peopling of the European continent: the mitochondrial DNA and Y chromosome perspectives

Università degli Studi di Pavia

Abstract

It has been recently suggested that global colonization by the human species is one of the “big questions” in human origins for both biological and non-biological disciplines. This project focuses its attention on the peopling of Europe - applying modern genetic methods to address this issue in finer detail than has ever before been possible. We will evaluate the sequence variation in the male-specific region of the Y chromosome (MSY) and the maternally-inherited mitochondrial DNA (mtDNA) - the only non-recombining genetic systems in humans - in order to unravel which aspects of contemporary human genetic variation in Europe are the result of primary colonization, late-glacial expansions from Ice-Age refugia, Neolithic dispersals or more recent events of gene flow. To achieve this objective we are going to utilize the sequence information contained in six mtDNA haplogroups (J1, J2, T1, T2, U3 and U7) and most European Y-chromosome haplogroups taking advantage of two major progresses in phylogeographic studies: (i) the possibility of carrying out human mtDNA studies at the highest level of molecular resolution - the sequencing of entire mitochondrial genomes - and (ii) the recent advances in mutation-detection technology allowing both a “relatively easy” search and a systematic survey at the population level of high-resolution markers (SNPs) on the MSY.

The project is complex but will be carried in the context of a long-standing collaboration among five Research Units that has already proven to be extremely productive at the international level in addressing questions on human origin at both continental and regional levels. The overall task has been divided among the five Units, each focusing on a specific geographic region of Europe (and on the most common haplogroups in that area) and addressing one specific issue. The questions are the following:

1) the legacy of the Neolithic transition in Europe (Unit 1); <br />2) the genetic imprint of the post-glacial expansion from the Iberian refuge (Unit 2);
3) the contributions from northern Asia and Caucasus (Unit 3);
4) gene flow events from the Near East and the Balkans (Unit 4);
5) the pre- and post-Neolithic peopling of Sardinia (Unit 5).

A common objective is the identification of novel and old markers through a sequencing analysis approach. Unit 1 will completely sequence 500 (selected) mtDNAs, while each of Units 2-5 will sequence a region of about 80 kb (the same for all Units) in the MSY, selecting 10-20 subjects for each of those haplogroups which, on the basis of the frequency in the area under study and the diversity of the associated microsatellites, will appear to be particularly interesting/informative.

This approach will dissect the six mtDNA haplogroups and the Y-chromosome haplogroups into a number of sub-haplogroups whose origins and patterns of distribution will be surveyed in a very large number of population samples already available in the five laboratories, and others of interest that can be provided by our numerous international collaborators. At least part of these novel sub-haplogroups should be rather young and harbor a rather restricted geographical and ethnic distribution. In the case of mtDNA, we expect this approach to detect all sub-haplogroups (within J1, J2, T1, T2, U3 and U7) that are older than 7,000 years - thus all "Neolithic branches" in that portion of the mtDNA tree should be identified. The scientific problem addressed by the "mtDNA Unit" is broad but very specific and might therefore seem not sufficient to play the role of "female counterpart" for the four different issues that the other Units are going to address from a male perspective. However, the molecular dissection of the six haplogroups, at a resolution boundary of about 7,000 years, will inevitably provide information not only on the “Neolithic branches” of the mitochondrial phylogeny, but also on the other questions addressed by the “Y chromosome“ Units.

In conclusion, the main and long-term aim of this work is to contribute to the reconstruction of the population history of Europe based purely on genetic grounds. This information will be useful to a wide audience including: historians and archeologists, ethnologist, anthropologists, forensic scientists, linguists, public health officers, teachers. Certainly the mtDNA and MSY data depict only two out of many possible lines of descent, and their results need to be compared to each other and interpreted in the light of the distribution of autosomal genes, but they are the genetic systems that, at least for the moment, provide by far the most detailed information. <<<

Principal Investigator

Antonio Torroni Università degli Studi di PAVIA

Research Objectives

Our objective is to determine which aspects of contemporary human genetic variation in Europe are due to primary colonization, late-glacial expansions from Ice-Age refugia, Neolithic dispersals or more recent events of gene flow. To achieve this objective we are going to utilize the sequence information contained in six mtDNA haplogroups (J1, J2, T1, T2, U3 and U7) and most European Y-chromosome haplogroups taking advantage of two recent major progresses in phylogeographic studies:

(1) Complete mtDNA sequencing has led to a greatly improved resolution of the maternal genealogy. Although complete sequencing is still expensive and time-consuming, the information from even a relatively limited complete-sequence database can be exploited to design high-resolution typing systems that focus on specific branches in the tree. For instance in the study of Olivieri et al. (2006), by sequencing only 81 entire mtDNAs, we were able to identify all of the informative markers of haplogroups M1 and U6 and their internal clades. This was followed by a large-scale screening of M1 and U6 mtDNAs from the entire distribution range of the two haplogroups, which demonstrated that after the “Out of Africa” exit – ~70,000 years ago– of modern humans through a “southern dispersal route” (Macaulay et al. 2005), a subsequent dispersal event from the Levant led to the peopling of both Europe and North Africa about 40-45,000 years ago.

(2) The identification of high-resolution markers (SNPs) on the MSY now allows for detailed studies of the male line of descent as well. To these Y-chromosome studies we have already extensively contributed. For instance, Semino et al. (2000) provided a first coarse picture of the genetic landscape of Europe based on a limited number of Y markers. Subsequent higher resolution studies, based on the distribution of E3b, I, and J sub-haplogroups (Cruciani et al. 2004, 2007; Rootsi et al. 2004; Semino et al. 2004) refined previous evolutionary hypotheses and also offered, in some cases, completely new insights into the peopling of the continent.

In this project, we will exploit the new developments in both fields. The “mtDNA Unit” will completely sequence 500 (selected) mtDNAs belonging to the six haplogroups J1, J2, T1, T2, U3 and U7, while each of the four “Y chromosome Units” will sequence a region of about 80 kb (the same for all Units) in the X-degenerate portion of the Y chromosome, selecting 10-20 subjects for each of those haplogroups which, on the basis of the frequency in the area under study and the diversity of the associated microsatellites, will appear to be particularly interesting / informative. Each new mutation will be inserted in the Y chromosome phylogenetic tree and its geographic distribution will be studied by all Units in the Y chromosomes belonging to that particular haplogroup/sub-haplogroup by either DHPLC, RFLP, allele-specific PCR, ASO hybridization, real-time-PCR or direct sequencing.

This will allow the subdivision of the six European mtDNA haplogroups and the major Y-chromosome haplogroups of our continent into a number of sub-haplogroups whose origins and patterns of distribution will be analyzed in detail in numerous population samples already available in the five laboratories, and others of interest that can be provided by our numerous international collaborators. At least part of these novel sub-haplogroups should be rather young and harbor a rather restricted geographical and ethnic distribution. In the case of mtDNA we expect this approach to detect all sub-haplogroups within J1, J2, T1, T2, U3 and U7 that are older than 7,000 years – thus all “Neolithic branches” in that portion of the mtDNA tree should be identified.

The scientific problem addressed by the “mtDNA Unit” is broad and controversial (the mtDNA legacy of the Neolithic transition in Europe), and it is also very specific and might therefore seem not sufficient to play the role of “female counterpart” for the four different issues that the other Units are going to address from a male perspective (see below). However, the molecular dissection of the six haplogroups, at a resolution boundary of about 7,000 years, will inevitably provide information not only on the Neolithic branches of the mitochondrial phylogeny, but also on those involved in the post-glacial expansions from European refuge areas, as well as those that arrived from northern Asia and the Caucasus, and those that arrived in Sardinia at the initial stages of human colonization of the island.

Similar or related issues will be addressed from a male perspective. Indeed, specific targets of the “Y chromosome” Units are the following geographic areas, populations and haplogroups:

a) the "Balkan" branches of haplogroups I and R and the J sub-haplogroups to elucidate, at a deep resolution level, the South East European population genetic structures and the relationships with the Near Eastern and Asian ones;
b) haplogroup R1b, which accounts, on average, for about 50% of the European paternal lineages, to elucidate the origin of this haplogroup in Europe and the evolutionary relationships among R1b chromosomes currently found in Europe, Africa and Asia;
c) populations from the Republics of the former Soviet Union, from the extreme west, to eastern Siberia and on both sides of the Caucasus to quantify the male-mediated genetic contribution from Asia to Europe as a whole;
d) the Sardinian population to determine the source of the founding gene pool(s), evaluate its variation in time and space and test for genetic heterogeneity at a micro-geographical level.

The main and long-term aim of this work is to contribute to the reconstruction of the population history of Europe based purely on genetic grounds that will be useful to a wide range of scientific audience including: historians and archeologists, ethnologist, anthropologists, linguists, public health officers, forensic scientists, teachers. Certainly the mtDNA and MSY data depict only two out of many possible lines of descent, and their results need to be compared to each other and interpreted in the light of the distribution of autosomal genes, but they are the genetic systems that, at least for the moment, provide by far the most detailed information. <<<

First Results

The added values of this research refer to both advancements in genetics (intrinsic value) and in interdisciplinarity (extrinsic value).

Intrinsically, this research will represent a leap forward in the description and comprehension of the genetic landscape of Europe. This achievement has been pursued since many decades, and the level of knowledge has been growing in parallel with theoretical and methodological developments. The main aim is to arrive at a "picture" of an entire "genetic pool" and to reconstruct the events that led to its present composition. Uniparental DNA markers (mtDNA and MSY) are here used to highlight features shared by the whole continent but also to spot specificities for some geographical regions or populations. From the genetic point of view, these data are important to draw inferences on particular regional gene pools, in which some autosomal variants may reach epidemiological relevance (e.g. lactase deficiency in the Baltic area). Furthermore, advancements in the methodologies must be considered. These refer to the best resequencing strategies, and the most reliable analyses for dating and phylogeographic inferences.

Moreover, the mtDNA and the MSY are not simply two pieces of DNA with peculiar features, informative for evolutionary studies. They contain genes with important functions and their sequence variation has been associated to a wide range of complex diseases / phenotypes (as for mtDNA, see for instance MITOMAP: http://www.mitomap.org/). Unfortunately, most of these associations remain provisional, since different studies have often reached different conclusions, depending on the population sample they considered. A detailed knowledge of the western Eurasian mtDNA and MSY phylogenies, pursued by this project, should provide solid bases to evaluate the results obtained until now, making them reproducible also in different population contexts.

As far as interdisciplinarity is concerned, the results of this research establish a benchmark for other disciplines such as archaeology, linguistics and storiography. An excellent example is the recent paper "Mitochondrial DNA variation of modern Tuscans supports the Near Eastern origin of Tuscans", Am J Hum Genet 80:759-768 (2007) that is co-authored by members of three Research Units of this project. The main added value consists in the possibility of defining the population history of Europe WITH ONLY GENETIC TOOLS, a history that condenses the contributions of all subjects who inhabited the continent and took part in the continuity of its human settlement. Only in this way cultural influences in the formation of linguistic and historical bodies of knowledge can be appropriately weighted. These aspects of the research will be strengthened by the existing collaborations between each of the Research Units and experts of the above-mentioned disciplines, and by their further development. <<<

Timescale

24 months

National and international background

Anatomically and behaviorally modern humans entered Europe from the Levant and rapidly dispersed into the continent between 46,000 and 41,000 years ago (Mellars 2006). This is considered to be the seeding event in the formation of the gene pool of modern Europeans. The rapid spread into Europe was probably facilitated by a major improvement in climatic conditions between about 43-41,000 years ago (the period of the Hengelo interstadial), which would inevitably have made a process of population expansion from southeast to northwest across Europe easier to achieve (Shackleton et al. 2004; Mellars, 1996, 1998).

After the arrival of first hunters and gatherers, the genetic structure of Europeans has been influenced by two important events: the retreat of European populations into refugia during the Last Glacial Maximum (LGM) about 20,000 years ago, followed by re-expansions starting from about 13,000 years ago, and the Neolithic revolution about 8-10,000 years ago when the agriculture spread all over Europe from the Near East. More recent events of gene flow must be also taken into account when dealing with the complexity of the European genetic landscape. Most of those probably did not have a demographic impact at the continental level, but certainly played an important role in shaping the gene pool of specific regions of Europe.

There are two models to explain the spread of agriculture: the demic diffusion model assumes that the Neolithic transition diffused in Europe from the Middle East by an important movement of population (Ammerman and Cavalli-Sforza, 1984), without substantial contact with local Palaeolithic populations; the cultural diffusion model assumes that the Neolithic transition occurred mainly through the transmission of agricultural techniques (Zvelebil and Zvelebil, 1988) without large movements of populations (and genes).

The work of Cavalli-Sforza and his colleagues (Cavalli-Sforza et al. 1994) on classical markers has become something of an icon in the debate that soon arose on the issue of the relative contributions of Paleolithic and Neolithic people to the gene pool of modern Europeans. Their map of the first principal component (PC) revealed a gradient with the Near East at one pole and Europe at the other, which was originally seen as a strong evidence for the demic diffusion hypothesis, implying that Neolithic farmers coming from the Near East and autochthonous European hunter-gatherers intermingled progressively (Fig. 1).



However, a growing body of evidence is questioning the scenario that the gene pool of modern Europeans is mainly attributable to the demic diffusion from the Near East. For instance, the initial dissection of one minor autochthonous European mtDNA clade, haplogroup V, and analyses of its geographic distribution (Torroni et al. 1998, 2001) showed that late glacial expansions of Paleolithic populations from refuge areas in southern Europe could have also had a major impact in repopulating the continent. Moreover, this scenario was compatible with only one of the two possible interpretations of the second PC obtained from classical genetic markers (Fig. 1). A detailed analysis at the level of complete mtDNA sequences of haplogroups H and U5 has provided further evidence of the importance of the Franco-Cantabrian glacial refuge in southwestern Europe for the repopulation of much of the western and northern part of the continent. Indeed, this is strongly supported by the age estimates and the geographic distributions of mitochondrial clades H1, H3, V and U5b (Fig. 2) (Achilli et al. 2004, 2005; Torroni et al. 2006), as well as by phylogeographic evidence provided by the human Y chromosome, extended in this case to other southern European refuge areas.



In particular, as shown in Fig. 3, haplogroups I, R1a and R1b are most likely signatures of postglacial population expansions from the Balkan, Ukrainian and Franco-Cantabrian refugia, respectively (Semino et al. 2000; Underhill et al. 2000, 2001; Rosser et al. 2000; Wells et al. 2001; Rootsi et al. 2004). In contrast, the Y chromosome haplogroups E and J, which characterize mainly the populations of southeast Europe, are of African/Near Eastern derivation and their distribution was initially interpreted as mainly due to the demic diffusion of the farmers (Semino et al. 1996; Hammer et al. 1998).



In the last years important efforts have been made in searching for new Y-chromosome markers. Different sub-lineages have been identified both for the main European haplogroups such as E (Cruciani et al. 2006, 2007), J (Sengupta et al. 2006), I (Rootsi et al. 2004; Underhill et al. 2007), and for some minor haplogroups (G, N, K, and H) of probable Asian origin (Sengupta et al. 2006; King et al. 2007; Rootsi et al. 2007). Indeed, gene flow from West Asia (either as a steady low-level exchange of migrants or episodic massive migrations) may have brought a substantial contribution of Asian genes (perhaps the third PC, Fig. 1) both after and before what is considered the European Neolithic. Since it is possible that even massive movements in the East-West direction were not associated with either major shifts in the subsistence style or with notable signatures in the archaeological record, the genetic signature of such movements from West Asia, if any, is likely to be more detectable in populations of Eastern Europe.

The possibility of reconstructing the nature and number of these contributions depends on the degree of genetic divergence between the ancestral donor populations and the recipient European populations. The question for the geneticist is whether a DNA polymorphism that is able to mark a specific episode indeed exists, and in which genetic system can it be detected. Thus, in each case, an appropriate level of phylogenetic resolution is required, and mitochondrial DNA and the male-specific region of the Y chromosome (MSY) remain for the moment the most informative genetic systems to track down human origins and dispersions. MtDNA is small (only 16,569 base pairs), but complete genome analyses at the population level in large number of (selected) subjects can be carried out (see form B from Unit 1 in this proposal). Thus, the resolution of the mtDNA tree has been improved many-fold (Macaulay et al. 2005; Olivieri et al. 2006) and the basal branching structure of mtDNA variation in many parts of the world is now rather well understood.

In contrast, the MSY is extremely large (almost 60 Mb) but it is slow evolving, so its variation at the population level can be evaluated only by a hierarchical survey of haplogroup diagnostic SNPs and analysis of microsatellite loci. However, the very large DNA content (in term of nucleotides) of the MSY ensures that abundant diversity exists to proceed a long way in this process of phylogeographic refinement, eventually leading in a close future to a level of resolution for human history comparable with, or even greater, than that currently achieved by mtDNA (Torroni et al. 2006).

In this project we propose to assess the peopling of the European continent from the perspectives of both mtDNA and Y chromosome. The reason to deal concomitantly with both genetic systems in a single project (like this) is that their phylogenies share many features. They: (a) developed from a common ancestor in a similar time frame; (b) were generated only by the sequential accumulation of new mutations along radiating male and female lineages, respectively, thus providing an independent views of the maternal and paternal contributions; (c) most likely experienced the same demographic processes; (d) their dissection can be achieved by using similar molecular approaches; and (e) the need of defining sub-haplogroups of younger age and more narrow geographical distribution is the same for both systems, especially in the European context.

In summary, it is clear that there is an urgent need of new markers able to delineate the internal subdivisions of European mtDNA and Y chromosome haplogroups. In this project, we propose to achieve an extremely high level of molecular and phylogenetic resolution for six mtDNA haplogroups (J1, J2, T1, T2, U3 and U7), commonly found in European populations, and for most Y chromosome haplogroups present in Europe. The task has been divided among the five Units participating in this project, each focusing on a specific geographic region of Europe (and on the most common haplogroups in that area) and addressing one specific question.

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