Mitochondrial genomes reveal an east to west cline of steppe ancestry in Corded Ware populations

From around 4,000 to 2,000 BC the forest-steppe north-western Pontic region was occupied by people who shared a nomadic lifestyle, pastoral economy and barrow burial rituals. It has been shown that these groups, especially those associated with the Yamnaya culture, played an important role in shaping the gene pool of Bronze Age Europeans, which extends into present-day patterns of genetic variation in Europe. Although the genetic impact of these migrations from the forest-steppe Pontic region into central Europe have previously been addressed in several studies, the contribution of mitochondrial lineages to the people associated with the Corded Ware culture in the eastern part of the North European Plain remains contentious. In this study, we present mitochondrial genomes from 23 Late Eneolithic and Bronze Age individuals, including representatives of the north-western Pontic region and the Corded Ware culture from the eastern part of the North European Plain. We identified, for the first time in ancient populations, the rare mitochondrial haplogroup X4 in two Bronze Age Catacomb culture-associated individuals. Genetic similarity analyses show close maternal genetic affinities between populations associated with both eastern and Baltic Corded Ware culture, and the Yamnaya horizon, in contrast to larger genetic differentiation between populations associated with western Corded Ware culture and the Yamnaya horizon. This indicates that females with steppe ancestry contributed to the formation of populations associated with the eastern Corded Ware culture while more local people, likely of Neolithic farmer ancestry, contributed to the formation of populations associated with western Corded Ware culture.


Pidlisivka barrow 1, Yampil Region, Vinnitsa Oblast, Ukraine
Research on the barrow was conducted in 2010. The site is located near the village of Pidlisivka, on the high plateau on the right bank of the River Jałanka, north-west of its mouth into the River Markivki, tributary of the Dniestersome 7 km from the corridor of the latter. At the commencement of barrow examination the tumulus was not a distinct form in the landscape. In the immediate surrounds of the mound the presence of vast signs of destruction caused by trenches from World War II was observed (features 2, 3 and 14). The recorded maximum height did not exceed 1 m in relation to the surrounding landscape and the recorded diameter of the tumulus on a west-east axis was measured at 30 m approximately.
Under the mound of Barrow 13 inhumations were discovered (Supplementary Fig S2) and samples taken from five of the features for DNA analysis (1A, 1B, 5,11,13), which in the case of 1B and 13 DNA was obtained successfully.

Grave 1B (Eneolithic) -poz090
Feature 1B is most likely the oldest central inhumation associated with Eneolithic communities. The male, aged 22 to 25, was interred along a north-west to south-east axis in a supine position with knees raised (Supplementary Fig S3). The deceased was laid to rest on a mat made out of organic materials and the only readable elements of grave furnishings were ochre and a flint flake. Over the rectangular grave pit measuring 1.45 x 0.7 m a roof of oak planks was constructed at an angle to the longer axis of the grave.

Grave 13 (Babyno culture) -poz094
Feature 13 is the grave of a woman aged around 25. Excavations at the southern edge of the tumulus mound were conducted for the phase of exploitation by the communities of the Babyno culture. The deceased lay on her right side in a crouched position on an east-west axis. No additional elements of grave furnishings were recorded near the human remains. From above the grave was secured by a fragment of a chalk slab.

Porohy 3A, Yampil Region, Vinnitsa Oblast, Ukraine
Barrow 3A in Porohy is situated in a concentration of 5 tumuli in the area of the Dniester watershed and Rusava River at a distance of 1.5 km from the former. At the time of commencing excavation work the actual form of the mound was already significantly eroded, its height amounting to approximately 1.2 m and its diameter approximately 40 m. Archaeological investigation at this site was conducted in 2011 and as a consequence 22 features associated with the Eneolithic were documented (4, 6, 7, 9, 13 14, 18), YC (1,2,10,11,12,15,16,17,19,20), NC (3,5,8,22) and that of the Iron Age -feature 21. For the purposes of DNA analysis samples were taken from the following features: 1, 3, 5, 7, 10, 11, 12, 14, 15, 19, 20 and 22. Only in the case of feature 19 DNA was obtained successfully.

Grave 19 (Yamnaya culture) -poz208
This is the grave of a deceased child no older than one and a half years (Supplementary Figure  S5). The individual lay supine along a west-east axis. The grave pit, measuring 0.9×0.4 m, was most likely secured by a wooden lid. Under the bones in the grave the remains of an organic mat were discovered as well as traces of ochre on the skull. The oldest central inhumation (grave 14) and two further graves (no. 5 and 15) were associated with the Neolithic creators of this settlement ( Supplementary Fig 6). Subsequent settlers of the area used for cult purposes are associated with the communities of the Babyno culture. The central inhumation for this stage is feature 1 and other associate inhumations: 2, 3, 6, 8, 10. The chronological sequence here is completed by the communities of the Noua culture for whom the major site was feature 13, while the remaining graves (features 7, 11, 12) were located at a significant distance away from the initial site, within the barrow ditch.
DNA was successfully obtained for all the samples taken for this purpose, forming a picture for each of the stages presented above (features 5, 14, 3, 11, 12).

Grave 3 (Babyno culture) -poz356
A deceased male aged 35-45 lay at the bottom of the pit, measuring 0.9 times a .6 m along an east-west axis (Supplementary Fig S7). The skeleton was poorly preserved and in a crouched position on its right side. The only element of furnishing was a small ceramic vessel deposited at the bones of the upper limbs.

Grave 5 (Eneolithic) -poz211
In a pit measuring 1.85×0.95 m, along a west-east axis, there lay a well preserved male skeleton aged 50-55, supine, crouched and head and lower limbs turned towards the left (Supplementary Fig  S8). The grave did not show any signs of furnishings and neither were there any wooden parts of construction for the roofing of the pit.

Grave 12 (Babyno culture) -poz213
The skeleton of a deceased child, aged 12 to 14, lay in a crouched position at the bottom of the pit, which measured 1.0×0.8 m along a north-south axis ( Supplementary Fig S9). From above the grave was secured by a layer of limestone.

Grave 14 (Eneolithic-central grave) -poz214
At the bottom of a deep pit, measuring 2.05×1.95 m, there lay a well preserved skeleton of a male aged 25-30 with signs of red ochre (Supplementary Figure S10). The deceased was placed in a crouched position on his left side and most likely rested on a mat of organic materials. On the occipital bone and the right parietal an irregular opening was present, the result of a blow (most likely the cause of death). Between the bones of the ribs a small flint flake was found. The pit could have been furnished with additional elements of construction made out of wood.

Prydnistryanske, site 1, Yampil Region, Vinnitsa Oblast
The cemetery complex was excavated in 2014 and is located on the crest of a long promontory in a north-west to south-east direction. From the western border with the Dniester Valley, about 1 km away from the corridor of this river, to the east of the valley of the Markovki. The most important point of this zone is Barrow mound number IV; its height at the commencement of excavation work was measured at about 2.4 m and the diameter at 35 m. About 80 m to the south along a line of a north-east to west-south axis three small mounds were situated (barrows I-III), whose diameter was measured at about 20 to 24 m and the preserved height merely at 0.15 to 0.3 m. The limitations of the surrounding terrain only allowed for the investigation of but one central and western part of barrow IV. Here the smaller mounds were identified entirely.
Within tumulus 1 four features were identified. One of the features was the hypothetical central grave, in which no traces of inhumation alas, were recordedonly in its fill did a fragment of ceramic ware and wood that were identified allow to categorize this grave to the Gordineşti group, Tripolye culture. The chronology of the other two features can be linked to the Iron Age. For the purposes of DNA analysis samples from the fourth feature were taken, which was sunk into the eastern part of the mound.

Barrow 1, Grave 4 (Catacomb culture) -poz220 and poz221
This is a double grave of the Catacomb culture. In the pit shaped oval and measuring 2.2×1.5 m, oriented south-north, on the western side a woman was laid to rest aged around 15 and on the eastern side a man aged 35-50. The only element of furnishing was a stone club with the remains of a wooden handle and copper mounting, placed at the shoulder of the deceased male.
Under tumulus mounds II and III seven features in all were found, which point to the fact that those responsible for construction were of Eneolithic communities and that subsequent phases of funerary exploitation were undertaken after the Bronze Age. No materials were gained from these graves for the purposes of DNA marking.
The initial, small mound of tumulus IV, measuring a diameter around 25 m was raised over Eneolithic feature of the Tripolye culture. From the subsequent two stages of its enlargement there originate inhumations of the Yamnaya culture from which samples were taken for DNA analysisfeatures 4 and 6 from the older phase and 3, 8 and 9 from the younger phase of the Yamnaya culture. The final results of these analyses provided DNA from skeletons from graves 4, 8 and 9.

Barrow IV, Grave 4 (Yamnaya culture) -poz222
The male interred in feature 4 died, aged 35-50. The deceased lay in a pit, measuring 0.9×2.85 m, in a supine position with raised knees and both arms along his body. Part of the furnishings entailed a mat of organic materials upon which the deceased rested and a small lump of ochre placed by the skull (Supplementary Figs S12, S13). The grave was secured by a wooden lid supported on stakes. In the upper part stone blocks were laid, thereby creating a slab measuring about 2.7×1.9 m that was in addition secured by an organic mat.

Barrow IV, Grave 8 (Yamnaya culture) -poz224
From the younger phase of tumulus enlargement by a community of the Grave Pit culture there originates grave 8 from the southern part of the tumulus. In the pit, measuring 1.6×1.3m, a male was placed in a supine position, aged 35-50 ( Supplementary Fig S14). Elements of grave furnishings for the deceased were a knife blade found near the pelvis, small lumps of ochre and preserved traces of an organic mat under the skeleton. The grave was secured by a wooden lid.

Barrow IV, Grave 9 (Yamnaya culture) -poz225
The pit of feature nine had very similar dimensions to that of grave 8 (1.85 x 1.4 m). The deceased male at the time of being laid to rest was aged 25-35 (Supplementary Fig S15). The deceased lay on a mat and was furnished with ochre. An animal bone was also found in the grave, while the upper part of the pit was covered by oak planks.

Corded Ware Culture individuals from Poland
In the period approximately 2800-2400 BC the system of cultural beliefs and patterns known as the Corded Ware culture characterized communities inhabiting the vast stretches of Europe. The eastern and southern part of this territory neighbored onto another great phenomenon in the third millennium BCthe Yamnaya culture. The regions of Małopolska also belonged to the broadly understood above mentioned zone, where relatively comprehensive grave finds originate (from Barrows and flat cemetery complexes). In this territory specific forms of funerary rites have been registered (among others catacomb graves), which could testify to ties with the Steppe culture circle of the north-west Black Sea region. The significance of this relationship is confirmed by some artifacts in the form of grave furnishings.
Małopolska Corded Ware culture sites can be said to have created several settlement concentrations located in the upland zone on territories covered with loess soils. The largest and best researched such concentration is in the western part, located on the left bank of the Vistula from where the graves presented below originate in Książnice and Malżyce. Other concentrations can be found in the Carpathian zone, Lublin Upland and the western part of the Volhynia Upland. It is the latter where the barrow from Hubinek it is located.

Hubinek, Ulhówek commune, district of Tomaszów Lubelski, site 2, barrow 1
The site is located amidst the concentration of Corded Ware culture barrows in the Sokal Hills, on the culmination of a loess rise. In 2011 the western and middle part of a large mound (about two thirds of its surface) was investigated, partially leveled as a result of farming practices. Under the mound two circular small ditches were discovered, the smaller located in the northern part, surrounding grave 9. The larger of the two surrounded feature 5 was most likely related to the second phase of mound construction and its significant enlargement. In the southern part of the barrow two human graves were subsequently excavated (features 4 and 8) and the inhumation of a horse (Feature 7).
In terms of funerary rites and their associated traits the finds from Hubinek differ in respect to discoveries from the south-east of Poland, which are clearly different from the numerous graves of the Corded Ware culture located in the Sokal Hills 3 . Their distinguishing characteristic is the non-ceramic nature of the inventory and the use of ochre to sprinkle the body of the deceased. Radiocarbon dating points to a relatively early age for grave 9 (3025 -2898 BC, Supplementary Table S1).

Grave 4 (Corded Ware culture) -poz235
The pit grave was sunk into the southern part of the mound and was in the shape of a rectangle, measuring 175 x 100 cm, and along a west-east axis. Within it a rectangular wooden construction was distinguished, measuring about 130 x 55 cm and traces of planks with a diameter of about 5cm. At its bottom there lay the remains of two individuals aged infans II (Supplementary Fig S16), placed in an antipodean position, most likely crouched (the anatomical arrangement was highly disturbed). Traces of ochre were identified on the bones and the grave had no furnishings.

Grave 8 (Corded Ware culture) -poz234
The pit grave was sunk into the southern part of the mound along a west-east axis and had a shape similar to a rectangle with rounded maculate corners, measuring 210 x 120 cm, with smudges of ochre with which the skeleton was also covered. The grave of the male (?), aged 25-30 rested at the bottom of the pit (Supplementary Fig S17), in a supine position with the head and lower limbs turned to the right. Both the bones and the bottom of the pit were profusely sprinkled with ochre, and the grave had no furnishings. Supplementary Fig S17. Grave 8, Hubinek site 2, barrow 1.
The pit grave in the northern part of the barrow, surrounded by a circular small ditch, ran along a west-east axis. It was in the shape of a rectangle with rounded corners, measuring 235 x 140 cm. At the bottom the skeleton of a male aged 20-25 was found, placed in a 'side 'position with the lower limbs contracted, towards the left side (Supplementary Fig S18).

Malżyce, Czarnocin commune, district of Kazimierza Wielka, site 30, barrow 1
The site is located in the Nida Trough, within the western Małopolska loess rise, which contains a cemetery complex that arose at barrows and long burial mounds of the megalithic Funnel Beaker culture. In the period 2004-2005 barrow 1 was investigatedentirely leveled as a result of intensive farming practices. The circular shape of the mound, measuring about 10-12 m in diameter, was marked only by a relevant darkening of the ground surface. In its centre a grave of the Final Beaker culture was discovered 4 . From the south-east, features of the Corded Ware culture were sunk into the mound: graves with a niche construction (nos. 2, 4 and 7), pit grave (no. 5) and a small pit with a vessel (no. 3). Moreover, at the edge of the mound, three inhumations of the Mierzanowice culture (nos. 8-10) were found. In respect to the construction traits of graves the placement of burials and characteristics of furnishings, the features from Malżyce represent a specific Małopolska variant of the Corded Ware culture known as the Kraków-Sandomierz group 5,6 .

Grave 2 (Corded Ware culture) -poz279
The niche grave was sunk into the central part of the barrow. The niche measured 280 x 220 cm. At its bottom (135 cm) a richly furnished inhumation of a male aged adultus/maturus was discovered (Supplementary Fig S19). The deceased was placed on his back with the lower limbs turned to the right. Only several teeth represented remains of the skull. The grave furnishing included a beaker, stone axe, flint artefacts (axes, 6 projectile points and two knife blade insets), stone polishing slab and a copper punch. The radiocarbon dating, taken from the bones in the grave reads: 3860±35 BP (Poz-59407), that is 2454-2236 BC.

Grave 4 (Corded Ware culture) -poz280
Feature with a niche construction sunk into the peripheral part of the barrow. The niche measured 190 x 175 cm and a depth of 145 cm. At its bottom there lay the skeleton of a female aged maturus/senilis, placed on the left side along a west-east axis (Supplementary Fig S20). By the skeleton a ceramic jug was discovered as well as two flint artefacts (axe and flake tools), whetstone and a bone awl.

Grave 5 (Corded Ware culture) -poz281
In a pit measuring 95 x 70 cm and a depth of 45 cm a poorly preserved skeleton of a child aged infans I/II was discovered (Supplementary Fig S21). The deceased was placed in a crouched position, most likely on the left side. A small beaker was found at its side. Supplementary Fig S21. Grave 5, Malżyce site 30, barrow 1.

Grave 7 (Corded Ware culture) -poz282
A niche grave discovered at the edge of the tumulus. The niche had an oval shape and measured 205 x 145 cm. At its bottom (105 cm), an adult deceased individual was found, sex unknown ( Supplementary Fig S22). The deceased was placed on their back, head and lower limbs turned to the left. Grave furnishings included a ceramic beaker, flint axe, a bone awl and five flint flakes. Supplementary Fig S22. Grave 7, Malżyce site 30, barrow 1.

Książnice, Pacanów commune, district of Busko Zdrój, site 2
The site in Książnice, located in the eastern end of the Pinczów Hummock, covers the peak of a small rise (200.15 m.a.s.l.), overlooking from the south and south-west over the valley of a nameless waterway.
During excavation research that had begun in 2001, conducted by Stanisław Wilk, a vast Neolithic and early Bronze Age sepulchral settlement complex was identified. The above complex contained a vast cemetery of the Lublin-Volhynia culture, Funnel Beaker Culture settlement, Złota culture cemetery complex and settlement, Corded Ware culture cemetery complex as well as a Mierzanowice culture cemetery complex and settlement.
The Kraków-Sandomierz group cemetery complex, Corded Ware culture contains nine graves, four of the first creating a concentration located in the southern part of the site and the other five were discovered in its eastern part. The graves subject to analysis, nos.1 and 2, were discovered in July and August respectively in 2003.

Grave 1 (feature 2/03) (Corded Ware culture) -poz287
A pit grave in the shape of an elongated oval along a north-south axis with a thickness of about 20 cm. At a depth of 60 cm a skeleton was found of a child (Infans I) placed in a crouched position on its back, the skull pointing north (Supplementary Fig S23). The grave furnishings included a flower pot beaker that was embellished in its upper section with horizontal cord imprints and placed in the southern part of the pit as well as five small flint products: a Volhynia flake, two chips (from Volhynia and Świeciechów flint), two fragments of flint splinters (from Volhynia and Jurassic flint) that were found in the fill of the pit grave. Supplementary Fig S23. Grave 1, Książnice site 2.

Grave 2 (feature 3 and 3a/03) (Corded Ware culture) -poz286
The grave was of a niche construction placed in a west-east axis with a rectangular entrance shaft (feature 3/03) and an oval niche grave along a north-south axis (feature 3a/03). In the western part of the shaft the entrance to the niche was obscured by a slab of loess. The niche was filled with a blackbrown humus and did not bear any signs of collapse.
At the bottom of the niche grave, at a depth of 125 cm, there lay a well preserved skeleton of an adult female, placed along a north-south axis in a crouched position on its left side with highly contracted lower limbs and the skull pointing north (Supplementary Fig S24). The grave furnishings contained three clay amphorae placed in the southern part of the grave, of which two (double-ear and quadruple-ear) are of middle size with necks embellished with horizontal cord imprints, while the third greater in size with a spherical belly, boasts six vertically attached ears and necks and an upper part of the belly covered with three rows of imprints bearing a vertical small stamp. Apart from the vessels, the grave revealed a semi-product of a bone awl and two entire awls, a small polishing stone made out of red sandstone and a four-sided axe made from Świeciechów flint. Moreover, the niche grave fill revealed nine flint products: seven flakes (from Volhynia, Jurassic and Chocolate flint), a Świeciechów flint knife blade insert with retouched convergent sides and a Volhynia flint unidirectional flake splinter.
On the basis of the recorded description of the funerary rite and typological analysis of artefacts found, the above described graves ought to be dated to the decline of the Corded Ware culture on the Małopolska Upland, c. 2400-2300 BC.

Graves 1 and 2 of the Moravian Corded Ware Culture, Držovice site, the Prostějov region, Czech Republic
During the rescue research (2014-2015) preliminary to the construction of 25 family houses, a part of the bi-ritual burial site of the Moravian CWC culture was found in the village Držovice (site called Pastviska) in the Prostějov region (middle Moravia), with 6 graves at considerable distances from each other (interval 17-48 m). The burials were located on a moderate SW slope above the Český potok brook terrace (100-200 m) at an altitude of 220-228 m 7 .
The two graves were found 17 m apart, composing part of a group burial ground. The burials can generally be dated to the period of local development of Moravian CWC, which in absolute terms represents the period between 2600/2500 -2300/2200 BC.

Preparation of biotinylated probes
Biotinylated probe DNA was generated by LR-PCR (PrimeSTAR GXL DNA Polymerase, Takara Bio) according to the manufacturer's protocol. The whole mitochondrial genome was amplified in multiple 25μl LR-PCR reactions using the two primer sets L06363/H14799 (8476 bp) and L14759/H06378 (8340 bp) following 1 . PCR reactions were pooled for each product and loaded onto 1% Agarose gels. Bands of the correct size were visualized with UV light and exercised with a clean scalpel blade. The gel slices were weighted and the DNA was extracted with the use of QIAquick Gel Extraction Kit (QIAGEN) following manufacturer's instructions. The concentration of the cleaned LR PCR products was than measured using NanoPhotometer™ Pearl (Implen). Acquired DNA fragments were pooled in equimolar amounts and adjusted to 20ng/μl. The DNA was then sheared on a Bioruptor Plus sonication device (Diagenode) for 120 cycles (30s on /30s off) with the output selector switched to (H)igh. The resulting fragmented DNA (∼100-200 bp average size, range 100-500) was subjected to end repair and dA-tailing by a NxSeq® AmpFREE Low DNA Library Kit (Lucigen) according to the manufacturer's protocol. Ligation was also performed with this kit, but with custom adapters prepared following 2 . Three microliters of 1:10 dilution of this T7 adaptor stock was used for the ligation reaction, according to the library preparation kit instructions. The libraries were then cleaned and size selected using AMPure® XP Reagents (Agencourt-Beckman Coulter) following the library preparation kit instructions, using 0.95x and 0.85x beads of original sample volume for size selection step, in order to select fragments ∼150-350 bp in length. The size selected libraries were PCR amplified in ten separate reactions with the following components: 5 μl 2× HiFi HotStart ReadyMix (KAPA), 20 μl H 2 O, 1 μl PCR, same as T7 oligo 1 2 , and 4 μl purified ligation mix. The cycling conditions were as follows: 98°C/3 min, 98°C/20 s; 18 cycles of 60°C/15 s, 72°C/30 s; 72°C/10 min. The reactions were pooled and purified with AMPure XP beads, eluting in 30 μl H 2 O. The concentrations and DNA fragment length distributions of the cleaned PCR products, were calculated using High Sensitivity D1000 Screen Tape assay on 2200 TapeStation system (AgilentTechnologies).
The bait libraries were transcribed into biotinylated RNA following 2 with minor modifications. Between ∼200 and ∼300 ng of amplified library was used for the in vitro transcription. The reaction product was then purified with an RNeasy Mini kit (QIAGEN) according to the manufacturer's instructions, using the same 30 μl of H 2 O twice for the final elution. The length distribution and concentration of the final RNA bait was than assessed using RNA Analysis ScreenTape on 2200 TapeStation system (Agilent Technologies).

Statistical analyses
PCA for mtDNA hgs frequencies was calculated using Python 3.5 and Scikit-learn v. 0.18.1 1 (Scikit-learn Machine Learning) package. We utilized Matplotlib 1.5.1 Python package 2 for plotting the PCA results and mtDNA hgs loadings.
We have used a centroid-based clustering approach to examine the PCA results and search for logical clusters within our data. We applied the k-means method as implemented in Scikit-learn v. 0.18.1 Python package 1 to the first 5 principal components coming from the PCA analysis. To access the quality of the clustering, we computed the silhouette parameter for k-means clustering with k = {2, 3, 4, …, 9}. For each k we ran the k-means algorithm 50 times and took the result with the best silhouette score. All k-means variants can be found in the Supplementary Material Text material (Supplementary Figure S27 and S28).
To further explore the relatedness of populations according to the mtDNA hgs frequencies, we have run the t-Distributed Stochastic Neighbor Embedding (t-SNE) analysis 3 as implemented in Scikit-learn (18.1) Python package. t-SNE is a modern dimension reduction method, which employs an iterative algorithm to visualize the high-dimensional data into 2D or 3D, retaining the local structure of the data while also revealing some important global structure (i.e. clusters). Following parameters were used in the t-SNE analysis: early_exaggeration: 4.0, learning_rate: 8, method: exact, metric: euclidean, n_iter: 500000, n_iter_without_progress: 500, perplexity: 5.0.
F ST values were computed in Arlequin 3.5 4 . The total of 21 populations were used in F ST and following AMOVA analyses, consisting only of ancient individuals with complete mt genomes (Table  S3), using Nei's average number of pairwise differences 5 and 10,000 permutations to estimate the pvalues. To visualize F ST values we employed multidimensional scaling (MDS) analysis with the use of Python Scikit-learn 0.18.1 package 1 .
We have used the traditional method of analysis of molecular variance (AMOVA) 6 to access the population division of the samples with complete mt genomes. We ran AMOVA as available in In the next step, we combined our populations of interest (CWW, CWPlM, CWBal, YAW, YAE) in turn with every group, and continued with combining of the two connected populations (CWW + CWPlM + CWBal, YAW + YAE) with all the groups. This approach allowed us to correctly assign the CWC and Yamnaya populations to appropriate groups.