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Home , Archaeogenetics, Human genetic variation

JASs Proceeding Paper Journal of Anthropological Vol. 88 (2010), pp. 93-112

Molecular Anthropology in the Genomic Era

Giovanni Destro-Bisol1,2, Mark A. Jobling3, Jorge Rocha4, John Novembre5, Martin B. Richards6, Connie Mulligan7, Chiara Batini2* & Franz Manni8

1) Istituto Italiano di Antropologia, Roma, Italy - e-mail: [email protected] 2) Department of Animal and , University of Rome “La Sapienza

3) Department of , University of Leicester, UK - e-mail: [email protected] 4) IPATIMUP - Institute of Pathology and Molecular Immunology of the University of Porto; Department of Biology, Faculty of Sciences, University of Porto - e-mail: [email protected] 5) Department of Ecology and Evolutionary Biology; Interdepartmental Program in Bioinformatics; University of California, Los Angeles, California, USA - e-mail: [email protected] 6) Institute of Integrative & Comparative Biology, Faculty of Biological Sciences, University of Leeds, UK e-mail: [email protected]

7) Department of Anthropology, University of Florida, USA - e-mail: [email protected] 8) CNRS UMR 7206 - USM 104 National of Natural History - Musée de l’Homme, Paris, France e-mail: [email protected]

9) Department of Genetics, University of Leicester, UK (current address) - e-mail: [email protected]

Summary – Molecular Anthropology is a relatively young field of research. In fact, less than 50 years have passed since the symposium ‘’Classification and Human ’’ (1962, Burg Wartenstein, Austria), where the term was formally introduced by Emil Zuckerkandl. In this time, Molecular Anthropology has developed both methodologically and theoretically and extended its applications, so covering key aspects of such as the reconstruction of the history of human and peopling processes, the characterization of DNA in extinct and the role of adaptive processes in shaping the of our . In the current scientific panorama, molecular anthropologists have to face a double challenge. As members of the anthropological community, we are strongly committed to the integration of biological findings and other lines of evidence (e.g. linguistic and archaeological), while keeping in line with methodological innovations which are moving the approach from the genetic to the genomic level. In this framework, the meeting “DNA Polymorphisms in Human Populations: Molecular Anthropology in the Genomic Era” (Rome, December 3-5, 2009) offered an opportunity for discussion among scholars from different disciplines, while paying attention to the impact of recent methodological innovations. Here we present an overview of the meeting and discuss perspectives and prospects of Molecular Anthropology in the genomic era.

Keywords – , Human populations, , Interdisciplinary approaches.

The dawn of Molecular Anthropology can, at Burg Wartenstein in Austria. In that context, at least formally, be traced back to the 1962 sym- the American biologist of Austrian origin Emil posium ‘’Classification and Human Evolution’’ Zuckerkandl first introduced the term “Molecular

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Anthropology” to designate the study of primate the origin of modern humans” (1988). Such con- phylogeny and human evolution through the tribution is also worth noting for the systematic genetic information decoded by proteins and comparison between theoretical expectations and polynucleotides (Sommer, 2008). findings of the RAO and multiregional models In the fifty years since that symposium, on modern human evolution, providing an alter- Molecular Anthropology has not only moved its native to most of the previous papers based on focus onto the molecule which encodes the genetic descriptive and circumstantial approaches. information, deoxyribonucleic acid (DNA), but The Human Diversity Project it has also extended its application well beyond (HGDP) may be regarded to as another turning the aspects initially implied by Zuckerkandl, so point for Molecular Anthropology. Promoted becoming one of the most promising and rap- by Luigi Luca Cavalli-Sforza and others in the idly growing sub-fields of Anthropology. In fact, early 1990s, HGDP aimed to explore human insights into several important issues have been differences and history by looking at obtained using a molecular approach, leading to from numerous indigenous populations across a substantial advance in our knowledge of various the globe, involving anthropologists, , key aspects of human evolution. These include, medical doctors, linguists, and other scholars among others, the reconstruction of the history (Cavalli-Sforza, 2005). This project was designed of human populations and peopling processes, to offer an opportunity for systematic research the characterization of DNA in extinct humans providing a shared set of DNA samples to labo- and ancient populations and the role of adaptive ratories working on , processes in shaping the genetic diversity of our which was obtained through the use of immor- species (Jobling et al., 2004). talized lymphoblastoid cells collected from pop- The pioneering study on mitochondrial ulations of particular anthropological interest variation in worldwide populations by Rebecca (Cann et al., 2002). Unfortunately, HGDP also Cann and coworkers in the late eighties is one raised important controversies, mostly of ethical of the most celebrated applications of Molecular (Ikilic & Paul, 2009), which slowed down Anthropology, due to its important implications the initiative. Nonetheless, HGDP played a key for the understanding of the origin and diffusion role in the change of perspective of Molecular of anatomically modern Homo sapiens (Cann et al., Anthropology from genetics to genomics, coher- 1987). Their findings were claimed to be a sub- ently with its mission to explore the mutual ben- stantial argument in favour of the recent African efits between groups involved in the initiatives origin (RAO) of our species and led to the spread of the Organization (HUGO) of the popular concept of “”. and the laboratories working on human diver- The initial results have been subsequently chal- sity. The most recent large-scale projects aimed lenged by further studies which have extended and at analysing human variation include HapMap improved sampling, increased genetic informa- and 1000Genomes (International HapMap tion and incorporated demographic aspects (e.g. Consortium, 2003; Via et al., 2010). Either Vigilant et al., 1991; Templeton, 1992; Relethford, through the analysis of common variants, in the 1998). Interestingly, it was soon understood that former, or through the discovery of rare vari- the genetic evidence, although powerful, needs ants within different genomes, in the latter, both to be considered jointly with paleontological and these initiatives are moving the focus of human archaeological evidence in order to achieve a more diversity studies to the genomic level. comprehensive view on the emergence of our spe- Those mentioned above can be considered cies and evaluate the relevant hypotheses more as paradigmatic examples of the double chal- carefully. This was thoroughly and elegantly pur- lenge that molecular anthropologists have to sued by Chris Stringer and Peter Andrews in their face even in the current scientific panorama. In seminal paper “Genetic and evidence for fact, as members of a discipline, Anthropology, G. Destro-Bisol et al. 95

which is strongly committed to the integra- poster presentations are available at http://www. tion of different forms of knowledge, we need isita-org.com/MolAnthroGenomics/2009.htm. to foster the debate with researchers belong- Here we summarize the contents of the ing to sister disciplines (e.g. linguists, archae- invited lectures from the Congress and comment ologists and primatologists). At the same time, on some issues raised during the meeting. This the increasing demand for exhaustive analyses report does not only aim to provide JASs read- of human genome variation requires constant ers with an overview of the Congress, but could methodological and theoretical updates, which also represent a useful reference for future initia- constitute a drive towards increasing specializa- tives designed to evaluate the state of the art and tion. While the promotion of interdisciplinary discuss perspectives and prospects of Molecular debate in Molecular Anthropology has already Anthropology in the genomic era. been at the centre of various initiatives, among which the series of conferences organized by Colin Renfrew are especially worth mentioning Molecular anthropology in the (Renfrew & Boyle, 2000; Bellwood & Renfrew, genomic era, an overview1 2002; Forster & Renfrew, 2006), the continuous development of genomic approaches to human Molecular Anthropology: past and present diversity is opening unprecedented opportuni- The first attempts to understand the his- ties and raising further issues which render a tory of human movement, demo- renewed focus necessary. graphic change and admixture through genetics Coherently with this background, the meeting used protein markers, such as blood groups and “DNA Polymorphisms in Human Populations: HLA (Cavalli-Sforza et al., 1994). We now sus- Molecular Anthropology in the Genomic Era”, pect that the diversity of these markers is strongly organized in Rome (December 3-5, 2009) by influenced by , and researchers the Istituto Italiano di Antropologia and the interested in investigating human history have National Museum of Natural History of Paris, since sought neutral markers, regarding pheno- offered an opportunity to foster dialogue among types and adaptive influences as a disturbance. researchers from different disciplines, while pay- Prominent amongst these markers have been the ing attention to the impact of recent innova- non-recombining region of the Y tions in theory and practice of molecular stud- and mitochondrial (mt)DNA, despite ongoing ies on human evolution. The first three sessions concerns about regional selection on the latter provided an updated view of the genetic vari- (Balloux et al., 2009), and most major questions ability continent-by-continent and highlighted and many populations have now been addressed the issues that still require investigation. Topics to some degree using small numbers of informa- under discussion included both results and infer- tive sites on these loci. Their uniparental modes ences obtained through “traditional” approaches of inheritance continue to illuminate sex-biased (e.g. data from unilinear markers) as well as new processes, and the coinheritance of Y and next-generation DNA sequencing methods. with patrilineal surnames allows the exploitation The closing session was dedicated to the dia- of these cultural labels in the investigation of past logue about theoretical and practical aspects of population structures (King et al., 2009). Issues interdisciplinary interactions in the Genomic of ascertainment bias of markers here are fading era, putting molecular anthropologists face-to- with the use of multiple Y-STRs and increasing face with researchers from Paleoanthropology, numbers of Y-SNPs, and with increased resolution , Linguistics and Medicine (see the of mtDNA analysis. The entire mtDNA (approxi- JASs forum “Molecular Anthropology in the mately 16.5kb) can be now readily sequenced in Genomic era: interdisciplinary perspectives” in this JASs issue). All the abstracts of oral and 1 lecture presented by Mark A. Jobling

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many individuals, whereas for the Y-chromosome From to (and back) only a small number of the available STRs are rou- By contrast, there are researchers who regard tinely analysed, and the resequencing of megabases and selection as the important issues, of this chromosome is now possible with the care- and population structure and history as the dis- ful application of new technologies. This reveals traction. Unfortunately, although the pheno- hundreds of new SNPs per chromosome analysed, types of humans are of particularly interest, our posing challenges for unifying datasets and stand- species is not a model organism. The kinds of ardizing methodology and nomenclature. Recent controlled experiments we might carry out on sequence analyses of Y separated by mice are impossible (Terwilliger & Lee, 2007). only a few generations have identified lineage-spe- so we must make do with the ‘experiments of cific markers (Xue et al., 2009), which represents nature’ represented by anthropologically inter- an important step towards reaching the phylo- esting populations, while at the same time trying genetic resolution needed to distinguish between to account for the complex influence of a com- different migration events which are very close plex environment that includes the epitome of in time. Members of the general public, through defining human complex phenotypes, culture. their obsessions with genetic , are help- Some anthropologically interesting phenotypes ing to provide useful scientific data. Genome-wide are yielding to the power of genetic and genomic SNP typing is now affordable and offers interest- analysis, including resistance or susceptibility to ing insights into the geographical patterning of some pathogens, dietary adaptation, pigmen- common autosomal variation (Novembre et al., tation, hair thickness and tooth morphology 2008). It suffers from the Eurocentric ascertain- (Kimura et al., 2009). Other traits promise to ment bias of common SNPs, and a similar bias in be less tractable, with the tractability depending the population distribution of available genome- on the often unknown underlying genetic archi- wide association study data (Need & Goldstein, tecture. Stature is a good example - in outbred 2009). Because of the tag-SNP-based designs of populations in the developed world, dozens of marker sets, it also lacks much of the potential loci have been identified in huge samples, but temporal resolution provided by the evolutionary each contributes only a tiny amount (a few mil- relationships among haplotypes. Conventional limetres) to the variance of the trait. Tellingly, resequencing of multiple specific X-chromosomal Francis Galton’s Victorian back-of-an-envelope and autosomal segments, and the typing of mark- approach to height prediction greatly outper- ers in low-recombination regions, can provide forms the technological might of twenty-first some of this resolution, and has thrown light on century genomics (Aulchenko et al., 2009). Here, the history of sex-specific behaviours (Hammer et the common-disease-common-variant hypoth- al., 2008). esis seems to be losing the battle to hypotheti- Using genetics to test hypotheses based on his- cal copy-number variants, rare , gene- torical, archaeological or linguistic evidence often gene interactions and (Manolio et al., uses a ‘cherry-picking’ approach when consider- 2009). Short stature among pygmy populations ing the other disciplines, which lacks objectivity. is a well-known example of an anthropologically Although most of the tractable questions seem interesting phenotype, but its elucidation falls likely to be those linked to relatively recent events, foul of the problem of unknown genetic archi- one of the most impressive findings of recent tecture, both within and between populations. If years has been the remarkable explanatory power one or a few loci explain it, and if candidate loci of simple distance from for patterns translate from to the rest of the world, of modern genetic diversity (Ramachandran et then simple approaches may bear fruit. But if, al., 2005), underscoring the importance of early as seems likely, the trait is complex and multi- events when populations were small. genic, then it will more difficult to understand. We may hypothesise a common origin of pygmy G. Destro-Bisol et al. 97

groups to explain the common phenotype (Patin would be able to learn everything that could be et al., 2009), but this would make it difficult to learned about the relationships among individu- pinpoint the specific locus or loci responsible als and populations, the processes of , for the phenotype amongst the loci shared sim- and the influence of selection. It seems likely that ply through recent common origin. Moreover, the quality of recording and classification of the the detection of phenotypically important loci environments and the phenotypes (Samuels et within populations will be difficult because of al., 2009), rather than the , will then small sample sizes, and grant applications (often become the crucial factor, and the anthropolo- damned by reviewers as ‘fishing expeditions’) gists (and the ethicists) will inherit the world. will tend to face the insoluble problem of power calculations. The role of natural selection in the development of short stature is mysterious, and The peopling of Africa2 certainly more complex that simple ‘Just So’ sto- ries based on the ease of moving about in forests Despite Africa’s central role in human evo- (Migliano et al., 2007 ) Even when we can see lution, African populations have been less well clear selective advantages in particular adapta- characterized than other groups in most studies tion, the problem of drift represents one of the addressing human genetic variation. Until recently, major difficulties of studies of poorly under- inferences about human population history typi- stood phenotypes. We can use genome-wide cally relied on few African populations that were approaches to seek segments of DNA showing assumed to be representative of the whole conti- frequency elevations in populations living, for nental diversity. While this limitation did not chal- example, at high altitude, but how do we dis- lenge the validity of general conclusions about the tinguish between adaptation and drift as expla- origins and global distribution of human genetic nations for frequency differences? And can we variability, insufficient sampling has certainly identify suitable control populations, in which hampered our perception of how human diversity drift has not also been a problem? If we want to was shaped within Africa. With the highest time support findings by ‘replication’ in other high- depth of human history and over 2000 ethnolin- altitude populations, we face the problem that guistic groups dwelling in landscapes that range the adaptation may have arisen independently, from the driest deserts to the most humid forests, and may even have a different physiological and Africa could hardly be understood without a more genetic basis. It seems likely that admixture-based comprehensive population sampling. approaches will be useful here. In the distance, In the last decade, improvements in sampling however, lies the brave and bright new world of coverage, together with the increasing availability whole genome sequences (www.1000genomes. of highly informative genetic markers and the use org; Via et al., 2010), uncompromised by ascer- of new approaches regarding data analysis, had a tainment bias and rich with rare variants – recent tremendous impact in the assessment of Africa’s investigations of African genome sequences are genetic variation. Although the amount and qual- already starting to show how much diversity will ity of genetic data is still far from being fully sat- be revealed (Schuster et al., 2010). Although the isfactory, the current genetic portrait of Africa has new methods are still too expensive to be applied reached an unprecedented level of precision. to most anthropologically interesting samples, this is likely to change soon, and molecular Unilinear markers anthropologists should learn how to mine and A significant part of our present understanding use such sequences, and think what questions of African genetic variation is based on the study they would like to address with them. Surely, of mitochondrial DNA (mtDNA) and the non- the more sequences, the better? If we knew the sequences of all the genomes of everyone, we 2 lecture presented by Jorge Rocha

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recombining portion of the Y chromosome (NRY) relevant population history parameters. Multilocus (Cruciani et al., 2002; Salas et al., 2002). Because approaches designed to overcome this difficulty of their uniparental patterns of inheritance and have received a remarkable boost with the recent lower effective population size, mtDNA and NRY publication of Tishkoff’s landmark study on 2,432 haplotypes provide complementary information individuals from 113 populations using a panel about female- and male-specific aspects of genetic of 1,327 polymorphic markers (Tishkoff et al., variation and are especially sensitive to the effects 2009). In brief, the study showed that most African of drift. MtDNA and NRY markers tend to be genetic variation can be sorted into 14 ancestral highly geographically structured and, due to lack population clusters and that most populations of recombination, phylogenies can be exhibited high levels of mixed ancestry, consistent easily reconstructed, providing a temporal frame- with historical migrations across the continent. work for mutation accumulation, which can be Consideration of geographic data along with clus- related to the geographic distribution of different tering analysis distinguished five major groups of lineages. Several NRY and mtDNA clusters, including (Fig. 1): i) a contiguous north- are particularly informative because their origins ern fringe encompassing Berber, Cushitic and appear to be geographically and temporally dis- Semitic Afroasiatic speakers from Saharan and tinct from each other. For example, the distribu- East Africa; ii) a widespread group correspond- tion of the oldest basal NRY- A-M91 ing to the distribution of the Niger-Kordofonian suggests an ancestral link of the southern African language family (paralleled by the distribution of Khoe-San click-speaking groups to East Africa. NRY haplogroup E3a-M2); iii) another group The relatively old NRY B2b-M112 haplogroup comprising Chadic and Nilo-Saharan-speaking points to the common ancestry of Khoe-San and populations from Nigeria, Cameroon, Chad and Pygmy hunter-gatherer groups. A lineage within southern Sudan (some of which share a lineage the younger E3b-M35* paragroup suggests that within NRY haplogroup R that may have been pastoralism might have been introduced to south- introduced into Africa by a back migration origi- ern African from East Africa prior to Bantu migra- nating in ; Cruciani et al., 2002); iv) a group tions. The relatively young E3a-M2 haplogroup with Nilo-Saharan and Cushitic-speaking popu- is widespread in Niger-Kordofonian-speaking lations from Sudan, Kenya and Tanzania; and v) a populations and provides a marker for the expan- group of noncontiguous geographic distribution sion of Bantu-speaking agriculturists. Among the consisting of Pygmy and Khoe- mtDNA haplotypes, the basal L0d clade is almost San populations, providing evidence for shared exclusive to the southern African Khoe-San but ancestry among hunter-gatherers (consistent with is also found in the click-speaking Sandwe from the distribution of NRY haplogroup B2b, but Tanzania confirming the ancient link of the Khoe- not with mtDNA, since the three main hunter- San to Eastern Africa. The younger haplogroup gatherer groups are characterized by very distinct L1c, which probably originated in , is haplogroups.) In spite of the major advances pro- crucial to assess the ancestral relationship between vided by this study, it is important to note that western Pygmy hunter-gatherers and their neigh- regions like the Sahel, the Atlantic , boring Bantu-speaking farmers. Namibia, Angola and the central corridor com- prising the DR of Congo, Central Zimbabwe and A multilocus approach the Zambia, remain sparsely sampled. On the An important limitation of studies based other hand, to make full use of the framework on the NRY and mtDNA markers is that they provided by Tishkoff’s investigation, it is crucial amount to the characterization of only two to generate increasingly comparable datasets. This genetic systems, which, due to the stochastic- could be achieved by defining a minimum subset ity of evolutionary processes, are insufficiently of highly informative markers to be used in future robust to generate meaningful estimates of works concerning other African populations. G. Destro-Bisol et al. 99

Prospects for future studies To disentangle the spatial-temporal processes that gave rise to the emergent portrait of African genetic diversity, it will be important to address both deep–time and more fine-scale questions, combining continent-wide studies with more detailed pictures provided by regional or local case studies. Moreover, an interesting approach to interpret the basic properties of the observed genetic variation is to focus on discordance among different sets of genetic data, or between genetic data and non-genetic aspects of human variation. For example, the discrepancy between the pat- terns of genetic variation in NRY and mtDNA has provided important insights about the influence of sociocultural factors in shaping differences in male and female migration rates and effective sizes (Destro-Bisol et al., 2004). Discordance between levels and patterns of genetic variation in nuclear Fig. 1 - Geographic location of major groups of and uniparental markers may be useful to reduce ancestral clusters within Africa. Patterns are the number of population history models that are identified by the number used in the text to describe the population composition of each compatible with the data. On the other hand, dif- group of clusters. WPYG=western Pygmies; ferences between geographic patterns at putatively EPYG=Eastern Pygmies; SAK= southern Africa selected loci and neutral loci may be used to eval- Khoe-San. For details see text (modified from uate the strength of selection and to analyze the Tishkoff et al., 2009). influence of demographic processes in spreading selected variants (Coop et al., 2009). Finally, dis- Pygmy and agricultural populations provide sociation of common trends in the relationships excellent examples of the usefulness of new com- between genetics, linguistics and lifestyles provide putational methods in addressing population unique opportunities to analyze the impact of history in Africa (Patin et al., 2009; Verdu et al., admixture between different populations and to 2009). With the rapid accumulation of multilo- analyze how major shifts in genetic and cultural cus genotype data and the significant increase in patterns occur. For example, interactions among sampling density, it is expected that similar infer- the peoples of southern Angola has generated ential frameworks will be successfully extended intriguingly discordant combinations of ethnicity, to explicit geographical modeling of human dis- language and lifestyle (Coelho et al., 2009). persals within Africa. A final aspect of the recent advances in understanding genetic diversity within Africa is related to data analysis. Datasets based on mul- Maps and migrations: insights to tiple, independently evolving genetic systems the genetic structure of Europe from are particularly well suited to simulation-based SNP data and PC analysis3 inferential frameworks which aim to distinguish between alternative models of population history The genetic variation of European individu- and to estimate key microevolutionary parame- als has been one of the most carefully character- ters under a given model. Recent applications of ized throughout the world and arguably across rejection algorithms and Approximate Bayesian Computation to infer the branching history of 3 lecture presented by John Novembre

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any species. Major pre-historic events invoked the fact that when doing genome-wide associa- to explain European genetic variation include the tion mapping for disease susceptibility loci, PC initial colonization of Europe, contractions and coordinates can be used as covariates to control expansions from glacial refugia due to Pleistocene for population stratification. Furthermore, indi- climate change, demographic expansions associ- vidual-based PCA has been argued to be attrac- ated with the Neolithic innovation of agriculture tive because it does not presume pre-defined in the Near East, and more recent population groups, nor does it assume a discrete set of ances- movements, such as those associated with the tral populations. early medieval period (Barbujani & Goldstein, An individual-based PCA plot of the 2004). Resolving which events had a predomi- European POPRES individuals shows a strik- nant effect on European genetic variation has ing resemblance to geographic maps of Europe been a long-standing goal of anthropological (Novembre et al., 2008; see Fig. 2). These results genetics, and is also relevant to the design and stand in contrast to alternative possibilities, such interpretation of genome-wide association stud- as clustering of European populations by lan- ies, tests of recent positive guage family (e.g. Romance, Slavic, Germanic selection, and personal ancestry testing. Despite languages). Notably, Hungarian individuals in the intensive attention, basic questions still the sample cluster with their geographic neigh- remain unanswered regarding what the domi- bours, a result which one might have found nant patterns are in European genetic variation surprising given they are local linguistic outliers and what ancestral events explain them. because they speak a non-Indo-European lan- Recent progress has been made due to the guage. PCA analyses by other groups at both advent of high throughput SNP genotyping tech- similar (e.g. Lao et al, 2008; Heath et al., 2008) nologies, which have made it possible to examine and finer spatial scales (e.g. within Finland and patterns of genetic variation in European samples Iceland, e.g. Lao et al., 2008; Sabatti et al., 2009) at an unprecedented scale. The application of SNP also evidence plots that resemble the geographic genotyping technology to European populations arrangement of populations (although in some has been facilitated by a growing recognition of cases the influence of relative sample sizes and/or the importance of population genetic variation for the presence of outlier populations distorts the mapping the variants underlying heritable disease basic pattern). traits and pharmacogenomic traits. For example, Why these PCA plots resemble geographic several thousand European individuals were sam- maps at all is an interesting question. Insight can pled and genotyped using the Affymetrix 500K be gained mathematically by considering cases in SNP genotyping platform as part of a collabora- which sampling is roughly uniform across space, tion between GlaxoSmithKline and academic sci- and the pattern of observed covariance amongst entists (the POPRES project, Nelson et al., 2008; individuals decays with geographic distance (an Novembre et al., 2008; Auton et al., 2009; data isolation-by-distance pattern). In these settings, available via dbGAP). PC coordinates will typically be a function of the To analyze patterns of variation in such a geographic position of each individual, and PC1 large set of polymorphic loci, researchers have and PC2 will form perpendicular gradients over been turning towards multivariate statistical geographic space (Novembre & Stephens, 2008). methods, chiefly principal components analysis This behaviour of PCA has been understood in (PCA). While PCA was first pioneered in the essence by some sub-disciplines of (e.g. 1970s to summarize patterns in sample fre- meteorology, image analysis) for some time, but quencies (e.g. Menozzi et al., 1978), a novel form their relevance was only recently noted within of individual-based PCA has recently become the population genetics community (Novembre popular for analyzing SNP data (e.g. Price et al., & Stephens, 2008). Importantly, the map-pro- 2006). This resurgence of PCA is mainly due to ducing behaviour of PCA is based on observed G. Destro-Bisol et al. 101

Fig. 2 – A principal component representation of genetic data from 1,387 Europeans (reprinted from Novembre et al., 2008). List of abbreviations: AL, Albania; AT, Austria; BA, Bosnia-Herzegovina; BE, Belgium; BG, Bulgaria; CH, Switzerland; CY, Cyprus; CZ, Czech Republic; DE, Germany; DK, Denmark; ES, Spain; FI, Finland; FR, France; GB, United Kingdom; GR, Greece; HR, Croatia; HU, Hungary; IE, Ireland; IT, Italy; KS, Kosovo; LV, Latvia; MK, Macedonia; NO, Norway; NL, Netherlands; PL, Poland; PT, Portugal; RO, Romania; RS, Serbia and Montenegro; RU, Russia, Sct, Scotland; SE, Sweden; SI, Slovenia; SK, Slovakia; TR, Turkey; UA, Ukraine; YG, Yugoslavia. See Novembre et al. (2008) for further details. patterns of spatial covariation in the data. Because broader context of European genetic diversity various processes or events may give rise to the same and what does it suggest about the peopling of pattern in which covariances decay with distance, Europe? An exciting arena of future research is it is still unclear which processes/events gives rise to use large panels of SNPs to understand the to the observed PCs in European populations. It fine-scale relationships of population isolates to certainly at some level must involve geographi- their geographic neighbours. Recent results from cally restricted mating, but how much of the spa- SNP studies in the Basque question whether the tial covariance is due to on-going geographically Basque are as isolated as previously supposed restricted mating versus more ancestral population (Laayouni et al., 2010; Garagnani et al., 2009). movements is unclear. On-going research is investigating the genetic Another major question that remains from origins of the Sorbs, a previously uncharacterized, this initial round of SNP studies is: how do puta- Slavic-speaking putative isolate from Eastern tive European population isolates fit into the Germany (Veeramah et al., in preparation).

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As studies of SNP diversity move forward, settled in by at least 50 kya, imply- one important caution is that while PCA is a ing that was already inhabited by this powerful tool for visualizing fine-scale popula- time, although unequivocal evidence from the tion structure, PCA can be dependent on rela- Subcontinent is more recent. Genetic estimates tive sample sizes (Novembre & Stephens 2009; are much less precise, but a recent new calibration McVean, 2009). As a result, the exact direction of the mtDNA mutation rate, which employs of PC1 can vary from study to study (contrast for the entire variation in the mtDNA genome for example Novembre et al., 2008 to Heath et al., maximum precision and makes allowance for the 2008). The expected PCA coordinates for each action of purifying selection, therefore also max- individual in a sample can be derived from aver- imising accuracy, provides at least one molecular age pair-wise coalescent times among individu- clock that can be employed for phylogeographic als in the sample (McVean, 2009), and doing so reconstructions (Soares et al., 2009). This sug- helps explain observations that PCA is dependent gests that modern humans first settled in Asia on relative sample-sizes (Novembre & Stephens, 60­–70 kya – somewhat earlier than the earliest 2009). In turn, we expect methods which are tai- widely accepted archaeological evidence, but lored to detect specific demographic signatures matching some less widely accepted evidence (e.g. the decay of diversity with distance from a from Australia and perhaps also China. putative origin) to be a powerful way forward in It was initially assumed that Eurasia had been illuminating the peopling of Europe. settled by modern humans via northeast Africa and the Levant, ~50 kya, and Y-chromosome evidence has been used to argue for a Central Asian “heart- and the peopling of land” from which much of the Old World was set- Asia4 tled (Wells et al., 2001). However, the aforemen- tioned dating evidence from Australia suggested Global patterns of human genetic diversity an earlier dispersal from the Horn of Africa across suggest that modern human variation is broadly the Red Sea and along the tropical southern Asian (albeit shallowly) structured at continental level, coastline. This was supported by the extremely with South Asia and (and probably high number of basal mtDNA haplogroup R and also Southeast Asia) forming genetic clusters or M lineages in India (Sun et al., 2006), and by simi- domains distinct both from each other and from larities between industries associated with modern (Native) America, Australasia, west Eurasia and humans in South Africa ~60 kya and South Asia at sub-Saharan Africa. This has been shown by ana- least 35 kya (Mellars, 2006). lysing multiple autosomal using the Analysis of complete mtDNA genomes STRUCTURE software (Rosenberg et al., 2006). sequences from so-called “relict” populations in However, evidence is accumulating, especially South Asia, Southeast Asia and Australasia have from the non-recombining marker systems, mito- been used to address this question. Modern non- chondrial DNA (mtDNA) and the non-recom- African populations throughout the world, with bining part of the Y chromosome (NRY), that this the exception of populations or regions with a is the result of sequential colonisation and expan- recent African ancestry, harbour mtDNAs from sion from very small founder groups who dis- just three major founder clades, M, N and (nested persed from an East African homeland within the closely within N) R, all of which belong to the last 70,000 years (ky) or so (Macaulay et al., 2005; L3 clade, which is of sub-Saharan African origin Metspalu et al., 2006; Richards et al., 2006). ~70 kya. Aboriginal populations in South Asia, Recent archaeological and fossil evidence Southeast Asia and Australasia display mtDNA suggests that anatomically modern humans were profiles that include basal lineages belonging to all three of the mtDNA founder clades, indicat- 4 lecture presented by Martin B. Richards ing that even the most ancient populations on the G. Destro-Bisol et al. 103

southern coast of Asia were part of the same, sin- Asia formed part of the mainland as the Sunda gle dispersal out of Africa (Macaulay et al., 2005). continent. Dental patterns, as well as genetic This pattern, and the molecular-clock tim- diversity, suggest that East Asia was initially set- ing of the dispersal to at least 60 kya, suggest tled from the south, although there is a suggestion that the primary expansion was along the south- in Y-chromosome patterns of an early offshoot ern coastal route, with the Asian continental from the southern route east of the Himalayas heartland (including Southwest Asia, and ulti- into the region of the Tibetan plateau, sometimes mately Europe) taking place subsequently along referred to as the “ steppe”. The north- various corridors as climatic conditions allowed, east Asian coast was reached at least 30 kya; some most likely after 50 kya. These dates seem to mtDNA and Y-chromosome lineages in Japan exclude the possibility, suggested on archaeologi- appear to trace to this time. Genetic and fossil cal grounds as well as on earlier genetic analy- data indicate discontinuities in the prehistory of ses, that the dispersal into South and Southeast East Asia; there are suggestions of subsequent re- Asia took place before the volcanic eruption of dispersals from north to south, which may be in Toba in Sumatra ~74 kya, which is therefore part due to Neolithic expansions, but seem likely unlikely to have had an impact on Asian popu- to also reflect the expansion of peo- lations. Moreover, the dispersal seems to have ple within the last 1,500 years or so. The impact been extremely rapid, within the space of a few of the is also likely to have thousand years, since it led to the divergence of been severe in continental East Asia, whereas refu- the distinct domains of basal mtDNA lineages gial areas existed within Southeast Asia. Sea-level in each region, rather than a pattern of nest- rises beginning ~19 kya had their maximal impact, ing (such as occurred in the settlement of the however, in Southeast Asia; the Sunda continent Americas from East Asia and the Remote Pacific was inundated leading to wide scale dispersals of from Southeast Asia/Near ). lineages across what is now Island Southeast Asia There is relatively little differentiation which may have had a much greater demographic between ethnic and language groups within impact than the subsequent Holocene spread of South Asia, which is similar to other parts of the Neolithic across Southeast Asia and into the Eurasia. The Indian Subcontinent has long been Pacific islands (Soares et al., 2008). seen as having been deeply affected by migra- tions from the north, and the non-recombining markers and autosomal SNP analysis indeed sug- A Genetic Perspective on Peopling of gest genetic gradients, but these have arisen from the Americas5 a variety of distinct prehistoric dispersals, with little or no impact attributable to the putative The colonization of the Americas represents Aryan migrations that are thought to have led to the most recent major human occupation of an the establishment of the caste system. There are uninhabited land mass on the planet. The recency mtDNAs in India that originated in Southwest of this event suggests that it may have left a sub- Asia but they probably arrived not long from the stantial signature in the genome. Therefore, we time of first settlement, and only a tiny minor- may be able to ask increasingly specific questions ity that appear to have arrived during historical and provide more detailed information about times. The demic impact of the Southwest Asian this process than for other older and more com- Neolithic appears to have been similarly minor plicated processes such as the initial migration for most of the Subcontinent, despite some of anatomically modern humans out of Africa. claims to the contrary (Chaubey et al., 2006). There are certain aspects of the colonization that Southeast Asia was settled by the south- are agreed upon by the scientific community, i.e. ern coastal route by ~55 kya according to the mtDNA clock, when much of Island Southeast 5 lecture presented by Connie Mulligan

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a single migration originated from an East Asian Once humans entered the Americas, it appears source and crossed over the Bering land bridge that their movement may have been very rapid before entering North America (summarized in based on archaeological evidence of human occu- Fig. 2 and Kitchen et al., 2008). This process pation at Monte Verde at the southern extent of created a strong population bottleneck such that South America ~14.5 kya (Dillehay, 2008). Simple modern Native Americans show significant reduc- simulation studies show that a rapid expansion is tions in genetic variation relative to other global necessary to maintain frequencies of the major populations and, furthermore, genetic variation mitochondrial haplogroups into the southern throughout the Americas shows evidence of sub- reaches of the Americas (Fix, 2004). Empirical stantial . Less consensus has been and simulation data suggest that genetic drift has reached for other parameters of the colonization played a significant role in determining patterns process such as the timing of the migration (both of Native American genetic diversity as evidenced leaving Asia and entering the Americas), size of by greater differentiation and population struc- the founding population, nature of the migration ture throughout the Americas relative to other from Asia (continuous movement versus several continents, reflecting the rapid dispersal, small short-range migrations), and migration route(s) population size, and genetic isolation of Native taken within the Americas. American groups. Native American genetic diver- sity also shows evidence of substantial admixture, Consensus on peopling of the Americas particularly through the incursion of European Y An East Asian source population for all indig- chromosomes (Wang et al., 2007). enous Native Americans, most likely around the Lake Baikal region, is widely accepted based on Debated points on peopling of the Americas mtDNA and Y chromosome data. The alterna- Of the issues still under active debate, the tive idea of an early European migration to the timing of the migration is a critical point. First, Americas prior to Columbus’ voyage in the 1490s it must be established that there are at least two to account for some Native American genetic relevant dates, the migration out of Asia and the diversity was once proposed based on presumed entry into the Americas. The first date is generally Caucasoid features of the famous ‘Kennewick based on the initial diversification of New World- Man’ discovered in the state of Washington; sup- specific haplogroups. For example, mtDNA data port for this idea has largely disappeared based support a date of ~30-40 kya (Bonatto & Salzano, on comparative skeletal analyses. The number of 1997), reflecting the initial diversification of migrations to the Americas was initially under New World genetic variation as the populations debate, but has converged on a single migration diverged from ancestral Asians but prior to their based on a wealth of data including mitochon- entry into the New World. The timing of entry drial DNA (mtDNA), Y chromosome markers, to the Americas is more debated and dates gener- short nuclear DNA sequences, and autosomal ally fall into periods that are pre- and post-LGM. markers (Mulligan et al., 2004; Different dates are frequently based on similar Wang et al., 2007; Fagundes et al., 2008) and mtDNA datasets but use different mitochondrial most recently, X chromosome sequence and genome substitution rates, i.e. ‘fast’ substitution nuclear single (SNP) rates (e.g. ~1.7 x 10-8 substitutions/site/year) sup- data (Bourgeois et al., 2009; Gutenkunst et al., port a post-LGM entry and ‘slow’ substitution 2009). Furthermore, most geneticists believe rates (e.g. ~1.26 x 10-8 substitutions/site/year) sup- there was virtually no ancient between port a pre-LGM entry. Endicott & Ho (2008) rec- Asia and the Americas after the initial migra- ommend that substitution rate estimates should tion, likely reflecting inundation of the exposed be based on an ‘internal calibration’ of the under- Bering land bridge after the last glacial maximum lying phylogeny used in the rate estimation; their (LGM) ~18-23 kya. estimates of the mitochondrial coding genome G. Destro-Bisol et al. 105

substitution rate generally support younger dates, thus, warrant attention. 1) Better estimates of sub- i.e. post-LGM entry. stitution rates, both mitochondrial and nuclear, The tempo of the migration has recently are necessary to provide robust support for age received widespread attention, e.g. Tamm et al. estimates of key events within the colonization 2007. This issue can be viewed as an investiga- process. This is particularly true for estimates of tion of the movement of people (was it a continu- entry to the Americas since a pre-LGM entry ous movement or a series of short-range migra- implies that the migrant population overcame tions?) or a focus on when (and where) did the severe climatic and geologic, i.e. North American genetic variation that is specific to and ubiquitous ice sheets, obstacles to survive that would not throughout the New World occur? There are have been present if their entry postdated the mitochondrial variants that define New World- LGM. 2) A better understanding of the period specific haplogroups, e.g. C1b, C1d, X2a (Tamm prior to entry into the Americas is also worthy of et al., 2007) prompting researchers to propose a study, i.e. Was Beringia the occupied land mass? period of population isolation prior to expansion How long was the occupation? What proportion into the Americas (first mentioned by Bonatto & of the population actually entered the Americas? Salzano in 1997). Mulligan et al. (2008) estimated 3) Continued investigation of patterns of genetic that ~7000-15,000 years were required to gener- variation within the Americas is necessary in ate the New World-specific variation. It has been order to better understand the various regional further proposed that the migrating population colonization events that occurred after the ini- occupied Beringia during this period of isolation. tial entry into the Americas. Studies that look for Paleoecological data from ancient eastern Beringia correlation between genetics and linguistics have are indicative of productive, dry grassland suggest- a checkered history in terms of providing general ing that Beringia was able to sustain at least small insights; most likely, correlation between linguis- populations of humans and other large mammals. tics and genetics will reflect unique regional his- The lack of archaeological data for human occu- tories and not general trends or processes during pation of Beringia most likely reflects the fact that the course of colonization. 4) There is a move the proposed occupation sites are now inundated. towards more simulation of data and modeling The size of the founding population has of alternative evolutionary scenarios in addition also been the subject of considerable study. New to continued collection of empirical data. The estimates based on mtDNA coding genomes simulation and modeling approaches have the and short nuclear sequences support an effec- advantage of statistically determining the good- tive population size of ~1,000-2,000 individuals ness of fit between empirical data and alternative (Fagundes et al., 2007; Mulligan et al., 2008). scenarios. For example, the support for a coastal Once the population entered the Americas, there and inland route within the Americas was sup- is considerable interest in determining the exact ported by the differential distribution of two route(s) taken by the migrants. The distribution distinctive mitochondrial haplogroups (Perego of two specific mtDNA haplogroups was used to et al., 2009); it would be informative to know support both coastal and inland routes (Perego et how often such a distribution occurs by random al., 2009), but simulation and empirical studies chance and, thus, if the actual distribution is suf- of whole mitochondrial genomes and hundreds ficiently unique to require explanation via sepa- of autosomal microsatellite markers strongly sup- rate migration routes within the Americas. 5) A port coastal routes over inland routes (Fix, 2004; broad perspective on the colonization process is Wang et al., 2007; Fagundes et al., 2008). also valuable. Comparison with other coloniza- tion processes, i.e. migration out of Africa, pro- Future research vides a complementary perspective and allows There are multiple aspects of the peopling of general inferences on the colonization process to the Americas that are still subject to debate and, be formulated.

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Perspectives and prospects for Molecular Anthropology in the Genomic Era

The congress “DNA Polymorphisms in Human Populations: Molecular Anthropology in the Genomic Era” offered an important opportunity to scholars and students to discuss some topical aspects of research in human evolu- tion. This initiative provided us with a picture of the various ways in which the genetic structure of human populations can be explored, show- ing the versatility of researchers in using dif- ferent sampling schemes, exploring variation at diverse geographic scales, looking at genes which are neutral or amenable to selection and focusing on whole genomes or specific lineages. A general impression we obtained from most of the presentations given in the course of the meeting is that modelling and comparison of evolutionary scenarios by data simulations are finally becoming a widespread alternative to the descriptive reports and ad-hoc explanations which have represented the standard for population studies up to a few years ago. In fact, many contributions have com- pared evolutionary histories using new computa- tional methods which are being developed to take a growing number of variables into account. This substantial change of perspective seems to dem- onstrate the consciousness, acquired by Molecular Anthropologists, of the importance of moving towards hypothesis testing approaches, and follows the path set out by Stringer and Andrews (1988), with the further advantage of using quantita- tive methodologies. It may also stimulate further Fig. 3 - Maps depicting a three-step coloniza- advancements, since the availability of multilocus tion model for the peopling of the Americas. (A) data and dense sampling will, hopefully, make it Divergence, then gradual population expan- sion of the Amerind ancestors from an East possible to test spatial models of human migrations Central Asian gene pool (blue arrow). (B) more carefully, which is another key issue in the Proto-Amerind occupation of Beringia with lit- reconstruction of the prehistory of our species. tle to no population growth for ~15,000 years. (C) Rapid colonization of the New World by a Thanks to the genomic approach, some founder group migrating southward through important results have been already achieved and the ice free, inland corridor between the east- further developments are to be expected. These ern Laurentide and western Cordilleran Ice include the recent calibration of the mtDNA Sheets (green arrow) and/or along the Pacific coast (red arrow). The lowest frame depicts mutation rate which takes into account the effect Beringia as it is today. Modified from Kitchen of purifying selection and makes phylogeographic et al. (2008). reconstructions more reliable (Soares et al., 2009). G. Destro-Bisol et al. 107

Furthermore, it has been pointed out that the set up Even more importantly, we have learned that of broad panels of genetic informative loci which making this integration more complete and fruit- have proved useful for the investigation of large ful will be crucial in achieving new targets and geographic areas inhabited by genetically heteroge- will extend applications to other anthropological neous populations, such as that studied by Tishkoff questions. We hope that “DNA Polymorphisms et al. (2009), provides a framework for optimizing in Human Populations: Molecular Anthropology cost/benefit ratios in future studies which aim to in the Genomic Era” will make a significant con- fill sampling gaps and gain a more complete pic- tribution in this direction. ture of genetic diversity and population history. Regarding future prospects, it has been envi- sioned that the power of genomic approaches Acknowledgements will also help Anthropology overcome some of its inherent limitations. This could material- The congress “DNA Polymorphisms in Human Pop- ize if the study of entire genomes opens up new ulations: Molecular Anthropology in the Genomic avenues for the identification of genetic determi- Era” was supported by The University of Rome “La nants underlying complex phenotypes of special Sapienza”, Ministero della Ricerca e dell’Istruzione, interest for human evolutionary biology, such as Ministero per i Beni e le Attività Culturali, Ateneo stature and high altitude adaptation. Federato della Scienza e della Tecnica and by the While the advent of genomics is already revo- journal “Human Biology” (Wayne State Univer- lutionizing research in Molecular Anthropology sity Press, Detroit MI, USA). GDB acknowledges and promises to continue to do so in the near funds from the MIUR (PRIN project 2007-2009 future, some interdisciplinary lines of anthropo- n. 2007TYXE3X) and the Università di Roma logical research have maintained all their relevance “La Sapienza” (prot. C26A09EA9C/2009). MAJ or seem to be destined to attract even more inter- is a Wellcome Trust Senior Fellow in Basic Bio- est. The lectures summarized here draw attention medical Science (grant no. 087576), and gratefully to the importance of studies of well defined pop- acknowledges support from the Wellcome Trust. JR ulations to help clarify issues of general interest, was partially financed by the research grants from such as the relations between cultural and bio- Fundação para a Ciência e a Tecnologia (FCT): logical changes or the assessment of hypotheses PPCDT/BIA-BDE/56654/2004 and PTDC/BIA- on routes of major migratory events in human BDE/68999/2006. We express our gratitude to the prehistory. This is the case of human groups with members of the scientific committee: Cristian Capelli, unusual combinations of genetic, linguistic and Oscar Lao Grueso, Andres Moreno-Estrada and Paul lifestyle features in Africa, relict populations in Verdu. Finally, we warmly thank Paolo Anagnostou, Asia and Australasia and European isolates (see Cinzia Battaggia, Valentina Coia, Vera Damiani, also Destro-Bisol et al., 2008 in this Journal). It Francesco Montinaro, Veronica Marcari and Nancy is also worth noting that new research avenues Wise for their help during the Congress. opened up by genomics revitalize interest in environmental aspects, viewed either as variables which act as co-determinants of phenotypic References variation in complex traits, or paleo-ecological changes which could have had a deep impact on Aulchenko Y.S., Struchalin M.V., Belonogova past human mass migrations. N.M., Axenovich T.I., Weedon M.N., Hofman In conclusion, our congress showed that A., Uitterlinden A.G., Kayser M., Oostra B.A., the combination between interdisciplinary van Duijn C.M., Janssens A.C. & Borodin P.M. approaches and methodological and theoretical 2009. Predicting human height by Victorian innovations has become an essential aspect for and genomic methods. Eur. J. Hum. Genet., 17: studies of human evolution at molecular level. 1070-1075.

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