Ancient DNA authentication and uniparental markers

The ancient individuals from this study originate from 13 Neolithic and Early Bronze Age sites in Switzerland (Fig. 1b), Southern Germany, and the Alsace region in France. All samples taken from the individuals were radiocarbon dated (Supplementary Note 2 and Supplementary Data 1). In a preliminary screening, 263 samples were enriched for mtDNA. We reconstructed complete mitochondrial genomes, used them to estimate DNA library contamination (Supplementary Data 1), and identified 96 samples that had less than 5% of contamination for further analyses. We determined mtDNA haplogroups using the software haplogrep (ref. 16, Supplementary Note 3, Supplementary Fig. 1, and Supplementary Data 1) and found the macrohaplogroups N1a, W, X, H, T2, J, U2, U3, U4, U5a, U5b, K, and U8 in our samples.

Fig. 1: Genetic, temporal, and spatial structure of individuals in this study. a Time ranges of calibrated radiocarbon dates of the archaeological sites. b Geographical distribution of the sites and samples sizes per sites in brackets. Map generated with R version 3.4.3 (R Core Team 2017) using the CIA World Data Bank II currently (mid 2003) available from http://www.evl.uic.edu/pape/data/WDB/. c PCA was reconstructed on 1960 modern European individuals of the POPRES dataset and ancient genomes were projected onto it. Full size image

For genome-wide analysis, we genotyped all selected individuals on ~1.2 million genomic SNPs2, also containing 49,704 SNPs on the X chromosome and 32,670 SNPs on the Y chromosome. SNPs on the X chromosome were used to estimate nuclear contamination in male individuals (Supplementary Data 1), and we again used a threshold of 5% to select clean libraries for further analysis (96 individuals). We also determined Y chromosomal haplogroups (Supplementary Note 4, Supplementary Fig. 2, Supplementary Data 1, and Supplementary Table 1).

Population turnover at the transition to the Bronze Age

We combined the genotype data of the new 96 individuals from this study that passed our contamination tests with 399 published ancient genomes from the same time period from Central and Western Europe as well as Neolithic individuals from Anatolia and the Pontic steppe (individuals annotated as Yamnaya Samara in ref. 6) and genotype data of modern individuals from the POPRES17 and the Human origins (HO)1 datasets for various analyses.

We projected our 96 new ancient genomes from Switzerland and surrounding regions and 52 published ancient genomes selected to reflect the genetic landscape of Europe at different time points onto the first two principal components constructed from 1960 individuals of 38 European populations from the POPRES dataset (Fig. 1c and “Methods”). Two distinct clusters can be identified and were also confirmed by ADMIXTURE analysis (Supplementary Note 5), one consisting of individuals dating to 4770–2500 calBCE, and one comprising individuals dating to 2900–1750 calBCE. The oldest individuals from the sites of Niederried (CH) and Lingolsheim (F) fall close to ancient individuals from Anatolia associated with early agricultural contexts. More recent individuals from the megalithic burials at Oberbipp (CH) and Aesch (CH) are shifted further towards Western Hunter-Gatherers (WHG) and close to modern-day Sardinian individuals, as well as towards Early and Middle Neolithic individuals from Iberia or the Middle Elbe-Saale (MES) region in Central Germany. This shift mirrors an increase of hunter-gatherer-related ancestry during the middle Neolithic that has been described previously for other parts of Europe.

The second distinct cluster is shifted towards the individuals associated with the “Yamnaya” complex, similar to other European groups younger than 2700 BCE, relative to individuals older than 2700 BCE. In this cluster, the oldest individuals are closest to Late Neolithic groups on the steppe, whereas more recent individuals are once again shifted towards the Middle/Late Neolithic cluster. All Final Neolithic and Early Bronze Age individuals fall within the range of modern-day Europeans, but none of the newly sequenced individuals of this study overlap with the present-day Swiss populations in this analysis, suggesting additional population changes in the region after the Middle Bronze Age.

Our individuals sequenced in this study fall in PC space between WHG individuals, Western Anatolian Neolithic Farmers (ANF) and steppe pastoralists from Samara (YAM), similar to other Late Neolithic individuals such as the Tyrolian Iceman18 and Bronze Age populations such as individuals of the Bell Beaker complex6 from Europe. Therefore, we modeled them as a three-way mixture between these three populations using qpAdm from the ADMIXTOOLS package (ref. 19, “Methods” and Supplementary Tables 2 and Supplementary Data 4). The overall pattern observed from this analysis matched previous analyses of that type9. Individuals from older sites (Early Neolithic and Middle Neolithic) are consistent with a two-way mixture between WHG and ANF ancestry, whereas individuals from younger sites after ~2700 BCE exhibit substantial amounts of ancestry related to YAM. Furthermore, the proportions of this component differ strongly between sites and tend to decrease over time (Fig. 2a). This trend is confirmed by further analysis of the ancestry components on an individual level.

Fig. 2: Genetic turnover at the transition to the Central European Bronze Age. a Three-way qpAdm models of the ancient individuals from Switzerland (number of individuals in brackets) sorted by sites in chronological order (bottom to top) with the source populations WHG, steppe pastoralists (Yamnaya Samara) and Anatolia Neolithic. Error bars represent standard error of the proportion of each component. b Relative proportion of the steppe-related ancestry component for each individual in four different regions, calculated with qpAdm and estimates of arrival times (error bars represent the range of C14 dating) and following decrease of the component (dashes lines). Red dots represent female individuals and blue dots male individuals. c An estimate of genetic diversity between individuals before 2700 BCE and after 2700 BCE and sites with individuals from both periods as well as modern European populations (German and French from the HO dataset). Full size image

Compared with previous studies2,9 analyzing Neolithic and Bronze Age individuals from present-day Germany and Great Britain, which do not report individuals dating to the transition period directly, in this study we analyze a gapless time-transect covering the Neolithic to Bronze Age transition. By viewing the YAM-related ancestry component estimated with qpAdm over time at an individual level, it becomes apparent that this ancestry was virtually absent before 2700 BCE, followed by a steep increase in parts of the population starting around 2700 BCE (Supplementary Note 7 and Supplementary Data 4). After this rapid increase in individual proportions of YAM-related ancestry from 0% to ~60%, a decrease down to 25–35% can be observed over the next thousand years. We also note four female individuals that can be modeled without any YAM-related ancestry even 1000 years after the appearance of that genetic component in the area. Comparing outgroup-f3 statistics between the autosomes and the X chromosome of Final Neolithic and Bronze Age individuals we find that autosomes are more closely related to YAM-related ancestry than the X chromosomes are (Supplementary Note 6), consistent with a model in which more males than females brought YAM-related ancestry into the region as already shown by previous studies20.

We analyzed pairwise genetic differences across all analyzed genomic positions between individuals before and after the genetic turnover and found that the mismatch rates increase, on average, by around 0.009 after 2700 BCE for all populations (Fig. 2c, see also “Methods”). This is more than twice the increase that can be attributed to the between-population variability in rates and indicates a significant increase in genetic diversity after the arrival of the YAM-related ancestry component in Central Europe. Modern populations would be expected to have higher levels on average but are not compared in this analysis since the modern individuals from published datasets usually do not originate from groups with the same background (e.g., being related distantly) as could be expected in multiple burials.

Comparing our newly analyzed individuals from Switzerland with ancient genomes from Great Britain, Iberia, and Germany2,9,21 we modeled the arrival time of the YAM-related ancestry in the different broadly defined European regions (Fig. 2b, see “Methods“). While our models indicate that the proportions of the YAM-related ancestry peaks earlier in the Swiss dataset (around 2750 BCE) compared with the comparative datasets from refs. 2,9,21 (around 2600 BCE), these differences fall within the uncertainty of the analysis (Supplementary Fig. 6), so may be considered suggestive of an earlier arrival of steppe-related ancestry, but not conclusive. We also caution that differences are likely affected by uneven sampling through time in the three different datasets, and so expect the precision of this analysis to improve with denser temporal sampling in the future.

Timing and duration of the genetic turnover

We used the software DATES (ref. 22, https://github.com/priyamoorjani/DATES) to estimate the admixture time between YAM-related and Late Neolithic ancestries in all Final Neolithic and Early Bronze Age individuals from Switzerland with substantial admixture proportion. Our estimates range between 3 and 60 generations ago, with substantial uncertainty. If the mixture occurred as one single event in the history of all individuals, we would expect more recent individuals to have a higher admixture time estimate (i.e., more generations ago) than individuals of older dating. However, we observe only a slight trend towards more generations in individuals with younger C14 dates (Fig. 3a), which suggests that the process of admixture with steppe-related ancestry occurred over several hundred years rather than as a single pulse.

Fig. 3: Estimated admixture times between the Yamnaya-like steppe component and the Middle Neolithic population using DATES software. a Admixture dates of single individuals are plotted against their calC14 dates (horizontal error bars indicate uncertainty in C14 dating and vertical error bars show 95% confidence interval of generation times) and b displayed as time range (C14 dates in red and estimated admixture dates in turquoise). c Admixture dates of grouped individuals according to their regions of origin were calculated (colors as above). Full size image

For comparison, we performed a similar analysis for published ancient Final Neolithic and Bell Beaker populations from the MES region in Germany, Great Britain, and Iberia using a mixture between the YAM-related steppe component and the corresponding Middle Neolithic population of the region (Fig. 3c). For the MES region from Germany, Late Neolithic and Early Bronze Age individuals were split in Bell Beaker and Corded Ware groups. Similar to this approach, we also split the Neolithic individuals from Switzerland into a group of individuals associated with the Corded Ware Complex from Spreitenbach and a group of younger individuals from the Bronze Age. All four regions (Iberia, Great Britain, MES and Switzerland) show similar ranges of admixture dates between the steppe-related component and the Neolithic component starting between 3000 and 2500 BCE.

The inferred admixture time describes the time when people of steppe-related ancestry encountered people with Late Neolithic ancestry but does not reveal the location. Admixture could have happened in Switzerland or elsewhere, with already admixed individuals moving to Switzerland.

Neolithic source population for the admixing event

We tested which Neolithic population likely admixed with the incoming people that carried large amounts of YAM-related ancestry, by adding Late Neolithic individuals from Switzerland, Globular Amphora, Iberia Middle Neolithic, and France Neolithic groups separately as additional right populations to the three-way model (WHG, Western Anatolia Neolithic and Yamnaya Samara) used in the above qpAdm analyses (ref. 19, Supplementary Data 3 and 4). The model remains fitting for Iberia Middle Neolithic and France Neolithic populations (p ≥ 0.01 and p ≥ 0.3, respectively) but it fails when we add Late Neolithic individuals from Switzerland or Globular Amphora as additional right population (p ≤ 2e−7 and p ≤ 2e−6, respectively). This suggests that both the local Swiss Late Neolithic population as well as people associated with the Late Neolithic Globular Amphora culture, located further east, are better proxies of the genetic sources for Final Neolithic and Bronze Age populations from Switzerland than Western ANF and steppe pastoralists.

Kinship before and after the genetic transition

In five burial sites, we identified first-degree relatives using the software lcmlkin23 and READ24, and by calculating pairwise mismatch rates across all analyzed genomic sites between individuals (see “Methods”). Four of these sites contained more than two closely related individuals, which allowed us to reconstruct family trees spanning three generations for Oberbipp, Aesch and Singen (Fig. 4). In these multiple burials, only a few female individuals (four individuals) were buried together with one of their parents or their sons, compared with a higher number (21 individuals) of males buried with their father, brothers or sons, indicating that males likely tended to stay where they were born, while females were likely mobile. This pattern is observed both before and after the arrival of the YAM-related ancestry and is indicative of patrilocal societies during Late Neolithic times in the studied region, consistent with previous results from Neolithic times throughout Northern and Western Europe25,26.

Fig. 4: Reconstructed family trees from different sites over time. All relationships between the single individuals were reconstructed from autosomal variants and confirmed by uniparentally inherited markers such as mtDNA haplotype and Y chromosomal haplogroup. Individuals with black outline were available for analysis and individuals with gray outline were not found within the burials and are missing. Same colors indicate identical mtDNA haplotypes and matching Y chromosomal haplogroups. Full size image

Population movement in Switzerland after the Bronze Age

We compared present-day Swiss people to regional Final Neolithic populations (Spreitenbach, Bad Zurzach, Wartau) to test whether there are additional ancestry components in present-day Swiss people. For that analysis, we made use of information available in the present-day dataset (POPRES) about the self-reported language group and split the individuals into three linguistic regions, as they were shown to be distinguishable genetically in previous studies27: German-speaking, French-speaking, and Italian-speaking Switzerland. To test for continuity between the ancient and present-day population we used the method qpWave from the ADMIXTOOLS package19 and found that a simple continuity can be rejected (p = 0.0003) for all three linguistic regions separately and the entire present-day Swiss population combined, consistent also with the PCA (Fig. 1c).

To assess whether ancient Swiss individuals from the Final Neolithic are symmetrically related to different linguistic present-day groups, or share an excess of alleles with any of them, we calculated D-statistics of the form D(Mbuti, Test, Swiss–French, Swiss–Italian), D(Mbuti, Test, Swiss–German, Swiss–Italian), and D(Mbuti, Test, Swiss–French, Swiss–German) where “Test” are the different Neolithic groups. The first two D-statistics were, with few exceptions, all negative with a |Z| ≥ 1.099 (maximum values to be found in D(Mbuti, Test, Swiss–French, Swiss–Italian) for Singen with −3.431 and Bad Zurzach −3.068) indicating the least genetic affinity of the Final Neolithic and Early Bronze Age individuals of this study to the Italian-speaking group in our present-day Swiss dataset. For D(Mbuti, Test, Swiss–French, Swiss–German) some variation between the sites can be found (Fig. 5) with the older sites sharing more alleles with the French-speaking group, and the younger sites being more similar with the German-speaking group.

Fig. 5: Genetic affinity between the ancient Individuals and the French-, German-, and Italian-speaking regions of Switzerland. Differences in genetic affinity of the ancient individuals from Switzerland to a Swiss–French and Swiss–German, b Swiss–Italian and Swiss–German, and c Swiss–French and Swiss–Italian. Maps generated with R version 3.4.3 (R Core Team 2017) using the CIA World Data Bank II are currently (mid 2003) available from http://www.evl.uic.edu/pape/data/WDB/. Full size image

Analysis of functional SNPs

We analyzed the frequencies of several phenotypic SNPs (Table 1, “Methods”). Derived alleles for SLC24A5 associated with light skin pigmentation in Europeans were found in all individuals with this position covered. The frequency of SLC45A2 also causing lighter skin pigmentation tends to increase and the frequency of HERC2 associated with light eye-color tends to decrease towards the Final Neolithic. A mutation associated with lactose tolerance in adulthood (LCT; rs4988235), which is of high frequency in Europe today, is absent in Late and Middle Neolithic samples. The only exception is one Final Neolithic individual from Spreitenbach dating to 2105–2036 calBCE, which is one of the earliest European individuals with this mutation found so far. The near absence of lactose tolerance in these ancient groups is in concordance with previous studies hypothesizing that this mutation arose in the Final Neolithic period and started to increase in frequency after the beginning of the Bronze Age2.