A genome sequence from a modern human skull over 45,000 years old from Zlatý kůň in Czechia

Abstract

Modern humans expanded into Eurasia more than 40,000 years ago following their dispersal out of Africa. These Eurasians carried ~2-3% Neanderthal ancestry in their genomes, originating from admixture with Neanderthals that took place sometime between 50,000 and 60,000 years ago, probably in the Middle East. In Europe, the modern human expansion preceded the disappearance of Neanderthals from the fossil record by 3,000-5,000 years. The genetic makeup of the first Europeans who colonized the continent more than 40,000 years ago remains poorly understood since few specimens have been studied. Here, we analyse a genome generated from the skull of a female individual from Zlatý kůň, Czechia. We found that she belonged to a population that appears to have contributed genetically neither to later Europeans nor to Asians. Her genome carries ~3% Neanderthal ancestry, similar to those of other Upper Palaeolithic hunter-gatherers. However, the lengths of the Neanderthal segments are longer than those observed in the currently oldest modern human genome of the ~45,000-year-old Ust'-Ishim individual from Siberia, suggesting that this individual from Zlatý kůň is one of the earliest Eurasian inhabitants following the expansion out of Africa.

Fig. 1. The Zlatý kůň fossil.

a, Locations of the Koněprusy cave, where the Zlatý kůň human remains were found, and of other fossils with an age of at least ~40,000 years that yielded genome-wide data (Ust’-Ishim, Oase 1 and Tianyuan) or mtDNA (Fumane 2 and Bacho Kiro). b, Micro-computed tomography-based virtual reconstruction of the Zlatý kůň skull in frontal and lateral view. The map in a was created with QGIS using Natural Earth vector data.

Fig. 2. Genetic relationship with present-day and ancient humans.

a, mtDNA haplogroup N of a maximum-parsimony phylogenetic tree of mtDNA from Zlatý kůň (red font), Upper Palaeolithic individuals ~40 ka or older (blue) or between ~37 and ~24 ka (orange), and present-day individuals (black) (the entire tree is presented in Extended Data Fig. 3). b, Analysis of nuclear sequences showing that Ust’-Ishim shares more alleles with European and Asian hunter-gatherers and later Eurasians than does Zlatý kůň. The error bars represent two standard errors. c, Admixture graph of the relationship inferred from the nuclear capture dataset. Zlatý kůň diverges earlier than Ust’-Ishim and the ancestors of later Eurasian populations represented here by the Upper Palaeolithic Tianyuan and Sunghir genomes (highest outlier |Z| = 3). A single gene flow event from Neanderthals into the ancestor of all tested modern humans fits the data. Colours for individuals follow the same scheme as in panel a.

Fig. 3. Neanderthal ancestry in Zlatý kůň and ancient Eurasian genomes.

a, Estimate of Neanderthal ancestry in ancient Eurasian hunter-gatherer genomes. The error bars indicate two standard errors. Individuals whose names are marked with an asterisk fall outside of the error bars for Zlatý kůň. b, Segments of Neanderthal ancestry in Zlatý kůň. The blue box shows the location of a desert of Neanderthal ancestry in present-day non-Africans. c, Length of the 100 largest Neanderthal segments in the genomes of Zlatý kůň and other Upper Palaeolithic and Mesolithic Eurasian hunter-gatherers. The y axis is logarithmic and the lines indicate a linear fit. The colours are as in a.

Extended Data Fig. 1. Skeletal remains from Zlatý kůň.

a, and b, frontal and dorsal view, respectively, of the skull, c, vertebrae and d, costae. Credit: Martin Frouz (a,b); Marek Jantač (c,d).

Extended Data Fig. 2. Four direct radiocarbon dates calibrated in years before present (calBP) with 68.3% and 95.4% probability.

OxA-38022 refers to the HYP dating performed in Oxford. The dates were calibrated using OxCal 4.4 and the IntCal20 calibration curve^,.

Extended Data Fig. 3. Maximum Parsimony tree of 54 modern-day worldwide mtDNA sequences, 28 Palaeolithic Eurasian mtDNAs, Zlatý kůň mtDNA and a Neanderthal mtDNA as outgroup.

Text color relates to radiocarbon date, that is red (undetermined), blue (45–40 kya) and yellow (40–24 kya). Numbers show the number of substitutions along lineages; capital letters on the right side of the tree indicate mtDNA haplogroups.

Extended Data Fig. 4. Coverage on sex chromosomes and autosomes.

Average coverage by shotgun sequences for chromosome X, Y (horizontal dashes) and autosomes (violin plot).

Extended Data Fig. 5. Characteristics of Bos taurus mtDNA sequences and genome.

a, C-to-T substitutions at the 5′ end and read length distributions of mtDNA sequences mapping specifically to the mtDNA references of Homo sapiens, Bos taurus, Capra hircus and Oryctolagus cuniculus. Note that a single-end 75-cycle chemistry kit was used for the retrieval of the reported sequencing reads and, therefore, DNA fragments of length 76 bp or above have not been sequenced at their full length and are binned here together. b, Maximum parsimony tree of 35 present-day and ancient cattle mtDNAs and the Bos taurus mtDNA reconstructed from the deep shotgun sequencing of the DNA extracted from the Zlatý kůň petrous bone. Cattle haplogroups are listed on the right side of the tree.

Extended Data Fig. 6. Comparable signal of Basal Eurasian ancestry in Zlatý kůň and Ust’-Ishim.

Zlatý kůň’s asymmetric sharing of alleles with some Europeans compared to Asians (x-scale) is also observed to a comparable degree in Ust’-Ishim (y-scale). The ancient genome from Tianyuan is used as a proxy for Asians. The solid and dash error bars correspond to one and two standard errors, respectively.

Extended Data Fig. 7. Heat-map matrix of pairwise f3-outgroup statistics with Mbuti as outgroup.

Zlatý kůň shows no closer affinity to Ust’-Ishim and Oase 1 or to any later hunter-gatherer individuals.

Extended Data Fig. 8. Neanderthal desert region on chromosome 1 in Zlatý kůň and 5 high-coverage ancient genomes.

Each panel shows Neanderthal-shared variants in red and non-shared variants in gray. The orange line indicates the posterior decoding probabilities for Neanderthal ancestry from the hidden Markov model approach. The Neanderthal ancestry segment in Zlatý kůň (chr1:112696231-112855758) is supported by 17 Neanderthal shared variants. A short region, spanning only 6 bp (chr1:102812075-102812081) is called in Loschbour and contains four sites that are Neanderthal shared. The short length and high density of sites do not support a true Neanderthal origin.

Extended Data Fig. 9. Fraction of the Zlatý kůň genome covered by the top 100 called introgressed regions sorted by length.

The fraction of the genome covered by Neanderthal ancestry (y-scale) was calculated by dividing the cumulative length in Morgan of introgressed regions up to and including the n^th longest region shown on the x-scale by two times the total length of the autosomes in Morgan. Results are shown for the African American or Decode recombination maps.

Extended Data Fig. 10. Pairwise comparison of low-coverage Neanderthals in their sharing of alleles with European and Asian hunter-gatherers.

Shown are the D-statistics of the form D(Mbuti, Hunter-Gatherer, Neanderthal1, Neanderthal2). The Hunter-Gatherer corresponds to the columns; rows show the two compared Neanderthals in order Neanderthal1: Neanderthal2. Error bars correspond to two standard errors.