Iain Mathieson

Songül Alpaslan-Roodenberg

Cosimo Posth

Anna Szécsényi-Nagy

Nadin Rohland

Swapan Mallick

Iñigo Olalde

Nasreen Broomandkhoshbacht

Francesca Candilio

Olivia Cheronet

Alexandrov 17

Kurt W Alt1

Radian Andreescu

Dragana Antonović

Abigail Ash

Nadezhda Atanassova

Krum Bacvarov

Mende Balázs Gusztáv

Hervé Bocherens

Michael Bolus

Adina Boroneanţ

Yavor Boyadzhiev

Alicja Budnik

Josip Burmaz

Stefan Chohadzhiev

Nicholas J Conard

Richard Cottiaux

Maja Čuka

Christophe Cupillard

Dorothée G Drucker

Nedko Elenski

Michael Francken

Borislava Galabova

Georgi Ganetsovski

Bernard Gély

Tamás Hajdu

Veneta Handzhyiska

Katerina Harvati

Thomas Higham

Stanislav Iliev

Ivor Janković

Ivor Karavanić

Douglas J Kennett

Darko Komšo

Alexandra Kozak

Damian Labuda

Martina Lari

Catalin Lazar

Maleen Leppek

Krassimir Leshtakov

Domenico Lo Vetro

Dženi Los

Ivaylo Lozanov

Maria Malina

Fabio Martini

Kath Mcsweeney

Harald Meller

Marko Menđušić

Pavel Mirea

Vyacheslav Moiseyev

Vanya Petrova

T Douglas Price

Angela Simalcsik

Luca Sineo

Mario Šlaus

Vladimir Slavchev

Petar Stanev

Andrej Starović

Tamás Szeniczey

Sahra Talamo

Maria Teschler-Nicola

Corinne Thevenet

Ivan Valchev

Frédérique Valentin

Sergey Vasilyev

Fanica Veljanovska

Svetlana Venelinova

Elizaveta Veselovskaya

Bence Viola

Cristian Virag

Joško Zaninović

Steve Zäuner

Philipp W Stockhammer

Giulio Catalano

Raiko Krauß

David Caramelli

Gunita Zariņa

Bisserka Gaydarska

Malcolm Lillie

Alexey G Nikitin

Inna Potekhina

Anastasia Papathanasiou

Dušan Borić

Clive Bonsall

Johannes Krause

Ron Pinhasi
email: ron.pinhasi@ucd.ie

David Reich
email: reich@genetics.med.harvard.edu

The genomic history of southeastern Europe

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Introduction

The southeastern quadrant of Europe was the beachhead in the spread of agriculture from its source in the Fertile Crescent of southwestern Asia. After the first appearance of agriculture in the mid-7 th millennium BCE [START_REF] Tringham | The Transition to Agriculture in Prehistoric Europe[END_REF][START_REF] Bellwood | First Farmers: The Origins of Agricultural Societies[END_REF] farming spread westward via a Mediterranean and northwestward via a Danubian route, and was established in both Iberia and Central Europe by 5600 BCE. [START_REF] Golitko | Ancient Europe, 8000 BC to AD 1000: An Encyclopedia of the Barbarian World[END_REF][START_REF] Vander Linden | Investigating Archaeological Cultures: Material Culture, Variability, and Transmission[END_REF] Ancient DNA studies have shown that the spread of farming across Europe was accompanied by a massive movement of people [START_REF] Bramanti | Genetic discontinuity between local hunter-gatherers and central Europe's first farmers[END_REF][START_REF] Skoglund | Origins and genetic legacy of Neolithic farmers and hunter-gatherers in Europe[END_REF][START_REF] Haak | Massive migration from the steppe was a source for Indo-European languages in Europe[END_REF][START_REF] Cassidy | Neolithic and Bronze Age migration to Ireland and establishment of the insular Atlantic genome[END_REF] closely related to the farmers of northwestern Anatolia [START_REF] Hofmanova | Early farmers from across Europe directly descended from Neolithic Aegeans[END_REF][START_REF] Mathieson | Genome-wide patterns of selection in 230 ancient Eurasians[END_REF][START_REF] Omrak | Genomic Evidence Establishes Anatolia as the Source of the European Neolithic Gene Pool[END_REF] but nearly all the ancient DNA from Europe's first farmers is from central and western Europe, with only three individuals reported from the southeast. [START_REF] Hofmanova | Early farmers from across Europe directly descended from Neolithic Aegeans[END_REF] In the millennia following the establishment of agriculture in the Balkan Peninsula, a series of complex societies formed, culminating in sites such as the mid-5 th millennium BCE necropolis at Varna, which has some of the earliest evidence of extreme inequality in wealth, with one individual (grave 43) from whom we extracted DNA buried with more gold than is known from any earlier site. By the end of the 6 th millennium BCE, agriculture had reached eastern Europe, in the form of the Cucuteni-Trypillian complex in the area of present-day Moldova, Romania and Ukraine, including "mega-sites" that housed hundreds, perhaps thousands, of people. [START_REF] Müller | Trypillia Mega-Sites and European Prehistory[END_REF] After around 4000 BCE, these settlements were largely abandoned, and archaeological evidence documents cultural contacts with peoples of the Eurasian steppe. [START_REF] Anthony | The horse the wheel and language[END_REF] However, the population movements that accompanied these events have been unknown due to the lack of ancient DNA.

Results

We generated genome-wide data from 225 ancient humans (216 reported for the first time), from the Balkan Peninsula, the Carpathian Basin, the North Pontic Steppe and neighboring regions, dated to 12,000-500 BCE (Figure 1, Supplementary Information Table 1, Supplementary Information Note 1). We extracted DNA from skeletal remains in dedicated clean rooms, built DNA libraries and enriched for DNA fragments overlapping 1.24 million single nucleotide polymorphisms (SNPs), then sequenced the product and restricted to libraries with evidence of authentic ancient DNA. [START_REF] Haak | Massive migration from the steppe was a source for Indo-European languages in Europe[END_REF][START_REF] Mathieson | Genome-wide patterns of selection in 230 ancient Eurasians[END_REF][START_REF] Fu | An early modern human from Romania with a recent Neanderthal ancestor[END_REF] We filtered out individuals with fewer than 15,000 SNPs covered by at least one sequence, or that had unexpected ancestry for their archaeological context and were not directly dated. We report, but do not analyze, nine individuals that were first-degree relatives of others in the dataset, resulting in an analysis dataset of 216 individuals. We analyzed these data together with 274 previously reported ancient individuals, [START_REF] Hofmanova | Early farmers from across Europe directly descended from Neolithic Aegeans[END_REF][START_REF] Mathieson | Genome-wide patterns of selection in 230 ancient Eurasians[END_REF][START_REF] Omrak | Genomic Evidence Establishes Anatolia as the Source of the European Neolithic Gene Pool[END_REF][START_REF] Allentoft | Population genomics of Bronze Age Eurasia[END_REF][START_REF] Fu | Genome sequence of a 45,000-year-old modern human from western Siberia[END_REF][START_REF] Fu | The genetic history of Ice Age Europe[END_REF][START_REF] Gallego Llorente | Ancient Ethiopian genome reveals extensive Eurasian admixture in Eastern Africa[END_REF][START_REF] Jones | Upper Palaeolithic genomes reveal deep roots of modern Eurasians[END_REF][START_REF] Keller | New insights into the Tyrolean Iceman's origin and phenotype as inferred by whole-genome sequencing[END_REF][START_REF] Kilinc | The Demographic Development of the First Farmers in Anatolia[END_REF][START_REF] Lazaridis | Genomic insights into the origin of farming in the ancient Near East[END_REF][START_REF] Lazaridis | Ancient human genomes suggest three ancestral populations for present-day Europeans[END_REF][START_REF] Olalde | Derived immune and ancestral pigmentation alleles in a 7,000-year-old Mesolithic European[END_REF][START_REF] Raghavan | Upper Palaeolithic Siberian genome reveals dual ancestry of Native Americans[END_REF][START_REF] Lazaridis | Genetic origins of the Minoans and Mycenaeans[END_REF][START_REF] Lipson | Parallel ancient genomic transects reveal complex population history of early European farmers[END_REF] 777 present-day individuals genotyped on the Illumina "Human Origins" array, [START_REF] Lazaridis | Ancient human genomes suggest three ancestral populations for present-day Europeans[END_REF] and 300 high coverage genomes from the Simons Genome Diversity Project (SGDP). [START_REF] Mallick | The Simons Genome Diversity Project: 300 genomes from 142 diverse populations[END_REF] We used principal component analysis (PCA; Figure 1B, Extended Data Figure 1), supervised and unsupervised ADMIXTURE (Figure 1D, Extended Data Figures 2&3), [START_REF] Alexander | Fast model-based estimation of ancestry in unrelated individuals[END_REF] D-statistics, qpAdm and qpGraph, [START_REF] Patterson | Ancient admixture in human history[END_REF] along with archaeological and chronological information (including 137 newly reported AMS [START_REF] Fu | An early modern human from Romania with a recent Neanderthal ancestor[END_REF] C dates) to cluster the individuals into populations and investigate the relationships among them.

We described the individuals in our dataset in terms of their genetic relatedness to a hypothesized set of ancestral populations, which we refer to as their genetic ancestry. It has previously been shown that the great majority of European ancestry derives from three distinct sources. [START_REF] Lazaridis | Ancient human genomes suggest three ancestral populations for present-day Europeans[END_REF] First, "hunter-gatherer-related" ancestry that is more closely related to Mesolithic hunter-gatherers from Europe than to any other population, and can be further subdivided into "Eastern" (EHG) and "Western" (WHG) hunter-gatherer-related ancestry. [START_REF] Haak | Massive migration from the steppe was a source for Indo-European languages in Europe[END_REF] Second, "NW Anatolian Neolithic-related" ancestry related to the Neolithic farmers of northwest Anatolia and tightly linked to the appearance of agriculture. [START_REF] Hofmanova | Early farmers from across Europe directly descended from Neolithic Aegeans[END_REF][START_REF] Mathieson | Genome-wide patterns of selection in 230 ancient Eurasians[END_REF] The third source, "steppe-related" ancestry, appears in Western Europe during the Late Neolithic to Bronze Age transition and is ultimately derived from a population related to Yamnaya steppe pastoralists. [START_REF] Haak | Massive migration from the steppe was a source for Indo-European languages in Europe[END_REF][START_REF] Allentoft | Population genomics of Bronze Age Eurasia[END_REF] Steppe-related ancestry itself can be modeled as a mixture of EHG-related ancestry, and ancestry related to Upper Palaeolithic hunter-gatherers of the Caucasus (CHG) and the first farmers of northern Iran. [START_REF] Jones | Upper Palaeolithic genomes reveal deep roots of modern Eurasians[END_REF][START_REF] Kilinc | The Demographic Development of the First Farmers in Anatolia[END_REF][START_REF] Lazaridis | Genomic insights into the origin of farming in the ancient Near East[END_REF] Hunter-Gatherer substructure and transitions Of the 216 new individuals we report, 106 from Paleolithic, Mesolithic and eastern European Neolithic contexts have almost entirely hunter-gatherer-related ancestry (in eastern Europe, unlike western Europe, "Neolithic" refers to the presence of pottery, [START_REF] Gronenborn | The Oxford Handbook of Neolithic Europe[END_REF][START_REF] Telegin | Neolithic cultures of Ukraine and their chronology[END_REF][START_REF] Telegin | Neolithic cemeteries and populations in the Dnieper Basin[END_REF] not necessarily to farming). These individuals form a cline from WHG to EHG that is correlated with geography (Figure 1B), although it is neither geographically nor temporally uniform (Figure 2, Extended Data Figure 4), and contains substructure in phenotypically important variants (Supplementary Information Note 2).

From present-day Ukraine, our study reports new genome-wide data from seven Mesolithic (~9500-6000 BCE) and 30 Neolithic (~6000-3500 BCE) individuals. On the cline from WHG-to EHG-related ancestry, the Mesolithic individuals fall towards the East, intermediate between EHG and Mesolithic hunter-gatherers from Scandinavia (Figure 1B). [START_REF] Haak | Massive migration from the steppe was a source for Indo-European languages in Europe[END_REF] The Neolithic population has a significant difference in ancestry compared to the Mesolithic (Figures 1B, Figure 2), with a shift towards WHG shown by the statistic D(Mbuti, WHG, Ukraine_Mesolithic, Ukraine_Neolithic); Z=8.5 (Supplementary Information Table 2). Unexpectedly, one Neolithic individual from Dereivka (I3719), which we directly date to 4949-4799 BCE, has entirely NW Anatolian Neolithic-related ancestry.

The pastoralist Bronze Age Yamnaya complex originated on the Eurasian steppe and is a plausible source for the dispersal of steppe-related ancestry into central and western Europe around 2500 BCE. [START_REF] Anthony | The horse the wheel and language[END_REF] All previously reported Yamnaya individuals were from Samara [START_REF] Haak | Massive migration from the steppe was a source for Indo-European languages in Europe[END_REF] and Kalmykia [START_REF] Allentoft | Population genomics of Bronze Age Eurasia[END_REF] in southwest Russia, and had entirely steppe-related ancestry. Here, we report three Yamnaya individuals from further West -Ukraine and Bulgaria -and show that while they all have high levels of steppe-related ancestry, one from Ozera in Ukraine and one from Bulgaria (I1917 and Bul4, both dated to ~3000 BCE) have NW Anatolian Neolithic-related admixture, the first evidence of such ancestry in Yamnaya-associated individuals (Figure 1B&D, Supplementary Data Table 2). Preceding the Yamnaya culture, four Copper Age individuals (I4110, I5882, I5884 and I6561; Ukraine_Eneolithic) from Dereivka and Alexandria dated to ~3600-3400 BCE have ancestry that is a mixture of hunter-gatherer-, steppe-and NW Anatolian Neolithic-related (Figure 1D, Supplementary Data Table 2).

At Zvejnieki in Latvia (17 newly reported individuals, and additional data for 5 first reported in Ref. 34) we observe a transition in hunter-gatherer-related ancestry that is opposite to that seen in Ukraine. We find (Supplementary Data Table 3) that Mesolithic and Early Neolithic individuals (Latvia_HG) associated with the Kunda and Narva cultures have ancestry intermediate between WHG (~70%) and EHG (~30%), consistent with previous reports. [START_REF] Jones | The Neolithic Transition in the Baltic Was Not Driven by Admixture with Early European Farmers[END_REF][START_REF] Mittnik | The Genetic History of Northern Europe[END_REF][START_REF] Saag | Extensive farming in Estonia started through a sex-biased migration from the Steppe[END_REF] We also detect a shift in ancestry between the Early Neolithic and individuals associated with the Middle Neolithic Comb Ware Complex (Latvia_MN), who have more EHG-related ancestry (we estimate 65% EHG, but two of four individuals appear to be 100% EHG in PCA). The most recent individual, associated with the Final Neolithic Corded Ware Complex (I4629, Latvia_LN), attests to another ancestry shift, clustering closely with Yamnaya from Samara, 7 Kalmykia 15 and Ukraine (Figure 2).

We report new Upper Palaeolithic and Mesolithic data from southern and western Europe. [START_REF] Fu | The genetic history of Ice Age Europe[END_REF] Sicilian (I2158) and Croatian (I1875) individuals dating to ~12,000 and 6100 BCE cluster with previously reported western hunter-gatherers (Figure 1B&D), including individuals from Loschbour [START_REF] Lazaridis | Ancient human genomes suggest three ancestral populations for present-day Europeans[END_REF] (Luxembourg, 6100 BCE), Bichon [START_REF] Jones | Upper Palaeolithic genomes reveal deep roots of modern Eurasians[END_REF] (Switzerland, 11,700 BCE), and Villabruna 17 (Italy 12,000 BCE). These results demonstrate that WHG populations [START_REF] Lazaridis | Ancient human genomes suggest three ancestral populations for present-day Europeans[END_REF] were widely distributed from the Atlantic seaboard of Europe in the West, to Sicily in the South, to the Balkan Peninsula in the Southeast, for at least six thousand years.

A particularly important hunter-gatherer population that we report is from the Iron Gates region that straddles the border of present-day Romania and Serbia. This population (Iron_Gates_HG) is represented in our study by 40 individuals from five sites. Modeling Iron Gates hunter-gatherers as a mixture of WHG and EHG (Supplementary Table 3) shows that they are intermediate between WHG (~85%) and EHG (~15%). However, this qpAdm model does not fit well (p=0.0003, Supplementary Table 3) and the Iron Gates huntergatherers show an affinity towards Anatolian Neolithic, relative to WHG (Supplementary Table 2). In addition, Iron Gates hunter-gatherers carry mitochondrial haplogroup K1 (7/40) as well as other subclades of haplogroups U (32/40) and H (1/40) in contrast to WHG, EHG and Scandinavian hunter-gatherers who almost all carry haplogroups U5 or U2. One interpretation is that the Iron Gates hunter-gatherers have ancestry that is not present in either WHG or EHG. Possible scenarios include genetic contact between the ancestors of the Iron Gates population and a NW Anatolian-Neolithic-related population, or that the Iron Gates population is related to the source population from which the WHG split during a reexpansion into Europe from the Southeast after the Last Glacial Maximum. [START_REF] Fu | The genetic history of Ice Age Europe[END_REF][START_REF] Maier | The Central European Magdalenian: Regional Diversity and Internal Variability[END_REF] A notable finding from the Iron Gates concerns the four individuals from the site of Lepenski Vir, two of whom (I4665 & I5405, 6200-5600 BCE), have entirely NW Anatolian Neolithic-related ancestry. Strontium and Nitrogen isotope data [START_REF] Borić | Strontium isotopes document greater human mobility at the start of the Balkan Neolithic[END_REF] indicate that both these individuals were migrants from outside the Iron Gates, and ate a primarily terrestrial diet (Supplementary Information section 1). A third individual (I4666, 6070 BCE) has a mixture of NW Anatolian Neolithic-related and hunter-gatherer-related ancestry and ate a primarily aquatic diet, while a fourth, probably earlier, individual (I5407) had entirely hunter-gathererrelated ancestry (Figure 1D, Supplementary Information section 1). We also identify one individual from Padina (I5232), dated to 5950 BCE that had a mixture of NW Anatolian Neolithic-related and hunter-gatherer-related ancestry. These results demonstrate that the Iron Gates was a region of interaction between groups distinct in both ancestry and subsistence strategy.

Population transformations in the first farmers

Neolithic populations from present-day Bulgaria, Croatia, Macedonia, Serbia and Romania cluster closely with the NW Anatolian Neolithic (Figure 1), consistent with archaeological evidence. [START_REF] Krauß | The rapid spread of early farming from the Aegean into the Balkans via the Sub-Mediterranean-Aegean Vegetation Zone[END_REF] Modeling Balkan Neolithic populations as a mixture of NW Anatolian Neolithic and WHG, we estimate that 98% (95% confidence interval [CI]; 97-100%) of their ancestry is NW Anatolian Neolithic-related. A striking exception is evident in 8 out of 9 individuals from Malak Preslavets in present-day Bulgaria. [START_REF] Bacvarov | Moments in time: Papers Presented to Pál Raczky on His 60th Birthday[END_REF] These individuals lived in the mid-6 th millennium BCE and have significantly more hunter-gatherer-related ancestry than other Balkan Neolithic populations (Figure 1B,D, Extended Data Figures 123, Supplementary Tables 234); a model of 82% (CI: 77-86%) NW Anatolian Neolithic-related, 15% (CI: 12-17%) WHG-related, and 4% (CI: 0-9%) EHG-related ancestry fits the data. This huntergatherer-related ancestry with a ~4:1 WHG:EHG ratio plausibly represents a contribution from local Balkan hunter-gatherers genetically similar to those of the Iron Gates. Late Mesolithic hunter-gatherers in the Balkans were likely concentrated along the coast and major rivers such as the Danube, [START_REF] Gurova | Pre-Neolithic' in Southeast Europe: a Bulgarian perspective[END_REF] which directly connects the Iron Gates with Malak Preslavets. Thus, early farmer groups with the most hunter-gatherer-related ancestry may have been those that lived close to the highest densities of hunter-gatherers.

In the Balkans, Copper Age populations (Balkans_Chalcolithic) harbor significantly more hunter-gatherer-related ancestry than Neolithic populations as shown, for example, by the statistic D(Mbuti, WHG, Balkans_Neolithic, Balkans_Chalcolithic); Z=4.3 (Supplementary Data Table 2). This is roughly contemporary with the "resurgence" of hunter-gatherer ancestry previously reported in central Europe and Iberia [START_REF] Haak | Massive migration from the steppe was a source for Indo-European languages in Europe[END_REF][START_REF] Mathieson | Genome-wide patterns of selection in 230 ancient Eurasians[END_REF][START_REF] Brandt | Ancient DNA reveals key stages in the formation of central European mitochondrial genetic diversity[END_REF] and is consistent with changes in funeral rites, specifically the reappearance around 4500 BCE of the Mesolithic tradition of extended supine burial -in contrast to the Early Neolithic tradition of flexed burial. [START_REF] Borić | The Oxford Handbook of Neolithic Europe[END_REF] Four individuals associated with the Copper Age Trypillian population have ~80% NW Anatolian-related ancestry (Supplementary Table 3), confirming that the ancestry of the first farmers of present-day Ukraine was largely derived from the same source as the farmers of Anatolia and western Europe. Their ~20% hunter-gatherer ancestry is intermediate between WHG and EHG, consistent with deriving from the Neolithic hunter-gatherers of the region.

We also report the first genetic data associated with the Late Neolithic Globular Amphora Complex. Individuals from two Globular Amphora sites in Poland and Ukraine form a tight cluster, showing high similarity over a large distance (Figure 1B,D). Both groups of Globular Amphora Complex samples had more hunter-gatherer-related ancestry than Middle Neolithic groups from Central Europe 7 (we estimate 25% [CI: 22-27%] WHG ancestry, similar to Chalcolithic Iberia, Supplementary Data Table 3). In east-central Europe, the Globular Amphora Complex preceded or abutted the Corded Ware Complex that marks the appearance of steppe-related ancestry, [START_REF] Haak | Massive migration from the steppe was a source for Indo-European languages in Europe[END_REF][START_REF] Allentoft | Population genomics of Bronze Age Eurasia[END_REF] while in southeastern Europe, the Globular Amphora Complex bordered populations with steppe-influenced material cultures for hundreds of years [START_REF] Szmyt | Transition to the Bronze Age[END_REF] and yet the individuals in our study have no evidence of steppe-related ancestry, supporting the hypothesis that this material cultural frontier was also a barrier to gene flow.

The movements from the Pontic-Caspian steppe of individuals similar to those associated with the Yamnaya Cultural Complex in the 3 rd millennium BCE contributed about 75% of the ancestry of individuals associated with the Corded Ware Complex and about 50% of the ancestry of succeeding material cultures such as the Bell Beaker Complex in central Europe. [START_REF] Haak | Massive migration from the steppe was a source for Indo-European languages in Europe[END_REF][START_REF] Allentoft | Population genomics of Bronze Age Eurasia[END_REF] In two directly dated individuals from southeastern Europe, one (ANI163) from the Varna I cemetery dated to 4711-4550 BCE and one (I2181) from nearby Smyadovo dated to 4550-4450 BCE, we find far earlier evidence of steppe-related ancestry (Figure 1B,D). These findings push back the first evidence of steppe-related ancestry this far West in Europe by almost 2,000 years, but it was sporadic as other Copper Age (~5000-4000 BCE) individuals from the Balkans have no evidence of it. Bronze Age (~3400-1100 BCE) individuals do have steppe-related ancestry (we estimate 30%; CI: 26-35%), with the highest proportions in the four latest Balkan Bronze Age individuals in our data (later than ~1700 BCE) and the least in earlier Bronze Age individuals (3400-2500 BCE; Figure 1D).

A new source of ancestry in Neolithic Europe

An important question about the initial spread of farming into Europe is whether the first farmers that brought agriculture to northern Europe and to southern Europe were derived from a single population or instead represent distinct migrations. We confirm that Mediterranean populations, represented in our study by individuals associated with the Epicardial Early Neolithic from Iberia 7 , are closely related to Danubian populations represented by the Linearbandkeramik (LBK) from central Europe [START_REF] Haak | Massive migration from the steppe was a source for Indo-European languages in Europe[END_REF][START_REF] Olalde | A Common Genetic Origin for Early Farmers from Mediterranean Cardial and Central European LBK Cultures[END_REF] and that both are closely related to the Balkan Neolithic population. These three populations form a clade with the NW Anatolian Neolithic individuals as an outgroup, consistent with a single migration into the Balkan peninsula, which then split into two (Supplementary Information Note 3).

In contrast, five southern Greek Neolithic individuals (Peloponnese_Neolithic) -three (plus one from Ref. 26) from Diros Cave and one from Franchthi Cave -are not consistent with descending from the same source population as other European farmers. D-statistics (Supplementary Information Table 2) show that in fact, these "Peloponnese Neolithic" individuals dated to ~4000 BCE are shifted away from WHG and towards CHG, relative to Anatolian and Balkan Neolithic individuals. We see the same pattern in a single Neolithic individual from Krepost in present-day Bulgaria (I0679_d, 5718-5626 BCE). An even more dramatic shift towards CHG has been observed in individuals associated with the Bronze Age Minoan and Mycenaean cultures, [START_REF] Lazaridis | Genetic origins of the Minoans and Mycenaeans[END_REF] suggesting gene flow into the region from populations with CHG-rich ancestry throughout the Neolithic, Chalcolithic and Bronze Age. Possible sources are related to the Neolithic population from the central Anatolian site of Tepecik Çiftlik, [START_REF] Kilinc | The Demographic Development of the First Farmers in Anatolia[END_REF] or the Aegean site of Kumtepe, [START_REF] Omrak | Genomic Evidence Establishes Anatolia as the Source of the European Neolithic Gene Pool[END_REF] who are also shifted towards CHG relative to NW Anatolian Neolithic samples, as are later Copper and Bronze Age Anatolians.

10,26

Sex-biased admixture between hunter-gatherers and farmers

We provide the first evidence for sex-biased admixture between hunter-gatherers and farmers in Europe, showing that the Middle Neolithic "resurgence" of hunter-gathererrelated ancestry [START_REF] Haak | Massive migration from the steppe was a source for Indo-European languages in Europe[END_REF][START_REF] Brandt | Ancient DNA reveals key stages in the formation of central European mitochondrial genetic diversity[END_REF] in central Europe and Iberia was driven more by males than by females (Figure 3B&C, Supplementary Data Table 5, Extended Data Figure 5). To document this we used qpAdm to compute ancestry proportions on the autosomes and the X chromosome; since males always inherit a maternal X chromosome, differences imply sex-biased mixture. In the Balkan Neolithic there is no evidence of sex bias (Z=0.27 where a positive Z-score implies male hunter-gatherer bias), nor in the LBK and Iberian_Early Neolithic (Z=-0.22 and 1.09). In the Copper Age there is clear bias: weak in the Balkans (Z=1.66), but stronger in Iberia (Z=3.08) and Central Europe (Z=2.74). Consistent with this, hunter-gatherer mitochondrial haplogroups (haplogroup U) [START_REF] Posth | Pleistocene Mitochondrial Genomes Suggest a Single Major Dispersal of Non-Africans and a Late Glacial Population Turnover in Europe[END_REF] are rare and within the intervals of genomewide ancestry proportions, but hunter-gatherer-associated Y chromosomes (haplogroups I2, R1 and C1) [START_REF] Fu | The genetic history of Ice Age Europe[END_REF] are more common: 7/9 in the Iberian Neolithic/Copper Age and 9/10 in Middle-Late Neolithic Central Europe (Central_MN and Globular_Amphora) (Figure 3C).

No evidence that steppe-related ancestry moved through southeast Europe into Anatolia

One version of the Steppe Hypothesis of Indo-European language origins suggests that Proto-Indo-European languages developed north of the Black and Caspian seas, and that the earliest known diverging branch -Anatolian -was spread into Asia Minor by movements of steppe peoples through the Balkan peninsula during the Copper Age around 4000 BCE. [START_REF] Anthony | The Indo-European Homeland from Linguistic and Archaeological Perspectives[END_REF] If this were correct, then one way to detect evidence of it would be the appearance of large amounts of steppe-related ancestry first in the Balkan Peninsula, and then in Anatolia. However, our data show no evidence for this scenario. While we find sporadic steppe-related ancestry in Balkan Copper and Bronze Age individuals, this ancestry is rare until the late Bronze Age. Moreover, while Bronze Age Anatolian individuals have CHG-related ancestry, [START_REF] Lazaridis | Genetic origins of the Minoans and Mycenaeans[END_REF] they have neither the EHG-related ancestry characteristic of all steppe populations sampled to date, [START_REF] Jones | Upper Palaeolithic genomes reveal deep roots of modern Eurasians[END_REF] nor the WHG-related ancestry that is ubiquitous in Neolithic southeastern Europe (Extended Data Figure 2&3, Supplementary Data Table 2). An alternative hypothesis is that the ultimate homeland of Proto-Indo-European languages was in the Caucasus or in Iran. In this scenario, westward movement contributed to the dispersal of Anatolian languages, and northward movement and mixture with EHG was responsible for the formation of a "Late Proto-Indo European"-speaking population associated with the Yamnaya Complex. [START_REF] Anthony | The horse the wheel and language[END_REF] While this scenario gains plausibility from our results, it remains possible that Indo-European languages were spread through southeastern Europe into Anatolia without large-scale population movement or admixture.

Discussion

Our study shows that southeastern Europe consistently served as a genetic contact zone. Before the arrival of farming, the region saw interaction between diverged groups of huntergatherers, and this interaction continued after farming arrived. While this study has clarified the genomic history of southeastern Europe from the Mesolithic to the Bronze Age, the processes that connected these populations to the ones living today remain largely unknown. An important direction for future research will be to sample populations from the Bronze Age, Iron Age, Roman, and Medieval periods and to compare them to present-day populations to understand how these transitions occurred.

Methods

Ancient DNA Analysis

We extracted DNA and prepared next-generation sequencing libraries in four different dedicated ancient DNA laboratories (Adelaide, Boston, Budapest, and Tuebingen). We also prepared samples for extraction in a fifth laboratory (Dublin), from whence it was sent to Boston for DNA extraction and library preparation (Supplementary Table 1).

Two samples were processed at the Australian Centre for Ancient DNA, Adelaide, Australia, according to previously published methods [START_REF] Haak | Massive migration from the steppe was a source for Indo-European languages in Europe[END_REF] and sent to Boston for subsequent screening, 1240k capture and sequencing.

Seven samples were processed [START_REF] Lipson | Parallel ancient genomic transects reveal complex population history of early European farmers[END_REF] at the Institute of Archaeology RCH HAS, Budapest, Hungary, and amplified libraries were sent to Boston for screening, 1240k capture and sequencing.

Seventeen samples were processed at the Institute for Archaeological Sciences of the University of Tuebingen and at the Max Planck Institute for the Science of Human History in Jena, Germany. Extraction [START_REF] Dabney | Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments[END_REF] and library preparation [START_REF] Meyer | Illumina sequencing library preparation for highly multiplexed target capture and sequencing[END_REF][START_REF] Rohland | Partial uracil-DNA-glycosylase treatment for screening of ancient DNA[END_REF] followed established protocols. We performed in-solution capture as described below ("1240k capture") and sequenced on an Illumina HiSeq 4000 or NextSeq 500 for 76bp using either single-or paired-end sequencing.

The remaining 199 samples were processed at Harvard Medical School, Boston, USA. From about 75mg of sample powder from each sample (extracted in Boston or University College Dublin, Dublin, Ireland), we extracted DNA following established methods [START_REF] Dabney | Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments[END_REF] replacing the column assembly with the column extenders from a Roche kit. [START_REF] Korlevic | Reducing microbial and human contamination in DNA extractions from ancient bones and teeth[END_REF] We prepared double barcoded libraries with truncated adapters from between one ninth and one third of the DNA extract. Most libraries included in the nuclear genome analysis (90%) were subjected to partial ("half") Uracil-DNA-glycosylase (UDG) treatment before blunt end repair. This treatment reduces by an order of magnitude the characteristic cytosine-to-thymine errors of ancient DNA data [START_REF] Briggs | Removal of deaminated cytosines and detection of in vivo methylation in ancient DNA[END_REF] , but works inefficiently at the 5' ends, [START_REF] Rohland | Partial uracil-DNA-glycosylase treatment for screening of ancient DNA[END_REF] thereby leaving a signal of characteristic damage at the terminal ends of ancient sequences. Some libraries were not UDG-treated ("minus"). For some samples we increased coverage by preparing additional libraries from the existing DNA extract using the partial UDG library preparation, but replacing the MinElute column cleanups in between enzymatic reactions with magnetic bead cleanups, and the final PCR cleanup with SPRI bead cleanup. [START_REF] Deangelis | Solid-phase reversible immobilization for the isolation of PCR products[END_REF][START_REF] Rohland | Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture[END_REF] We screened all libraries from Adelaide, Boston and Budapest by enriching for the mitochondrial genome plus about 3,000 (50 in an earlier, unpublished, version) nuclear SNPs using a bead-capture [START_REF] Maricic | Multiplexed DNA sequence capture of mitochondrial genomes using PCR products[END_REF] but with the probes replaced by amplified oligonucleotides synthesized by CustomArray Inc. After the capture, we completed the adapter sites using PCR, attaching dual index combinations [START_REF] Kircher | Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform[END_REF] to each enriched library. We sequenced the products of between 100 and 200 libraries together with the non-enriched libraries (shotgun) on an Illumina NextSeq500 using v2 150 cycle kits for 2x76 cycles and 2x7 cycles.

In Boston, we performed two rounds of in-solution enrichment ("1240k capture") for a targeted set of 1,237,207 SNPs using previously reported protocols. [START_REF] Haak | Massive migration from the steppe was a source for Indo-European languages in Europe[END_REF][START_REF] Fu | An early modern human from Romania with a recent Neanderthal ancestor[END_REF][START_REF] Lazaridis | Ancient human genomes suggest three ancestral populations for present-day Europeans[END_REF] For a total of 34 individuals, we increased coverage by building one to eight additional libraries for the same sample. When we built multiple libraries from the same extract, we often pooled them in equimolar ratios before the capture. We performed all sequencing on an Illumina NextSeq500 using v2 150 cycle kits for 2x76 cycles and 2x7 cycles. We attempted to sequence each enriched library up to the point where we estimated that it was economically inefficient to sequence further. Specifically, we iteratively sequenced more and more from each individual and only stopped when we estimated that the expected increase in the number of targeted SNPs hit at least once would be less than about one for every 100 new read pairs generated. After sequencing, we trimmed two bases from the end of each read and aligned to the human genome (b37/hg19) using bwa. [START_REF] Li | Fast and accurate long-read alignment with Burrows-Wheeler transform[END_REF] We then removed individuals with evidence of contamination based on mitochondrial DNA polymorphism [START_REF] Fu | A revised timescale for human evolution based on ancient mitochondrial genomes[END_REF] or difference in PCA space between damaged and undamaged reads [START_REF] Skoglund | Separating endogenous ancient DNA from modern day contamination in a Siberian Neandertal[END_REF] , a high rate of heterozygosity on chromosome X despite being male [START_REF] Skoglund | Separating endogenous ancient DNA from modern day contamination in a Siberian Neandertal[END_REF][START_REF] Korneliussen | ANGSD: Analysis of Next Generation Sequencing Data[END_REF] , or an atypical ratio of X-to-Y sequences. We also removed individuals that had low coverage (fewer than 15,000 SNPs hit on the autosomes). We report, but do not analyze, data from nine individuals that were first-degree relatives of others in the dataset (determined by comparing rates of allele sharing between pairs of individuals).

After removing a small number of sites that failed to capture, we were left with a total of 1,233,013 sites of which 32,670 were on chromosome X and 49,704 were on chromosome Y, with a median coverage at targeted SNPs on the 216 newly reported individuals of 0.90 (range 0.007-9.2; Supplementary Table 1). We generated "pseudo-haploid" calls by selecting a single read randomly for each individual at each SNP. Thus, there is only a single allele from each individual at each site, but adjacent alleles might come from either of the two haplotypes of the individual. We merged the newly reported data with previously reported data from 274 other ancient individuals [START_REF] Hofmanova | Early farmers from across Europe directly descended from Neolithic Aegeans[END_REF][START_REF] Mathieson | Genome-wide patterns of selection in 230 ancient Eurasians[END_REF][START_REF] Omrak | Genomic Evidence Establishes Anatolia as the Source of the European Neolithic Gene Pool[END_REF][START_REF] Allentoft | Population genomics of Bronze Age Eurasia[END_REF][START_REF] Fu | Genome sequence of a 45,000-year-old modern human from western Siberia[END_REF][START_REF] Fu | The genetic history of Ice Age Europe[END_REF][START_REF] Gallego Llorente | Ancient Ethiopian genome reveals extensive Eurasian admixture in Eastern Africa[END_REF][START_REF] Jones | Upper Palaeolithic genomes reveal deep roots of modern Eurasians[END_REF][START_REF] Keller | New insights into the Tyrolean Iceman's origin and phenotype as inferred by whole-genome sequencing[END_REF][START_REF] Kilinc | The Demographic Development of the First Farmers in Anatolia[END_REF][START_REF] Lazaridis | Genomic insights into the origin of farming in the ancient Near East[END_REF][START_REF] Lazaridis | Ancient human genomes suggest three ancestral populations for present-day Europeans[END_REF][START_REF] Olalde | Derived immune and ancestral pigmentation alleles in a 7,000-year-old Mesolithic European[END_REF][START_REF] Raghavan | Upper Palaeolithic Siberian genome reveals dual ancestry of Native Americans[END_REF][START_REF] Lazaridis | Genetic origins of the Minoans and Mycenaeans[END_REF][START_REF] Lipson | Parallel ancient genomic transects reveal complex population history of early European farmers[END_REF] , making pseudo-haploid calls in the same way at the 1240k sites for individuals that were shotgun sequenced rather than captured.

Using the captured mitochondrial sequence from the screening process, we called mitochondrial haplotypes. Using the captured SNPs on the Y chromosome, we called Y chromosome haplogroups for males by restricting to sequences with mapping quality ≥30 and bases with base quality ≥30. We determined the most derived mutation for each individual, using the nomenclature of the International Society of Genetic Genealogy (http:// www.isogg.org) version 11.110 (21 April 2016).

Population genetic analysis

To analyze these ancient individuals in the context of present day genetic diversity, we merged them with the following two datasets:

1.

300 high coverage genomes from a diverse worldwide set of 142 populations sequenced as part of the Simons Genome Diversity Project 28 (SGDP merge).

2.

777 West Eurasian individuals genotyped on the Human Origins array [START_REF] Lazaridis | Ancient human genomes suggest three ancestral populations for present-day Europeans[END_REF] , with 597,573 sites in the merged dataset (HO merge).

We computed principal components of the present-day individuals in the HO merge and projected the ancient individuals onto the first two components using the "lsqproject: YES" option in smartpca (v15100) [START_REF] Price | Principal components analysis corrects for stratification in genome-wide association studies[END_REF] (https://www.hsph.harvard.edu/alkes-price/software/).

We ran ADMIXTURE (v1.3.0) in both supervised and unsupervised mode. In supervised mode we used only the ancient individuals, on the full set of SNPs, and the following population labels fixed:

• Anatolia_Neolithic • WHG • EHG • Yamnaya
For unsupervised mode we used the HO merge, including 777 present-day individuals. We flagged individuals that were genetic outliers based on PCA and ADMIXTURE, relative to other individuals from the same time period and archaeological culture.

We computed D-statistics using qpDstat (v710). D-statistics of the form D(A,B,X,Y) test the null hypothesis of the unrooted tree topology ((A,B),(X,Y)). A positive value indicates that either A and X, or B and Y, share more drift than expected under the null hypothesis. We quote D-statistics as the Z-score computed using default block jackknife parameters.

We fitted admixture proportions with qpAdm (v610) using the SGDP merge. Given a set of outgroup ("right") populations, qpAdm models one of a set of source ("left") populations (the "test" population) as a mixture of the other sources by fitting admixture proportions to match the observed matrix of f 4 -statistics as closely as possible. We report a p-value for the null hypothesis that the test population does not have ancestry from another source that is differentially related to the right populations. We computed standard errors for the mixture proportions using a block jackknife. Importantly, qpAdm does not require that the source populations are actually the admixing populations, only that they are a clade with the correct admixing populations, relative to the other sources. Infeasible coefficient estimates (i.e. outside [0,1]) are usually a sign of poor model fit, but in the case where the source with a negative coefficient is itself admixed, could be interpreted as implying that the true source is a population with different admixture proportions. We used the following set of seven populations as outgroups or "right populations":

• Mbuti.DG • Ust_Ishim_HG_published.DG • Mota.SG • MA1_HG.SG • Villabruna • Papuan.DG • Onge.DG • Han.DG
For some analyses where we required extra resolution (Supplementary Data Table 4) we used an extended set of 14 right (outgroup) populations, including additional Upper Paleolithic European individuals [START_REF] Fu | The genetic history of Ice Age Europe[END_REF] :

• ElMiron • Mota.SG • Mbuti.DG • Ust_Ishim_HG_published.DG • MA1_HG.SG • AfontovaGora3 • GoyetQ116-1_published • Villabruna • Kostenki14 • Vestonice16 • Karitiana.DG • Papuan.DG • Onge.DG • Han.DG
We also fitted admixture graphs with qpGraph (v6021) [START_REF] Patterson | Ancient admixture in human history[END_REF] (https://github.com/DReichLab/ AdmixTools, Supplementary Information Note 3). Like qpAdm, qpGraph also tries to match a matrix of f-statistics, but rather than fitting one population as a mixture of other, specified, populations, it fits the relationship between all tested populations simultaneously, potentially incorporating multiple admixture events. However, qpGraph requires the graph relating populations to be specified in advance. We tested goodness-of-fit by computing the expected D-statistics under the fitted model, finding the largest D-statistic outlier between the fitted and observed model, and computing a Z-score using a block jackknife.

For 114 individuals with hunter-gatherer-related ancestry we estimated an effective migration surface using the software EEMS (https://github.com/dipetkov/eems) [START_REF] Petkova | Visualizing spatial population structure with estimated effective migration surfaces[END_REF] . We computed pairwise differences between individuals using the bed2diffs2 program provided with EEMS. We set the number of demes to 400 and defined the outer boundary of the region by the polygon (in latitude-longitude co-ordinates) [ (66,60), (60,10), (45,-15), (35,-10), (35,60)]. We ran the MCMC ten times with different random seeds, each time with one million burn-in and four million regular iterations, thinned to one in ten thousand.

To analyze potential sex bias in admixture, we used qpAdm to estimate admixture proportions on the autosomes (default option) and on the X chromosome (option "chrom: 23"). We computed Z-scores for the difference between the autosomes and the X chromosome as

Z = p A -p X σ A 2 + σ X 2
where p A and p X are the hunter-gatherer admixture proportions on the autosomes and the X chromosome, and σ A and σ X are the corresponding jackknife standard deviations. Thus, a positive Z-score means that there is more huntergatherer admixture on the autosomes than on the X chromosome, indicating that the huntergatherer admixture was male-biased. Because X chromosome standard errors are high and qpAdm results can be sensitive to which population is first in the list of outgroup populations, we checked that the patterns we observe were robust to cyclic permutation of the outgroups. To compare frequencies of hunter-gatherer uniparental markers, we counted the individuals with mitochondrial haplogroup U and Y chromosome haplogroups C1, I2 and R1, which are all common in Mesolithic hunter-gatherers but rare or absent in Anatolian Neolithic individuals. The Iron Gates hunter-gatherers also carry H and K1 mitochondrial haplogroups so the proportion of haplogroup U represents the minimum maternal huntergatherer contribution. We computed binomial confidence intervals for the proportion of haplogroups associated with each ancestry type using the Agresti-Coull method [START_REF] Brown | Interval Estimation for a Binomial Proportion[END_REF][START_REF] Agresti | Approximate is better than "exact" for interval estimation of binomial proportions[END_REF] implemented in the binom package in R.

Given autosomal and X chromosome admixture proportions, we estimated the proportion of male and female hunter-gatherer ancestors by assuming a single-pulse model of admixture.

If the proportions of male and female ancestors that are hunter-gatherer-related are given by m and f, respectively, then the proportions of hunter-gatherer-related ancestry on the autosomes and the X chromosome are given by m + f 2 and m + 2 f 3 . We approximated the sampling error in the observed admixture proportions by the estimated jackknife error and computed the likelihood surface for (m,f) over a grid ranging from (0,0) to (1,1).

Direct AMS 14 C Bone Dates

We report 137 new direct AMS [START_REF] Fu | An early modern human from Romania with a recent Neanderthal ancestor[END_REF] C bone dates for 136 individuals from multiple AMS radiocarbon laboratories. In general, bone samples were manually cleaned and demineralized in weak HCl and, in most cases (PSU, UCIAMS, OxA), soaked in an alkali bath (NaOH) at room temperature to remove contaminating soil humates. Samples were then rinsed to neutrality in Nanopure H 2 O and gelatinized in HCL. [START_REF] Longin | New method of collagen extraction for radiocarbon analysis[END_REF] The resulting gelatin was lyophilized and weighed to determine percent yield as a measure of collagen preservation (% crude gelatin yield). Collagen was then directly AMS [START_REF] Fu | An early modern human from Romania with a recent Neanderthal ancestor[END_REF] C dated (Beta, AA) or further purified using ultrafiltration (PSU, UCIAMS, OxA, Poz, MAMS). [START_REF] Brown | Improved collagen extraction by modified longin method[END_REF] It is standard in some laboratories (PSU/UCIAMS, OxA) to use stable carbon and nitrogen isotopes as an additional quality control measure. For these samples, the %C, %N and C:N ratios were evaluated before AMS [START_REF] Fu | An early modern human from Romania with a recent Neanderthal ancestor[END_REF] C dating. [START_REF] Kennett | Archaeogenomic evidence reveals prehistoric matrilineal dynasty[END_REF] C:N ratios for well-preserved samples fall between 2.9 and 3.6, indicating good collagen preservation. [START_REF] Van Klinken | Bone collagen quality indicators for paleodietary and radiocarbon measurements[END_REF] For 119 of the new dates, we also report δ 13 C and δ 15 N values (Supplementary Table 6).

All [START_REF] Fu | An early modern human from Romania with a recent Neanderthal ancestor[END_REF] C ages were δ 13 C-corrected for mass dependent fractionation with measured 13 C/ [START_REF] Müller | Trypillia Mega-Sites and European Prehistory[END_REF] C values [START_REF] Stuiver | Discussion: Reporting of 14 C data[END_REF] and calibrated with OxCal version 4.2.3 70 using the IntCal13 northern hemisphere calibration curve. [START_REF] Ramsey | OxCal 4.23 Online Manual[END_REF] For hunter-gatherers from the Iron Gates, the direct [START_REF] Fu | An early modern human from Romania with a recent Neanderthal ancestor[END_REF] C dates tend to be overestimates because of the freshwater reservoir effect (FRE), which arises because of a diet including fish that consumed ancient carbon, and for these individuals we performed a correction (Supplementary Information Note 1), [START_REF] Cook | A freshwater diet-derived 14 C reservoir effect at the Stone Age sites in the Iron Gates gorge[END_REF]

Extended Data

Extended Data Figure 1.

PCA of 486 ancient individuals, projected onto principal components defined by 777 present-day West Eurasian individuals (grey points). This differs from Figure 1B in that the plot is not cropped and the present-day individuals are shown.

Extended Data Figure 2.

Supervised ADMIXTURE analysis modeling each ancient individual (one per row), as a mixture of populations represented by clusters that are constrained to contain Anatolian Neolithic (grey), Yamnaya from Samara (yellow), EHG (pink) and WHG (green) populations. Dates in parentheses indicate approximate range of individuals in each population. This differs from Figure 1D in that it contains some previously published samples, and includes sample IDs.

Extended Data Figure 3.

Unsupervised ADMIXTURE plot from k=4 to 12, on a dataset consisting of 1099 presentday individuals and 476 ancient individuals. We show newly reported ancient individuals and some previously published individuals for comparison.

Extended Data Figure 4.

Spatial structure in hunter-gatherers. Estimated effective migration surface (EEMS). [START_REF] Petkova | Visualizing spatial population structure with estimated effective migration surfaces[END_REF] This fits a model of genetic relatedness where individuals move (in a random direction) from generation to generation on an underlying grid so that genetic relatedness is determined by distance. The migration parameter m defines the local rate of migration, varies on the grid and is inferred. This plot shows log 10 (m), scaled relative to the average migration rate (which is arbitrary). Thus log 10 (m)=2, for example, implies that the rate of migration at this point on the grid is 100 times higher than average. To restrict as much as possible to huntergatherer structure, the migration surface is inferred using data from 116 individuals that date to earlier than ~5000 BCE and have no NW Anatolian-related ancestry. Though the migration surface is sensitive to sampling, and fine-scale features may not be interpretable, the migration "barrier" (region of low migration) running north-south and separating populations with primarily WHG from primarily EHG ancestry seems to be robust, and consistent with inferred admixture proportions. This analysis suggests that Mesolithic hunter-gatherer population structure was clustered and not smoothly clinal, in the sense that genetic differentiation did not vary consistently with distance. Superimposed on this background, pies show the WHG, EHG and CHG ancestry proportions inferred for populations used to construct the migration surface (another way of visualizing the data in Figure 2, Supplementary Table 3.1.3 -we use two population models if they fit with p>0.01, and three population models otherwise). Pies with only a single color are those that were fixed to be the source populations.

Extended Data Figure 5.

log-likelihood surfaces for the proportion of female (x-axis) and male (y-axis) ancestors that are hunter-gatherer-related for the combined populations analyzed in Figure 3C, and the two populations with the strongest evidence for sex-bias. Numbers in parentheses give the number of individuals in each group. Log-likelihood scale ranges from 0 to -10, where 0 is the feasible point with the highest likelihood. Inferred ancestry proportions for populations modeled as a mixture of WHG, EHG and CHG (Supplementary Table S3.1.3). Dashed lines show populations from the same geographic region. Percentages indicate proportion of WHG+EHG ancestry. Standard errors range from 1.5-8.3% (Supplementary Table S3.1.3).

Figure 1 .

 1 Figure 1. Geographic and genetic structure of 216 newly reported individuals A: Locations of newly reported individuals. B: Ancient individuals projected onto principal components defined by 777 present-day West Eurasians (shown in Extended Data Figure1). Includes selected published individuals (faded circles, labeled) and newly reported individuals (other symbols, outliers enclosed in black circles). Colored polygons cover individuals that had cluster memberships fixed at 100% for supervised admixture analysis. C: Date (direct or contextual) for each sample and approximate chronology of southeastern Europe. D: Supervised ADMIXTURE analysis, modeling each ancient individual (one per

Figure 2 .

 2 Figure 2. Structure and change in hunter-gatherer-related populations

Figure 3 .

 3 Figure 3. Structure and change in NW Anatolian Neolithic-related populations A: Populations modeled as a mixture of NW Anatolia Neolithic, WHG, and EHG. Dashed lines show temporal relationships between populations from the same geographic region. Percentages indicate proportion of WHG+EHG ancestry. Standard errors range from 0.7-6.0% (Supplementary TableS3.2.2). B: Z-scores for the difference in hunter-gathererrelated ancestry on the autosomes compared to the X chromosome when populations are modeled as a mixture of NW Anatolia Neolithic and WHG (N=126 individuals, group sizes in parentheses). Positive values indicate more hunter-gatherer-related ancestry on the autosomes and thus male-biased hunter-gatherer ancestry. "Combined" populations merge all individuals from different times from a geographic area. C: Hunter-gatherer-related ancestry proportions on the autosomes, X chromosome, mitochondrial DNA (i.e. mt haplogroup U), and the Y chromosome (i.e. Y chromosome haplogroups I2, R1 and C1). Points show qpAdm (autosomes and X chromosome) or maximum likelihood (MT and Y

  .2.2). B: Z-scores for the difference in hunter-gathererrelated ancestry on the autosomes compared to the X chromosome when populations are modeled as a mixture of NW Anatolia Neolithic and WHG (N=126 individuals, group sizes in parentheses). Positive values indicate more hunter-gatherer-related ancestry on the autosomes and thus male-biased hunter-gatherer ancestry. "Combined" populations merge all individuals from different times from a geographic area. C: Hunter-gatherer-related ancestry proportions on the autosomes, X chromosome, mitochondrial DNA (i.e. mt haplogroup U), and the Y chromosome (i.e. Y chromosome haplogroups I2, R1 and C1). Points show qpAdm (autosomes and X chromosome) or maximum likelihood (MT and Y chromosome) estimates and bars show approximate 95% confidence intervals (N=109 individuals, group sizes in parentheses).

  assuming that 100% FRE = 545±70 yr, and δ 15 N values of 8.3% and 17.0% for 100% terrestrial and aquatic diets, respectively.

EEMS: https://github.com/dipetkov/eems/ bwa: http://bio-bwa.sourceforge.net OxCal: https://c14.arch.ox.ac.uk/oxcal.html

Nature. Author manuscript; available in PMC 2018 August 21.

Acknowledgments

We thank David Anthony, Iosif Lazaridis, and Mark Lipson for comments on the manuscript, Bastien Llamas, Alan Cooper and Anja Furtwängler for contributions to laboratory work, Richard Evershed for contributing 14 C dates and Friederike Novotny for assistance with samples. Support for this project was provided by the Human Frontier Science Program fellowship LT001095/2014-L to I.M.; by DFG grant AL 287 / 14-1 to K.W.A.; by Irish Research Council grant GOIPG/2013/36 to D.F.; by the NSF Archaeometry program BCS-1460369 to DJK (for AMS 14 C work at Penn State); by MEN-UEFISCDI grant, Partnerships in Priority Areas Program -PN II (PN-II-PT-

PCCA-2013-4-2302) to C.L.; by Croatian Science Foundation grant IP-2016-06-1450 to M.N.; by European Research Council grant ERC StG 283503 and Deutsche Forschungsgemeinschaft DFG FOR2237 to K.H.; by ERC starting grant ADNABIOARC (263441) to R.P.; and by US National Science Foundation HOMINID grant BCS-1032255, US National Institutes of Health grant GM100233, the Howard Hughes Medical Institute, and an Allen Discovery Center grant from the Paul Allen Foundation to D.R.

Data Availability

The aligned sequences are available through the European Nucleotide Archive under accession number PRJEB22652. The pseudo-haploid genotype dataset used in analysis and consensus mitochondrial genomes are available at https://reich.hms.harvard.edu/datasets.

Code Availability

Software used to analyze the data is available from the following sources: smartpca, qpAdm, qpDstat, qpGraph: https://github.com/DReichLab/AdmixTools/ ADMIXTURE: https://www.genetics.ucla.edu/software/admixture/

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Authors

Iain Mathieson †,1 , Songül Alpaslan Roodenberg 1 , Cosimo Posth 2,3 , Anna Szécsényi-Nagy 4 , Nadin Rohland 1 , Swapan Mallick 1,5 , Iñigo Olalde 1 , Nasreen Broomandkhoshbacht 1,5 , Francesca Candilio 6 , Olivia Cheronet 6,7 , Daniel Fernandes 6,8 , Matthew Ferry 1,5 , Beatriz Gamarra 6 , Gloria González Fortes 9 , Wolfgang Haak 2,10 , Eadaoin Harney 1,5 , Eppie Jones [START_REF] Omrak | Genomic Evidence Establishes Anatolia as the Source of the European Neolithic Gene Pool[END_REF][START_REF] Müller | Trypillia Mega-Sites and European Prehistory[END_REF] , Denise Keating 6 , Ben Krause-Kyora 2 , Isil Kucukkalipci 3 , Megan Michel 1,5 , Alissa Mittnik


Thomas Oliver Pryce

Myo Aung Aung Kyaw

Tin Tin Win Min Kyaw

Thu Thu Win

Khin Htwe

Mar Mar May Myat Mon

Aye

Hlaing Su

Aye Htay

Aye Mar

Thomas Oliver

Pryce 1+2

Khin Htwe Win3

Su Hlaing Htay

Aye Aye Mar

Bérénice Bellina

Alex Bentley

Andrew Bevan

Louis Champion

Camille Colonna

Amanda J Cook

Aude Favereau

Dorian Fuller

Cloé Georjon

Charles Higham

Kalayar Myat

Myat Htwe

Philip J Piper

Yoshiyuki Iizuka

Jitlada Innanchai

Xavier Peixoto

Peter Petchey

Ron Pinhasi

Baptiste Pradier

Fréderique Valentin

Anna Willis

Barbara Wohlfarth

Antoine Zazzo

Affiliations

A first absolute chronology for Late Neolithic to Early Bronze Age Myanmar: new AMS 14 C dates from Nyaung'gan and Oakaie

Keywords: Southeast Asia, Myanmar, Late Neolithic, Bronze Age

d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Myanmar's location makes it pivotal to tracing cultural, genetic, linguistic and technological links between South, East and Southeast Asia. Investigations of its prehistory, however, are in their infancy, with virtually no sites investigated in the uppermost and lowermost thirds of the country. Even the more intensively studied central area lacks a coherent culture-historical sequence until the appearance of the Pyu city-states in the early centuries AD. Recent research in archaeology and related disciplines in Thailand and Vietnam has fundamentally advanced our understanding of the Neolithic and Bronze Age transitions in Mainland Southeast Asia, both of them thought to be linked to migrations and/or stimuli from present-day China. Of the approximately 2500km-long border between China and the continental Association of Southeast Asian Nations states (ASEAN), approximately 1600km is with Myanmar. Characterising Myanmar's Neolithic and Bronze Age periods may therefore offer important complementary information to that obtained from its eastern neighbours-perhaps identifying additional routes, timings and mechanisms of biological and/or cultural transmission. This paper lays out chronological building blocks from our investigation of two sites in central Myanmar: Nyaung'gan and Oakaie (Figure 1).

Since excavations of the cemetery at Nyaung'gan in 1998-1999, the site has been virtually synonymous with Myanmar's late prehistoric period. Relative dating of grave goods associated with the 43 excavated burials attributed the cemetery to the Bronze Age-the first site of this date to be found in Myanmar [START_REF] Moore | Nyaung-gan: a preliminary note on a Bronze Age cemetery near Mandalay, Myanmar (Burma)[END_REF][START_REF] Tayles | Bronze Age (Myanmar):a report on the people from the cemetery of Nyaunggan, Upper Myanmar[END_REF]. No charcoal was recovered, and the radiocarbon dating of human remains failed due to a lack of extractable collagen (using late 1990s techniques). The major aims of the Franco-Myanmar project were to provide an absolute chronology for Nyaung'gan and the surrounding area, and to contribute towards a national culture-historical framework and comparative bioarchaeological, material culture and environmental data for Thailand's and Vietnam's rich archaeological datasets, as well as those being developed in Cambodia and Laos. This paper addresses the first and essential issue-the chronology.

Archaeological sites

Nyaung'gan is located 10km north-east of the Chindwin River, on the edge of a Cenozoic volcanic crater (95.0600° east, 22.4110° north). The environment is semi-arid, averaging 675mm of rainfall per annum. Modern, active copper mines 30km to the south [START_REF] Maung Khin | Geology and copper deposit of Sabetaung in Monywa district[END_REF] were previously assumed relevant to Nyaung'gan's Bronze Age phase (Moore & Pauk 2001: 37-38) (Figure 1). The Franco-Myanmar project began by investigating the extensive ceramic and lithic surface scatters south of the modern village of Oakaie. Ten excavations at four different loci with a combined surface area of 348m² were conducted over three seasons. These revealed mortuary, occupation and industrial evidence.

The OAI1 cemetery was partially exposed during two contiguous excavations in 2014 (80m²) and 2015 (105m²). Forty single and six multiple burials of adults-both males and females-and subadults were cut into a sterile volcanic tuff at varying depths and at orientations that suggest at least two and possibly three phases of use (Figure 2). Funerary offerings included bivalve shells, pottery, stone beads and bracelets, bone bracelets, shell beads, spindle whorls, a cowrie shell and a dog burial. A single bronze axe was found in S15, an upper level burial. Due to complex grave intercutting, superpositioning and recent disturbance, it is currently impossible to attribute definitively OAI1 burials devoid of metal to the Bronze Age or preceding periods (cf. [START_REF] Pryce | Dating the Myanmar Bronze Age: preliminary 14 C dates from the Oakaie 1 cemetery near Nyaung'gan[END_REF]. Twenty radiocarbon dates from OAI1 have been obtained from human bone, human tooth enamel and bivalve shell.

OAI2 is a settlement area centred around 95.0420° east, 22.3905° north, approximately 750m west of OAI1. Excavation here aimed to provide a wide area perspective of habitation deposits containing domestic and industrial material culture. All five excavation squares, totalling 114m², revealed a dense, sherd-packed, 300-500mm-thick occupation deposit, representing two phases according to ceramic analysis. Postholes were numerous, as were lithic debitage and artefacts, animal bone and flecks of copper-base corrosion products. A single adult male inhumation was found, as well as seven under-floor infant jar burials, one of which contained two stone bracelets (Figure 3). Eight radiocarbon dates from OAI2 have been obtained from human bone, human tooth enamel and charcoal.

OAI3 is located approximately 200m south-west of OAI2, at 95.0403° east, 22.3893° north. The aim in excavating OAI3 in 2016 was to explore the sequence of occupation, as opposed to the horizontal variation identified at OAI2. Six cultural layers were found above the sterile layer at a depth of 2.1m (Figure 4). The first cultural layer below plough soil comprised a dark brown soil matrix with numerous postholes and large quantities of ceramics, lithics, animal bone, nine copper-base fragments and a single infant jar burial. The intact domestic deposits were extremely dense, representing floors or middens, and one feature had been heated sufficiently to cause some ceramics to deform. Lithics were present as finished and near-finished adzes, beads, bracelets, grindstones and hammer stones, as well as related debitage. A single bloated and vitrified copperstained crucible fragment was found in the first layer [START_REF] Pryce | Metallurgical traditions and metal exchange networks in late prehistoric central Myanmar, c. 1000 BC to c. AD[END_REF]. The third layer consisted of a lighter brownmatrix that contained similar but fewer features and material than the second cultural layer, except for the complete absence of copper-base artefacts. The interface of the third and fourth layers consisted of a well-defined floor surface covered with horizontally aligned ceramic fragments. Beneath this was a 200-500mm-thick deposit with fewer artefacts and a near absence of lithic tools or debitage. The boundary between the fourth and fifth layers sloped to the south-east and was followed by 200-650mm of a dark brown matrix containing some ceramic fragments, but far fewer cut features than the second and third cultural layers. The sixth layer comprised a lightcoloured, hard volcanic tuff, as per the sterile seventh layer, but containing very few ceramic sherds. Seven charcoal radiocarbon determinations were obtained from the upper metal-bearing phase, and eight from lower contexts devoid of metal artefacts.

OAI4 was a settlement and industrial site at 95.0465° east, 22.3955° north, approximately 950m north-east of OAI3. Excavation in 2016 revealed two cultural layers 700-850mm in depth. Compared to OAI3, the density of material was far lower than expected, given the plough soil rich in lithics, animal bone and ceramics. A sterile volcanic tuff was encountered at a depth of 850-1000mm. Further excavation 50m to the southeast encountered two undisturbed cultural deposits beneath the plough soil. The upper phase was 300mm-thick and the second, lighter in colour, was 600mm thick, with gravel inclusions. Both layers contained lithics (artefacts and production debris), animal bone and ceramics, the latter having morpho-stylistic differences between phases comparable to those at OAI2 and 3. Two charcoal radiocarbon determinations come from the upper phase and one from the lower phase.

We view the four excavated loci, OAI1-4, as components of one settlement. This is supported by an intensive systematic pedestrian survey of the type developed in the Eastern Mediterranean and Mesoamerica, and previously tested in Thailand [START_REF] Banning | Archaeological survey[END_REF][START_REF] Bevan | GIS, archaeological survey, and landscape archaeology on the island of Kythera, Greece[END_REF][START_REF] Pryce | Intensive archaeological survey in tropical environments: methodological and metallurgical insights from Khao Sai On, central Thailand[END_REF]. Two square kilometres covering all four loci were walked with a spacing of 20m. All artefacts were recorded and collected every 10m, and subsequently assessed by the project's find specialists. The presence of prehistoric material decreased towards the east, but no significant attenuation was found to the north, south or west. Current data suggest the surface area of this late prehistoric site to be at least 1km², which is exceptionally large for Southeast Asia.

Nyaung'gan

As the 1998-1999 excavations at Nyaung'gan were intended to provide a site museum, the uppermost burials and their grave goods were partially exposed and then pedestalled for ease of viewing. This permitted wide exposure of the archaeology but limited diachronic perspective. Our 2016 excavations investigated the full time-depth of the cemetery, and recorded and removed each burial. Targeting the highest extant ground surface within the cemetery, a 4 × 4m square trench (NYG1) was placed between former trenches 4 and 5 (Figure 5). Two burials-S1 and 2-oriented north were found at a depth of 0.5m, accompanied by pottery vessels. Three layers could be distinguished stratigraphically, but due to the friable ashy fill, it was not possible to identify the grave cuts (Figure 6). A further 4 × 4m square (NYG2) was placed 12m to the south, in which three individuals were exposed (Figure 7). The first, S3a, was represented only by a skull and had disturbed S3b, a previous burial, immediately below. S3b was oriented north-north-west and was associated with a dog burial, and funerary deposits including a turtle, a bone bracelet, stone beads and pottery. The third burial, S4, was oriented north-west and also incorporated pottery grave goods. All burials were found approximately 0.8m below the current ground surface, but no grave cuts could be identified.

A final trench (NYG3) covering 2 × 11m was placed immediately adjacent to test pit 1 from 1998-1999, which had produced the majority of the copper-base artefacts. The upper layer, approximately 100mm thick, was highly disturbed and yielded a single leadedcopper arrowhead or spearhead. While its context is not secure, we have assumed, based on the absence of later period sites in the area, that it does relate to the Bronze Age cemetery [START_REF] Pryce | Metallurgical traditions and metal exchange networks in late prehistoric central Myanmar, c. 1000 BC to c. AD[END_REF]. A highly disturbed burial-S5-was found in NYG3 just below the current ground surface in the western part of the excavation. Towards the centre, one individual-S6-was found at approximately 0.4m below the current ground surface, oriented north-west, and accompanied by pottery vessels, a cattle skull and ribs, and stone beads. The final individual-S7-oriented north-north-west, was uncovered below S6, and was accompanied by pottery vessels and stone beads (Figure 8).

Ceramic techno-typology

The methodology applied to the Oakaie and Nyaung'gan ceramic assemblages from the 2014-2016 excavation and survey seasons relies on the concept of the chaîne opératoire and its application as a descriptive and analytical tool to every sherd recovered [START_REF] Roux | Identification of wheel-fashioning methods: technological analysis of 4 th -3 rd millennium BC Oriental ceramics[END_REF], 2007;[START_REF] Roux | Lecture anthropologique des assemblages céramiques: fondements et mise en oeuvre de l'analyse technologique[END_REF]Roux , 2016)). Most of the pottery analysed, whether from funerary or settlement contexts, belongs to the same technical tradition: roughedout using coils and discontinuous pressure, followed by shaping with paddle and anvil for medium and large closed forms. As such, we consider that there was social continuity in the population during the period in question, with the differences in the ceramic assemblage limited almost exclusively to morphometry and decoration.

These differences allow us to identify two main phases (Figure 9). The youngest of these is the copper-/bronze-bearing level at OAI1, currently only represented with certainty by individual S15, the burials excavated at Nyaung'gan in 1998-1999 and 2016's upper level burials S1 and S6. This phase is also represented by large paddled and impressed burial jars in the upper copper-/bronzebearing layers at OAI2 and 3. The second phase, which sees a significant drop in the proportion of decorated wares, is represented in the 2016 season's burials S2, S3b, S4 and S7 at Nyaung'gan, and the non-copper-/bronze-bearing layers at the occupation/industrial sites OAI2-4. It is possible that OAI3's layers 4-6 represent an even older phase. Likewise, pottery from OAI1 burials devoid of metal could potentially be differentiated on the criteria of carination and paddle-impressed decoration, but we currently lack chronostratigraphic resolution for this cemetery.

Chronology

The Franco-Myanmar team's 52 new radiocarbon determinations allow us to provide some absolute dating for what has been a critical chronological lacuna for over 20 years, not only for the Nyaung'gan area, but also for central Myanmar and the country as a whole [START_REF] Pryce | Dating the Myanmar Bronze Age: preliminary 14 C dates from the Oakaie 1 cemetery near Nyaung'gan[END_REF]. Radiocarbon (AMS) dating was performed at a number of laboratories in the UK, France, the USA and New Zealand, using their respective methodologies [START_REF] Brock | Current pretreatment methods for AMS radiocarbon dating at the Oxford Radiocarbon Accelerator Unit[END_REF][START_REF] Hoper | The Queen's University, Belfast: laboratory protocols used for AMS radiocarbon dating at the 14 CHRONO Centre[END_REF][START_REF] Dumoulin | Status report on sample preparation protocols developed at the LMC14 Laboratory, Saclay, France: from sample collection to 14 C AMS measurement[END_REF]). The combined dataset was calibrated using OxCal v4.24 (Tables S1-5, see online supplementary material (OSM); Bronk Ramsey 2009; [START_REF] Reimer | IntCal13 and Marine13 radiocarbon age calibration curves 0-50,000 years cal BP[END_REF]. Our radiocarbon methodology is not without issues and we were obliged to experiment substantially. Despite archaeobotanical flotation of grave fills, stomach cavities and pot contents, we were unable to recover any charcoal from mortuary contexts. Following unsuccessful attempts to extract collagen from 13 OAI1 femur samples, five of these samples were re-submitted for bone apatite dating, including S15-the only grave containing a copper-base artefact. In 2015, teeth and ribs from OAI1 and OAI2 burials were sampled for apatite dating. These determinations, which were all successful apart fromthe tooth inOAI1/S29, indicated that the ribs were consistently more susceptible to postdepositional diagenesis and that the teeth gave more reliable dates. All of our apatite dates, however, risk being young-as is often the case (Zazzo 2014)-and we use them cautiously.

Eight bivalve shells found in association with OAI1 burials were also dated. This approach was used with great success in Thailand using freshwater species [START_REF] Higham | A new chronological framework for prehistoric Southeast Asia, based on a Bayesian model from Ban Non Wat[END_REF]). We have thus far been unable to identify the shell species, but some of the dates are suspiciously early (in particular OAI1/S26 & S45). This may be due to species-specific marine or freshwater reservoir effects. Until there is full identification of shell species, we are likewise inclined to discount these dates (Table S1, OSM). Flotation provided numerous charcoal samples from OAI2-4, and teeth samples were taken from the adult burial at OAI2, as well as at NYG1-3 (Tables S2-5, OSM). As per OAI1, the NYG enamel apatite dated later than was expected for burials assigned stratigraphically and from material-culture to the Neolithic (S2-5 & S7), and was indistinguishable indeed from those burials ascribed to the Bronze Age (S1 & S6), with all of them falling in the ninth to fifth centuries BC at two standard deviations.We note that the NYG1-3 matrices of friable volcanic ash would have allowed for easy passage of water, thereby increasing the risk of diagenesis. Due to these issues, we rely heavily upon the charcoal dates (notwithstanding the 'old wood effect') and use dates derived from other materials as relative guidance at present. While the 14C determinations provide the numbers, it is their combination with the stratigraphic, bioarchaeological and particularly the ceramic data that provide the framework for our chronological reconstruction of the late prehistoric Nyaung'gan/Oakaie area. The excavation ofOAI3 was especially critical, as our strategy was to achievemaximum depth, rather than the large open area exposure as at OAI2. The absence of metal in layer 3 and below at OAI3 implies that the majority of the deposit was potentially 'Neolithic', and only the upper 300-400mm were Bronze Age sensu stricto. Ceramic analysis also indicated a clear shift from proportionally less to more decoration in the OAI3 Neolithic to Bronze Age layer transition, a distinction also seen at OAI2 and OAI4.

But what of the cemeteries? OAI1's sole metal-bearing burial, S15, cuts burial S18 and has a ceramic assemblage highly comparable to those from the 1998-1999 excavations at Nyaung'gan, as well as NYG3/S6. In particular, a large paddle-impressed jar found in OAI1/S15 is very similar to those used for underfloor infant burials in metal-bearing levels at OAI2 and OAI3, as well as one found during the excavation of OAI1/S9. This was probably invasive, as its placement destroyed the individual's lower limbs.We thus consider it reasonable to interpret OAI1/S15 as representing Early Bronze Age burial activity, despite its later first-millennium BC bone apatite direct date (Table S2, OSM). Intercutting, superpositioning and pottery assemblages at OAI1 strongly suggest that there is at least one, and possibly two, burial phases prior to that of S15. Not only do all these burials lack metal, but from extrapolation from OAI2-4 using pottery techno-typologies, it is probable that a large proportion are Neolithic.

At Nyaung'gan, the bronze-bearing burials excavated in 1998-1999 have comparable pottery assemblages to individuals NYG1/S1 and NYG3/S6 excavated in 2016. In NYG3, S6 superposes S7, which has a similar pottery assemblage to individuals S2, S3b and S4 (S3a and S5 were incomplete and disturbed burials, respectively), suggesting that these metal-less burials are also potentially Neolithic. The relation between these older burials and the lower-level burials at OAI1 is not yet clear. We recognise that our Neolithic burials at Nyaung'gan and Oakaie 1 all have direct 14C dates that are regionally unacceptably late, but given the complementary ceramic and stratigraphic evidence, we are inclined to attribute this discrepancy to elevated apatite diagenesis (Table S5, OSM). The Neolithic and Bronze Age phases evidenced at Nyaung'gan and Oakaie 1 were probably not widely separated in time.

Discussion and conclusion

To make sense of our local chronology we have leant upon the relatively secure charcoal dating from the settlement/industrial sites-which may suffer from the 'old wood' effect-and extrapolated them to the cemeteries, as described above, to support the teeth and bone apatite dates, which are probably artificially young. As such, a precise date for the debut of the Bronze Age in the Nyaung'gan area cannot yet be assigned. Figure 10, however, shows that a line plotted between charcoal dates attributed to copper-/bronze-bearing and copper-/bronze-devoid phases indicates a transition during the tenth to ninth centuries BC. For the copper-/bronze-devoid phase, the lack of incised and impressed ('i&i') pottery generally considered typical of the initial Mainland Southeast Asian Neolithic is notable [START_REF] Rispoli | The incised & impressed pottery style of Mainland Southeast Asia following the paths of neolithization[END_REF][START_REF] Chi | The emergence of agriculture in southern China[END_REF]. Concomitantly with the earliest 14C charcoal determinations in which we have confidence dating only to the thirteenth to twelfth centuries BC, it is clear that the investigated area is, at the oldest, Late/Terminal Neolithic. Thus, our entire local chronology can be constrained between the thirteenth to eighth centuries BC (or 600 years) at a maximum, and probably significantly less, in the order of 400-500 years. As there is no indication of Iron Age or historical-era activity in the area, the question remains as to why the Nyaung'gan/Oakaie sites were abandoned by the mid first millennium BC.

No other Bronze Age culture area in Myanmar has been dated radiometrically, hence comparisons at the national scale are not yet possible. Excavations at Halin, approximately 80km east of Nyaung'gan, and Ywa Gon Gyi, approximately 100km south of Mandalay [START_REF] Pautreau | Level of Neolithic occupation and 14 C dating at Ywa Gon Gyi, Samon Valley (Myanmar)[END_REF][START_REF] Hudson | Earthernware from a firing site in Myanmar (Burma) dates to more than 4500 years ago[END_REF], however, are claimed to have yielded examples of the 'i&i' pottery characteristic of early Mainland Southeast Asian immigrant farming populations, in association with a total of five radiocarbon dates. The three Halin dates fall in the early to mid third millennium BC, while the two from Ywa Gon Gyi fall in the mid to late third and early to mid second millennia BC respectively. We have not been able to examine the ceramic samples to check the 'i&i' claims, but note that four of the five dates offered (the exception being the youngest Ywa Gon Gyi date, on burnt animal bone) [START_REF] Pautreau | Level of Neolithic occupation and 14 C dating at Ywa Gon Gyi, Samon Valley (Myanmar)[END_REF], are significantly older than those currently accepted for the debut of the Mainland Southeast Asian Neolithic, at c. 2000-1800 BC (Higham et al. 2011a;Oxenham et al. 2011).

The Nyaung'gan/Oakaie chronology presented above documents the transition to copper-base metallurgy rather than the introduction of the first agricultural practices, and it is appropriate to compare this with the few other reliably dated regional sequences. The sites in question-Ban Chiang, Ban Lum Khao, Ban NonWat, Non Nok Tha, Non PaWai and Tha Kae-are all located in central and north-east Thailand (Figure 1) and have recently been subjected to a comprehensive radiocarbon dating or re-dating programme to establish an authoritative late prehistoric cultural sequence [START_REF] Higham | A new chronological framework for prehistoric Southeast Asia, based on a Bayesian model from Ban Non Wat[END_REF]Higham et al. 2011bHigham et al. , 2014[START_REF] Higham | A new chronology for the Bronze Age of northeastern Thailand and its implications for Southeast Asian prehistory[END_REF][START_REF] Rispoli | Establishing the prehistoric cultural sequence for the Lopburi region, central Thailand[END_REF][START_REF] Higham | The Mun Valley and central Thailand in prehistory: integrating two cultural sequences[END_REF]. The necessity for this effort derives from controversial claims made in the 1960s and 1970s concerning a fourth-millennium BC (Bronze Age) designation for contexts at the Ban Chiang and Non Nok Tha sites (e.g. [START_REF] Solheim | Early bronze in northeastern Thailand[END_REF][START_REF] Bayard | Early Thai bronze: analysis and new dates[END_REF]. While substantial chronological revisions have been subsequently proposed (e.g. [START_REF] White | Early East Asian metallurgy: the southern tradition[END_REF][START_REF] White | Early East Asian metallurgy: the southern tradition[END_REF], there remained until recently a division in opinion between those who advocated a 'Long Chronology Model (LCM)' with copper-base metallurgy appearing in the early second millennium BC, and those proposing a 'Short Chronology Model (SCM)', with a late second-millennium BC Bronze Age transition. The difference between these positions was not merely chronological but also concerned the mechanism of cultural transmission from or via present-day China. The LCMadvocates metal-using migrants from northwest China, while the SCM proposes a 'trade and exchange' model with the Chinese Central Plains and the south-east [START_REF] Pigott | Metals and mines: studies in archaeometallurgy[END_REF][START_REF] White | The transmission of early bronze technology to Thailand: new perspectives[END_REF]Higham et al. 2011b). Higham et al.'s (2015) new dating programme has established that a thirteenth-to tenthcentury BC Bronze Age transition occurred in central and north-eastern Thailand. These results are supported by technological and geochemical studies of contemporary metal production and consumption behaviours [START_REF] Pryce | Prehistoric copper production and technological reproduction in the Khao Wong Prachan Valley of Central Thailand[END_REF][START_REF] Pryce | More questions than answers: the Southeast Asian Lead Isotope Project 2009-2012[END_REF]. At present, there are no reliable data for Laos, and the chronology for northern Vietnam remains unclear [START_REF] Oxenham | Mainland Southeast Asia: towards a new theoretical approach[END_REF]. But what of Myanmar's pivotal geographic position, with regard to the diachronic movement of people, materials and culture over its long terrestrial borders with South and East Asia?

There is, of course, no reason why Myanmar's late prehistoric chronology should exactly match that proposed by [START_REF] Higham | A new chronology for the Bronze Age of northeastern Thailand and its implications for Southeast Asian prehistory[END_REF] for Thailand. Indeed, our objectives at Nyaung'gan/Oakaie specifically included searching out variation in regional and interregional cultural transmission. We note that although a stimulus for the Bronze Age of Myanmar could have come from the secondmillennium BC northern Indian Copper Hoard Culture [START_REF] Coningham | Archaeology of South Asia: from the Indus to Asoka, c. 6500 BCE-200 CE[END_REF], there is a dearth of evidence for making such an argument east of Bengal. Similarly, the late prehistoric chronology of the mountainous Yunnan province in China is relatively poorly understood compared to Guangxi and Guangdong [START_REF] Ciarla | Rethinking Yuanlongpo: the case for technological links between the Lingnan (PRC) and central Thailand in the Bronze Age[END_REF], and thus a direct transmission from south-west China remains possible. It is remarkable, however, that the Neolithic to Bronze Age transition of the tenth to ninth centuries BC proposed for central Myanmar should be so consistent with the Thai evidence, which falls perhaps only 100-300 years older. It should also be noted that at Ban Non Wat, incised and impressed ceramics were absent from later Neolithic contexts [START_REF] Higham | A new chronological framework for prehistoric Southeast Asia, based on a Bayesian model from Ban Non Wat[END_REF], which seemingly mirrors the situation at Nyaung'gan/Oakaie. Furthermore, the archaeometallurgical evidence itself is highly suggestive of a strong connection with Myanmar's eastern neighbours, and thus the SCM. From the very outset of central Myanmar's Bronze Age, copper and bronze artefacts with lead isotope signatures consistent with copper production at Non Pa Wai in central Thailand and the Vilabouly Complex in central Laos were appearing in Nyaung'gan/Oakaie area deposits. This is in addition to as yet unidentified sources that do not correspond to the Monywa mineralisation [START_REF] Pryce | Metallurgical traditions and metal exchange networks in late prehistoric central Myanmar, c. 1000 BC to c. AD[END_REF]. This implies that the exchange networks necessary for the transport of copper-base raw materials or finished products to central Myanmar over more than 1000 geodesic kilometres, and thus far longer following suitable river valleys, mountain passes or coastal routes, were active within several generations of the Thai Bronze Age transition, if indeed they were not already active for other materials or population movements.

Many of the specialist studies (e.g. archaeobotany, bioarchaeology (archaeothanatology, strontium isotopes, dietary stable isotopes, aDNA), lithic analysis and archaeozoology) for Nyaung'gan and Oakaie are ongoing, and thus more detail will be forthcoming.With what we argue to be the beginnings of a reliable chronology in place, we can nevertheless be reasonably certain that this area presents evidence of late second-/early first-millennium BC activity at a scale practically unknown in Southeast Asia, and a late prehistoric trajectory intimately intertwined with its eastern neighbours. The pressing need is to detect the transition to agriculture in central Myanmar. The Franco-Myanmar team is currently attempting this with excavations of suspected Early Neolithic through to historic Pyu deposits at the UNESCO-listed site of Halin (Figure 1). We thus hope to advance the national culture-historical sequence for late prehistory and to enrich regional syntheses.

Figure 2 .

 2 Figure 2. Plan of the 2014 and 2015 excavations at OAI1, including human remains in situ. Late season flooding prevented the excavation of six tombs (hashed).

Figure 3 .

 3 Figure 3. Neolithic/Bronze Age living surfaces at OAI2/TP6A-C, showing dense sherd-rich deposits, extensive postholes and sub-floor burials (e.g. OAI2/TP6/S4).

Figure 4 .

 4 Figure 4. Eastern and southern sections of OAI3 showing cultural layers and features, including context numbers for the 1 × 1m² column.

Figure 5 .

 5 Figure 5. The 1998/1999 (TP1-5) and 2016 (NYG1-3) excavations at Nyaung'gan, including human remains indicated in their tombs.

Figure 6 .

 6 Figure 6. Plan and north and east sections of NYG1.

Figure 7 .

 7 Figure 7. Plan and south and east sections of NYG2.

Figure 8 .

 8 Figure 8. Plan and north and south sections of NYG3.

Figure 9 .

 9 Figure 9. The ceramic techno-typology for the Oakaie-Nyaung'gan sites.

Figure 10 .

 10 Figure 10. Charcoal radiocarbon determinations from OAI2-4 settlement industrial sites, metal-bearing vs metal-devoid layers clearly identified at OAI2 and OAI3, showing the c. 1000 BC Late Neolithic to Early Bronze Age transition. Determinations for OAI3 contexts 30054, 30060 and 30061 are considered suspect, but we present full data.

Acknowledgements

We are extremely grateful to U Aung Ko, Minister for Religious Affairs and Culture, and his predecessor, U Aye Myint Kyuu,Minister of Culture, and to U Kyaw Oo Lwin, Director General of the Department of Archaeology in Naypyidaw, and their support staff for permitting us to work at these sites and for providing assistance. We also warmly thank M. Charles Bonhomme, former Cultural Attaché at the French Embassy in Yangon, M. Sebastian Bris, his deputy, and Daw Yi Mon, their administrator, for providing essential in-country support. The 'Mission Archéologique Française auMyanmar' is financed primarily by the Archaeological Consultative Commission of the French Ministry for Europe and Foreign Affairs. The many institutions affiliated to the Mission via our extensive co-authorship have also contributed generously in-kind with analytical support. In particular, we are grateful to Christine Oberlin and the CNRS InSHS ARTEMIS committee for supporting our dating project, and to the LMC14 team for the AMS measurements. Finally, we thank the two reviewers for their helpful comments. Any remaining errors are our own.

Supplementary material

To view supplementary material for this article, please visit https://doi.org/10. 15184