During the Neolithic, the North Pontic Region (NPR) was home to major prehistoric cultural conglomerates, among them—the Dnieper-Donets cultural complex (DD). The DD culture has been studied in approximately 200 sites in Ukraine and Byelorussia, including settlements and large collective cemeteries of the Mariupol-type (M-t).1 The main feature of M-t cemeteries is inhumation burial in the supine position. This burial rite differs from most local Mesolithic burial traditions and is characteristic of the ‘Euro-Siberian’ zone of extended burials, which are found from Lake Baikal and the forest and forest-steppe zones of the East European Plain to the northern part of Central Europe and Scandinavia.2, 3

The distinct burial tradition of Neolithic populations of the NPR as well as their anthropological characteristics suggests an influx of external population sources. We set out to quantify the extent of gene flow into the NPR during the Neolithic. Recent advances in mitochondrial DNA (mtDNA) analysis have revealed global patterns of neutral marker variation that appear to correlate with the geographic origin of the source population.4, 5 To elucidate the maternal genetic lineages of the population that constructed the M-t cemeteries, we undertook a genetic analysis of ancient mtDNA extracted from specimens from two Neolithic NPR cemeteries2 (Figure 1).

Figure 1
figure 1

Map of the North Pontic region showing locations of the Neolithic Mariupol-type cemeteries (Nikolskoye and Yasinovataka) from which specimens were selected for mtDNA analysis.

Materials and Methods

Origin of samples

Samples chosen for DNA analysis came from the anthropological collections of the Department of Bioarchaeology of the Institute of Archaeology in Kyiv, Ukraine. In most cases, only small cranial fragments were available for DNA extraction.

Authenticity criteria

There have been a variety of techniques suggested to minimize contamination with modern DNA, including amplifying small DNA fragments, cloning of PCR products and maintaining the strict conditions of sterility.6 These and other precautions were followed, such as minimizing the number of individuals handling the specimens and determining the mtDNA polymorphism pattern of the researchers who had the most direct contact with the specimens (Table 1). While such precaution controls the most likely source of modern DNA contamination, the possibility of contamination by other exogenous sources that came into contact with the specimens prior to DNA extraction may still persist.

Table 1 Specimen description, radiocarbon dates and mtDNA haplogroup designation for specimens from Mariupol-type cemeteries for which comprehensive haplogrouping information was obtained, along with mtDNA haplogroups of the researchers who handled and processed the osteological material

Ancient mtDNA extraction, amplification and analysis

All sample preparation, DNA extraction and initial amplification was performed by a single person (AGN) in facilities dedicated to DNA work with spatially separated cleanup, DNA extraction, amplification and post-PCR processing.

Two temporary separated DNA extractions were performed per each osteological fragment. The surface of each specimen was treated with 253.7 nm ultraviolet for 45 min on all sides. Then, approximately 1 mm of the surface was sanded off, after which the ultraviolet treatment was repeated. Then, 300–400 mg of the bone were cut off per extraction and ground with mortar and pestle. The powder was washed with 0.5 M EDTA pH 8.0 then rinsed with water. DNA was extracted using a QIAGEN QIAmp DNA Investigator Kit (QIAGEN Inc., Valencia, CA, USA). Multiple blank extractions were performed for each specimen. DNA was eluted in 50 μl of water.

DNA was typed by amplifying the first hypervariable segment (HVS1) of the control region of mtDNA in overlapping segments (Table 2). Amplifications of haplogroup-diagnostic segments of the coding region were subjected to restriction fragment length polymorphism (RFLP) analysis. Haplogroup designation based on coding region RFLPs was produced from at least three independent PCRs from two different extractions.

Table 2 DNA primers used to amplify the control and diagnostic coding regions of mtDNA

DNA amplification was done using a QIAGEN Fast Cycling PCR kit, following the conditions optimized for fragments in the 10 to 100 copy range. Each HVS1 segment was amplified up to 4 times per extraction or until two independent amplification products were obtained per extraction. The HVS1 amplicons were cloned and subsequently sequenced.

Results and Discussion

Out of 18 samples analyzed, 14 produced mtDNA amplification. Comprehensive haplotyping information was obtained for seven specimens (Table 1). In seven specimens, the replicative analysis of ancient mtDNA could not be accomplished. DNA sequences have been deposited in GenBank ( under accession numbers JN873355–873361.

Of the 286 HVS1 clones sequenced, only four (1.4%) could be identified as belonging to one of the three principal researchers. Most clones displayed amplification-replica-specific random non-repeatable nucleotide misincorporations occurring at non-phylogenetically relevant positions, which is consistent with the expected post-mortem nucleotide damage.7, 8

Two specimens were designated as members of haplogroup H, two were members of the U clade and three shared the 16223–16298–16327 HVS1 sequence motif characteristic of the root sequence of haplogroup C.9, 10 Specimen Ya34 carried a transition at 16357, characteristic of the C4a2 subbranch of the C clade.10

While the majority of mtDNA lineages found in the Neolithic remains from the NPR can be ascribed to the haplogroups of West Eurasian origin and are in agreement with those previously reported for prehistoric haplogroups in West Eurasia,11, 12, 13, 14, 15, 16, 17, 18 our finding of East Eurasian lineages is uncommon in ancient European remains and has only been observed in the neighboring populations of the Carpathian Mountains.19, 20

Haplogroup C is a derivative of the M8 subclade of the Asian-specific M clade. The frequency of M-derived lineages in the modern mtDNA pool of Eastern Europe is marginal, with the exception of an isolated population of Carpathian highlanders.21 The haplogroup C HVS1 root sequence motif was frequent in ancient populations of the Tarim Basin.22 Modern Siberian populations as well as Neolithic populations of the Northern Baikal region and Northeastern Siberia have been found to harbor polymorphisms found in the C-bearing DD specimens.23, 24, 25 A C5-bearing specimen has been recently found in Neolithic Hungarian remains,20 extending the presence of the C clade in Neolithic Europe from the northern coast of the Black Sea to the Carpathians.

The overall conclusion about the genetic composition of the builders of M-t cemeteries is that they were a genetically heterogeneous population that contained admixtures of mtDNA lineages from neighboring geographic regions as well as from the territories stretching far east. The noticeable anthropological influences of DD on local post-Neolithic populations suggest the possibility of genetic continuity in populations succeeding the people who built the M-t cemeteries. The genetic relationship between Neolithic DD populations and Copper-Bronze Age inhabitants of the North Pontic steppe is the subject of an ongoing investigation.