Analysis of Ancient Human Mitochondrial DNA from Verteba Cave, Ukraine: Insights Into the Late Neolithic-Chalcolithic Cucuteni–Tripolye Culture Ryan W

Anthropological Science Vol. 128(1), 1–10, 2020

Analysis of ancient human mitochondrial DNA from Verteba Cave, Ukraine: insights into the Late Neolithic-Chalcolithic Cucuteni–Tripolye culture Ryan W. Schmidt1,2*, Ken Wakabayashi1, Daisuke Waku6, Takashi Gakuhari1,3, Kae Koganebuchi1, Motoyuki Ogawa 1, Jordan K. Karsten4, Mykhailo Sokhatsky5, Hiroki Oota1,6** 1Department of Anatomy, Kitasato University, 1-15-1 Kitasato, Sagamihara, Kanagawa 252-0374, Japan 2School of Archaeology, Earth Institute, University College Dublin, Belfield, Dublin 4, Ireland 3Kanazawa University, Center for Cultural Resource Studies, Kakuma, Kanazawa, Ishikawa 920-1164, Japan 4Department of Anthropology and Religious Studies, University of Wisconsin-Oshkosh, Oshkosh, WI 54901, USA 5Borschiv Regional Museum, Ministry of Culture and Arts, Shevchenka St. 9, Bilche Zolote, Ukraine 6Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Received 6 February 2019; accepted 5 February 2020

Abstract Verteba Cave (VC) in western Ukraine dates to the Eneolithic period (c. 5500 YBP), and contains the largest collection yet found of human skeletal remains associated with the Cucuteni–Tripolye culture. The subsistence economy of this people was based on agropastoralism, and included some of the largest and densest Middle Neolithic settlement sites in all of Europe. To understand further the evolutionary history of the Tripolye people, we examined population genetics patterns in mitochondrial DNA from ancient human remains excavated from VC chambers. From five commingled and secondary burial sites within the cave, we obtained 368 bp mtDNA HVR1 sequences from 22 individuals assignable to eight haplogroups: H (three haplotypes), HV (two haplotypes), W, K, and T. Overall nucleotide diversity is low (π = 0.00621). The two largest samples, from Chamber G3 and Site 7, were significantly differentiated with respect to haplotype composition: G3 (n = 8) is dominated by haplotype W (π = 0), whereas Site 7 (n = 15) is dominated by H haplotypes (π = 0.00439). Tajima’s D as an indication of population expansion was not significantly negative for the complete sample (D = –1.37) or for sites G3 (D = –0.973) and 7 (D = –1.35), which were analyzed separately. Individuals from the Tripolye culture buried at VC c. 5500 YBP had predominantly haplogroup H and related haplotypes. This contrasts with predominantly haplogroup U individuals in preEneolithic peoples from the same area, which suggests lack of genetic continuity in a site that has been dated to the Mesolithic. Peoples of the Tripolye culture are more closely related to other early European farmers than to Mesolithic hunter-gatherers and/or pre Eneolithic cultures.

Key words: mtDNA, ancient DNA, Tripolye, Eneolithic, Ukraine

2015; Mathieson et al., 2015). A key question in this debate Introduction posits whether these cultural changes were the results of Profound cultural transitions accompanied the Neolithic movements of people (demic diffusion model), or the move- and Bronze Age in Europe. A continuing question of debate ment of ideas and artifacts (cultural diffusion model) among researchers is how migrations from West Asia or the (Ammerman and Cavalli-Sforza, 1984). That is, were these Pontic-Caspian steppe affected the genetic composition of large-scale migrations a process of cultural diffusion, with modern-day Europe (Haak et al., 2010; Bollongino et al., little or no genetic admixture among early Neolithic farmers 2013; Allentoft et al., 2015; Brandt et al., 2015; Jones et al., and Mesolithic hunter-gatherers? Or, is a model of demic diffusion (Ammerman and Cavalli-Sforza, 1984; Cavalli- * Correspondence to: Ryan W. Schmidt, CIBIO – InBIO, University Sforza, 2000; Pinhasi and von Cramon-Taubadel, 2009; of Porto, Campus de Vairão, Rua Padre Armando Quintas, nº 7, Haber et al., 2016) more appropriate, whereby different re- 4485-661 Vairão, Portugal. gions of Europe were more or less affected by admixture E-mail: [email protected] with early farmers, and later steppe herders during the ** Correspondence to: Hiroki Oota, Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1 Bronze Age? Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. The transition to farming from a foraging lifestyle first E-mail: [email protected] appeared in the Near East c. 10500 years before present Published online 20 March 2020 (YBP) in modern-day southeastern Anatolia and Syria. Ar- in J-STAGE (www.jstage.jst.go.jp) DOI: 10.1537/ase.200205 chaeologists have described two major and contemporane-

© 2020 The Anthropological Society of Nippon 1 2 R.W. SCHMIDT ET AL. Anthropological Science ous routes of expansion, namely the Continental (Danubian) of this region differently than in other parts of Europe. and Mediterranean routes. By 9500 YBP, farming spread Following the establishment of farming communities in into parts of Central Europe through the migration of peo- the Balkan Peninsula, a series of complex societies formed, ples associated with the Linear Pottery culture (or Linear- culminating in large settlements. By 6500 YBP, agriculture bandkeramik, LBK). These LBK cultures originated in had reached Eastern Europe, in the form of the Cucuteni– Hungary and Slovakia (the Carpathian Basin) and then Trypillian (C-T) complex in the area of present day Moldo- spread rapidly as far as the Paris Basin and Ukraine. A lin- va, Romania, and Ukraine. This culture spanned close to gering question among archaeologists has always been 2000 years and influenced much of SE Europe and the Baltic whether these first farmers were descendants of local hunt- regions. It is known for elaborate anthropomorphic and ani- er-gatherers or whether they migrated from the Near East. mal figurines, as well as distinct, elegantly painted pottery. Paleogenetic studies have generally suggested these early Around 5000 YBP, these societies began to change, with the farmers were migrants, though in some places peoples con- large settlements being abandoned, and archaeological evi- tinued to admix after the adoption of agriculture (Sampietro dence suggesting contact with nomadic steppe populations et al., 2007; Lacan et al., 2011a, b; Hervella et al., 2012; from the East. Brandt et al., 2015). We address the complex process of Neolithisation Southeastern Europe (SE Europe) has not been as exten- (Ammerman and Cavalli-Sforza, 1984) in SE Europe by sively investigated as southwestern Europe, Central Europe, examining an Eneotlithic (Chalcolithic) Cucuteni–Tripolye or southern Scandinavia, in terms of their ancient DNA var- site from Ukraine. For brevity, we will use the term Tripolye, iation. In contrast to Central Europe, the area of what is as it is known in Ukraine. Tripolye culture (7100–5000 cal- modern Ukraine saw the adoption of agriculture late. Al- BP) is defined as Eneolithic based on the presence of copper though features of the Neolithic package are visible in artifacts and the onset of metallurgy, and ends at the begin- Ukraine as early as 8500–7500 YBP [we use calBP (calibrat- ning of the Bronze Age (Ledogar et al., 2019). The Tripolye ed before present) to indicate radiocarbon dating], agricul- culture occupied a large area from the Carpathian Mountains ture was not adopted as a primary subsistence economy until in the west to the Dnieper River in the east, and extended as the Eneolithic or Chalcolithic period (c. 6500 YBP) far south as the Black Sea and north to Kiev (Figure 1). Rel- (Zvelebil and Dolukhanov, 1991). Whereas Central Europe ative and absolute chronologies divide the Tripolye culture saw mostly demic diffusion (Pinhasi and von Cramon- into several phases (Table 1), which normally accompany Taubadel, 2009), SE Europe seems to have adopted agricul- changes in pottery manufacture and decoration (Ryzhov, ture through innovative subsistence strategies as a result of 2012). The people associated with this culture are known as transfer of ideas, with little genetic influence and genetic Trypillians. continuity from the Mesolithic to the Neolithic (Richards et In the present study, we investigate human remains found al., 2002). Therefore, the Neolithic transition occurred at a at a single Tripolye site known as Verteba Cave (VC), with slower pace, thus perhaps shaping the genetic composition human and faunal remains dating to the Eneolithic (Ryzhov,

Figure 1. Map showing location of Verteba Cave in western Ukraine and the locations of the sites where samples were collected. Note that Sites G1 and G2 have not been mapped, but the sites from which data were analyzed (г3 (G3), 7, and 20) are highlighted. Vol. 128, 2020 ANCIENT DNA ANALYSIS ON HUMAN REMAINS FROM VERTEBA CAVE 3

Table 1. Chronology for Cucuteni-Tripoloye period Oblast, Ukraine (Figure 1). Most samples date to the Tripol- Phase Date ye CII period (c. 5500 YBP) based exclusively on associated A 5100/5000–4700/4600 calBP pottery found in the same cultural layer as the human re- BI 4700/4600–4400/4300 calBP mains. Nikitin et al. (2010, 2017) and Ledogar et al. (2019) BI/II 4400/4300–4200/4100 calBP radiocarbon dated human and animal remains, as well as BII 4200/4100–3900 calBP pottery sherds from Verteba, and found that the dates corre- CI 3900–3450/3350 calBP spond to transitional phases in pottery decoration, with peak CII 3450/3300–3000/2900 calBP activity placed c. 5500 calBP, although some remains date to before or after this date. All skeletal samples excavated within VC are commingled and individual burials are difficult to identify. Therefore, in order to avoid sequencing the same 2012; Kadrow and Pokutta, 2016). VC has been excavated individual twice, one of us (J.K.) collected second right met- as an archaeological site since the 1820s, though more inten- acarpal bones for analysis from a single chamber (Site 7). sive excavation has been ongoing since 1996 under the di- These samples were collected over several excavation field rection of coauthor M. Sokhatsky (Borschiv Regional Muse- seasons (2008–2014) by J.K. The remains were initially kept um of the Ukrainian Ministry of Culture and Arts). Verteba at the University of Wisconsin-Oshkosh, and later trans- is a gypsum cave located in western Ukraine, in the boreal ferred to Kitasato University for processing. R.W.S. then forest-steppe zone of the East European Plain. It is one of collected bone and teeth samples on site and in situ using many vast underground cave systems in the region. sterile sampling methods (wearing coveralls, gloves, and Interpretations surrounding the use of the cave during facemask) from four additional chambers (20, G1, G2, G3) these periods vary. Some believe the cave was used as a during field seasons 2015–2016. We attempted to collect temporary shelter, while increasing archaeological evidence samples from these other chambers that did not overlap ele- suggests use as a ritual site or a mortuary function (Kadrow ments from a single individual and we are confident this is and Pokutta, 2016; Ledogar, 2017). There is also evidence to the case for sites 20, G1, and G2 as samples were collected support the idea that individuals buried in the cave, which from different levels and from different parts of the site; are largely secondary in nature, are victims of warfare or however, site G3 had several skeletal elements within a sacrifice, due to the high frequency of blunt force trauma small chamber and thus these samples could derive from a (Anthony, 2007; Madden et al., 2018). The cave contains the single individual or only a few individuals—though they largest accumulation of human remains associated with the may likely be members of the same family. Samples from Tripolye culture found to date. Very few Tripolye culture sites 20, G1, G2, and G3 were deposited into sterile bags on human remains exist, making the cave one of the most im- site and are now stored at the University of Vienna, Vienna, portant sites for the investigation of the diet, health, patholo- Austria under the curation of R. Pinhasi. A total of 63 skele- gy, and population history of Trypillian peoples (Karsten et tal elements were analyzed for this study. al., 2014, 2015). Using data obtained from the mitochondrial HVR-I re- Ancient DNA extraction gion, we ask several interrelated questions about individuals Prior to extraction, samples were wiped with a 10% buried at VC. First, is there evidence for a maternal genetic bleach solution, rinsed in DNA-free water, and UV irradiat- continuity, and thus a degree of cultural diffusion, with local ed in a cross-linker for 30 minutes on each side before dry- Mesolithic hunter-gatherers, as suggested in several recent ing. DNA extraction was carried out with ~100–300 mg of studies? If there is no evidence for this, how are these indi- bone powder using a modified silica-column based protocol viduals related to earlier farmer groups from SE or Central (Yang et al., 1998). First, the surface of the bone was cleaned Europe? Is there any indication of a steppe influence for the using a diamond drill bit at low speed. Then, we either used Trypillian peoples? And lastly, can we infer something about the drill bit to obtain powder, or powdered the bone in a the collapse of the Trypillian people from maternal lineages? mixer mill (ShakeMaster Auto v. 2.0; BioMedical Science Several studies (Nikitin, 2011; Nikitin et al., 2010, 2017) Inc.). For samples VC001–VC035 and VC046–VC063, we have briefly addressed two of these three questions, indicat- used the following protocol: bone powder was incubated for ing a link to early Neolithic farmers with some possibility of 24 h at 55°C followed by 24 h at 37°C in 2 ml tubes with a link with Mesolithic hunter-gatherers. However, in those 1 ml of lysis buffer in final concentrations of Tris–HCl studies, the sample sizes tended to be smaller and the human (pH 7.4), 20 mM; 0.7% Sarkosyl NL; 0.5 M EDTA (pH 8.0), remains used came from only a single chamber. Here, we 47.5 mM; 0.65 U/ml Proteinase K; with shaking at 300 rpm analyze mitochondrial (mt) DNA data from several cham- in a Thermomixer (Thermomixer Comfort Eppendorf). bers in different locations found throughout the cave in an Samples were then centrifuged at 13000 rpm for 10 m and attempt to answer these questions. the supernatant was removed. Fresh lysis buffer (1 ml) was then added to the pellet, vortexed, and the incubation and Materials and Methods centrifugation steps were repeated. The second supernatant was then transferred to an Ami- Samples con Ultra-4 Centrifugal Filter Unit 30K (Merck), diluted Human remains for DNA analyses come from several ex- with 3 ml of TE, and centrifuged at ~2500 rpm until a final cavation sites located within VC. VC is a mortuary site lo- concentration of ~100 μl was obtained. This volume was cated outside the modern village of Bilche Zolote, Ternopil then transferred to a silica column (MinElute PCR Purifica- 4 R.W. SCHMIDT ET AL. Anthropological Science tion Kit, QIAGEN) and purified according to manufacturer’s amplified with three sets of overlapping primers (Adachi et instructions, except at the final step adding TWEEN 20 (at al., 2004, 2013, 2014) (Table 1). This region also includes 0.05% final concentration) to 60 μl EB buffer preheated to part of the tRNAPro gene, although none of our samples ex- 60°C. hibited mutations in this region, and thus this did not affect For samples VC036–VC044, our second protocol fol- our results. PCR amplification was carried out using 2 μl lowed the first with the following modifications based on a extract in a 50 μl reaction mixture containing Ex Taq Hot ‘predigestion’ step recommended in Gamba et al. (2016). Start (TaKaRa, Japan), 10× Ex Taq buffer (containing This protocol was used in an effort to increase DNA yield MgCl2), 2.5 mM dNTPs, and 10 μM of each primer. PCR from degraded samples. It has been shown that a prediges- conditions were 94°C for 5 min followed by 40 cycles of tion step generally improves endogenous DNA from degrad- 94°C for 30 sec, 55°C and 61.5°C for Primer Set 1 and Prim- ed archaeological bone (Damgaard et al., 2015). Fresh lysis er Sets 2/3, respectively, annealing and extension tempera- buffer was added to the powder and incubated in a Thermo- ture for 5 min 30 sec, and a hold at 4°C. PCR products were mixer for 1 h at 56°C, shaking at 1200 rpm. After 1 h, the visualized on 2% agarose gel containing ethidium bromide, sample was centrifuged for 2 min at 13000 rpm and the su- and purified using the MinElute PCR Purification Kit. Am- pernatant was discarded. Fresh lysis buffer was then added plification products were sequenced using Applied Biosys- to the pellet and incubated at 56°C for 1 h followed by 37°C tems Big Dye protocols and analysed on an Applied Biosys- overnight, shaking at 1200 rpm. After ultrafiltration in an tems 3130 Genetic Analyzer at Kitasato University. Amicon Ultra-4 Centrifugal Filter Unit 30K, the sample was transferred to a silica column (MinElute PCR Purification Data analysis Kit) with the following modifications: after adding the PB Sequences were visually inspected, corrected, and com- buffer, the centrifugation speed was reduced to 8000 rpm pared against the revised Cambridge Reference Sequence and the elution step included a 5 min incubation at room (rCRS; Andrews et al., 1999) using DNASTAR Lasergene temperature. software (SeqMan Pro). A maximum-parsimonious phy- logenetic network was constructed manually. HVR-I haplo- Contamination controls types were classified into possible haplogroups and sub- To control for contamination, all pre-PCR procedures haplogroups using MITOMAP (Lott et al., 2013). Although were conducted in a controlled-access, positive-pressure we understand that the resolution of haplotype motifs could laboratory with HEPA-filtered air that is used exclusively for be resolved using HVR-II, most of the haplotypes defined in ancient DNA analysis at Kitasato University. Disposable this study would not be improved with the analysis of HVR- protective clothing was worn during all sampling and extrac- II as our intention is to define broader haplogroups to com- tion procedures. Pipettes with aerosol-resistant tips were pare with Mesolithic and Neolithic peoples, and therefore used. The lab is cleaned prior to all procedures with DNA- our results are generally unaffected by not including data OFF (Takara, Japan), and exposed to UV irradiation for at from HVR-II. We generated population genetic statistics, least 2 hours after each procedure. All PCR reactions and number of sequence types, number of polymorphic sites, post-PCR procedures were performed in a separate laborato- nucleotide diversity (π), and Tajima’s D using the software ry. The movement of laboratory materials and personnel was DnaSP 5 (Librado and Rozas, 2009). always unidirectional, from the ancient to modern facilities. The mtDNA of all researchers with access to the clean lab Results were typed and compared with the results. At least two extractions and two amplifications were performed at separate We began with 63 specimens (53 bones and 10 teeth) from times to assess the authenticity of our results. All batches of VC (Figure 1), from which we made 156 DNA extractions. extractions included a negative control blank (no template Of these, we were successful in PCR amplification of 38 DNA), and PCR runs included a water blank to monitor rea- specimens with three overlapping primer sets each (Table 2). gent contamination. All negative controls were clean with We sequenced all 114 amplicons using the Sanger method lack of observable amplifiable bands in the gels. on an ABI 3130 Genetic Analyzer, and obtained contigs based on all three amplicon sequences from 34 of the 38 PCR amplification and direct sequencing specimens. We removed 6 specimens where the amplicon The hypervariable region I (HVR-I) of the mitochondrial sequences were mutually incompatible, so as to produce a D-loop (368 bp, nucleotide positions 15999–16366) was final set of 28 specimens with consensus sequences over a

Table 2. Primers for PCR amplification and Direst sequencing Primer set Primer Primer sequence Nucleotide position Product size (bp) 1 L15998 5'-CCATTAGCACCCAAAGCTA-3' 15980–16161 182 H16142 5'-ATGTACTACAGGTGGTCAAG-3' 2 L16120 5'-TTACTGCCAGCCACCATGAA-3' 16101–16258 158 H16239 5'-TGGCTTTGGAGTTGCAGTTG-3' 3 L16208 5'-CCCCATGCTTACAAGCAAG-3' 16190–16384 195 H16367 5'-CTGAGGGGGGTCATCCAT-3' Vol. 128, 2020 ANCIENT DNA ANALYSIS ON HUMAN REMAINS FROM VERTEBA CAVE 5

Table 3. mtDNA nucleotide sequences of the Verteba Cave specimens nucleotide position (+16000 bp) Sequence Bone or Haplogroup

Chamber Sample 70 93 311 type tooth 126 192 209 223 224 256 292 294 296 304 324 356 362 assignment rCRS A T T C T C T C C C C T T T T T K.W. ・・・・ C T ・・・・・・C ・・ M7a R.S. ・・・・・・C ・ HV12b VC002 B ・・・・・・VC003 B ・・・・・・VC016 B ・・・・・・Site 7 I VC019 B ・・・・・・H(rCRS) VC050 B ・・・・・・VC051 B ・・・・・・G3 VC040 T ・・・・・・II Site 7 VC001 B ・・・・・・C ・ HV12b VC008 B G ・・・・・・VC013 B G ・・・・・・Site 7 VC035 B G ・・・・・・III VC052 B G ・・・・・・H36 VC054 B G ・・・・・・VC056 B G ・・・・・・Undefined VC055 B G ・・・・・・VC015 B ・・・・・・C ・・・ IV Site 7 H2a VC021 T ・・・・・・C ・・・ VC028 T ・・・・・ T ・・ T ・・・・・・VC034 B ・・・・・ T ・・ T ・・・・・・VC041 T ・・・・・ T ・・ T ・・・・・・V G3 VC046 B ・・・・・ T ・・ T ・・・・・・W VC047 B ・・・・・ T ・・ T ・・・・・・VC048 T ・・・・・ T ・・ T ・・・・・・VC049 T ・・・・・ T ・・ T ・・・・・・VI G2 VC038 B ・・・ T ・・・・・・C ・ HV12b G2 VC025 B ・ C ・・・・ C ・・・・・ C ・・・ VII K1a 20 VC032 B ・ C ・・・・ C ・・・・・ C ・・・ VIII Site 7 VC004 B ・・ C ・・・・ T ・ T T C ・・・ C T2b

368 bp region of HVR-1. VIII] among 15 individuals, whereas Chamber G3 was al- Eight distinct HVR-1 sequences were found in the 28 most exclusively type V (7 out of 8). The other two sequenc- specimens. We also include the sequence types for two of us es were confined to sites G2 and Site 20 (VI and VII). We (K.W. and R.S.) who worked on DNA extraction. K.W. and were unable to produce any sequence data from site G1. Al- R.S. sequence types were not identical to the eight sequence though we have a total of 28 samples represented in our se- types, except for Seq type II from one bone specimen that quence assemblage, the possibility exists that only 22 indi- was identical to the sequence type of R.S. This sequence viduals are present in our dataset. This reasoning is based on type corresponds to mitochondrial haplogroup HV12b, the following: we know that all of the samples from Site 7 which is common in modern European populations. We are different individuals (each is represented by a single could not eliminate the possibility that it is contamination right second metacarpal bone, n = 15); the samples from the and so did not include it in subsequent population genetic ‘undefined chamber’ were also two right metacarpal bones analyses. In addition to Seq type II, sequence type VI was (n = 2); samples from sites G2 and 20 derive from different assignable to be haplogroup HV. Sequence types I, III, and layers and are not in close approximation to each other, i.e. IV were assignable to be haplogroup H, while sequence sites are spatially separated (n = 3); and site G3 contains types V, VII, and VIII were assignable to be haplogroups W, different skeletal elements that may come from a single indi- K, and T, respectively (Table 3). Thus, all the haplotypes vidual, though we observe two different haplotypes, so it is found in the VC specimens are also found in modern Euro- possible that n = 2 rather than n = 8. This gives us a mini- pean populations, as expected (Richards et al., 2002). mum of 22 individuals in our dataset for population genetic The distribution of haplotypes varies among spatially dis- analyses. tinct areas of the cave (Table 3). Most notably, Site 7 had We tested for nucleotide diversity (π) and Tajima’s D five different haplotypes [I, II (similar to R.S. and thus re- (Table 4). A significantly negative Tajima’sD would suggest moved from further population genetic analyses), III, IV, and that a population has experienced a demographic expansion 6 R.W. SCHMIDT ET AL. Anthropological Science

Table 4. Genetic diversity statistics for Verteba Cave Number of Number of Nucleotide Name of population Size Tajima’s D sequence types segregating sites (S) diversity (π) Verteba Cave 22 7 14 0.00621 –1.36690 P = 0.078 Site 7 14 4 8 0.00439 –1.34944 P = 0.085 Chamber G3 5 2 2 0.00217 –0.97256 P = 0.097 Haplogroup W of Site G3 4 1 0 0 0

(Tajima, 1989); otherwise, there is no evidence of departure haplotypes occur in haplogroup H, which is the most com- from constant population size. Tajima’s D values of VC mon haplogroup among modern-day Europeans and peoples specimens were negative (–1.36690) but not significantly of the West Asia, accounting for around 40% in Europeans different from zero (P = 0.078). Several values for nucleo- (Van Oven and Kayser, 2009), including approximately 44% tide diversity were inferred for the different Seq types (apart of modern Ukrainians (Malyarchuk and Derenko, 2001). from Seq Type II) (Table 4) and show relatively low diversi- This suggests a possible continuity between the Tripolye and ty, although the sample sizes for each are small. modern Ukrainians. The VC haplotypes included one T2b individual (Table 3), which is a possible marker of Anatolian Discussion expansion (Brandt et al., 2015) that has also been found at high frequency in the Carpathian Mountains (Nikitin et al., In this study we have attempted to answer a number of 2009). In Nikitin et al. (2009), an individual from Bilche related questions surrounding Trypillian population history Zolote was found to have haplogroup T2b. Bilche Zolote is using data gleaned from maternal ancestry. We include a only 3 km from the VC site, indicating some degree of local minimum of 22 individuals buried at VC, an Eneolithic site continuity with the Trypillian people. associated with the Tripolye culture. Although the data are Previous ancient DNA studies showed that hunter-gatherers limited, our findings may offer some insight into aspects of before 6500 YBP in Europe commonly had haplogroups U, Trypillian people’s genetic affiliation with other Neolithic U4, U5, and H, whereas hunter-gatherers after 6500 YBP groups. in Europe had a lower frequency of haplogroup H than be- To address the complexities associated with the transition fore (Brandt et al., 2015). Haplogroups T and K appeared in to farming (or the process of Neolithisation), a wealth of hunter-gatherers only after 6500 YBP, indicating a degree ancient mtDNA data has been amassed from the Late Meso- of admixture in some places between farmers and hunter- lithic to the Late Bronze Age (Brandt et al., 2015; Haak et gatherers. Farmers before and after 6500 YBP in Europe had al., 2015). To explain these genetic changes in various re- haplogroups W, HV*, H, T, K, and these are also found in gions of Europe, it is important to fully understand the ge- individuals buried at VC (Deguilloux and Mendisco, 2013). netic substratum spanning the Mesolithic–Neolithic transi- Therefore, our data point to a common ancestry with early tion. Studies have suggested that the maternal signature of European farmers. local hunter-gatherer groups in many parts of Europe is ho- Our data may suggest a degree of population replacement mogeneous, with a relatively small population size and hap- from the Mesolithic to the Neolithic. Mathieson et al. (2018) logroups dominated by lineage haplogroup U, such as U2, analyzed a number of Neolithic Ukrainian samples (petrous U4, U5a, U5b, and U8 (Haak et al., 2015; Mathieson et al., bone) from several sites in southern, northern, and western 2015, 2018). In contrast, Neolithic mtDNA arrived in Cen- Ukraine, dating to c. 8500–6000 YBP, and found exclusively tral Europe c. 8000 years BP along with cultures associated U (U4 and U5) mtDNA lineages. It should be noted that with LBK farmers, comprising largely haplogroups N1a, T2, ‘Neolithic’ in this context does not mean the adoption of K, J, HV, V, W, and H. These lineages replaced local signa- agriculture, but is simply coincident with a change in mate- tures of haplogroup U from peoples associated with Meso- rial culture. They also analyzed several Trypillian individu- lithic cultures, over large areas of Central Europe. als from VC (different samples than those included in this We first explored whether individuals buried at VC are study), among whom they found a wider diversity of mtDNA more closely related to earlier Neolithic farmers from Cen- lineages, including H5a, HV, and T2b. One individual tral Europe, or perhaps have some connection with local (I3151) had haplotype U8b1b. This finding is similar to an hunter-gatherers, thus emphasizing the role of cultural diffu- earlier study by Nikitin et al. (2017), who analyzed the same sion in the adoption of agriculture. mtDNA haplogroup data samples for only mtDNA variation. Although found as early for modern and ancient populations in Europe and West Asia as the Paleolithic, haplotype U8b1 has also been discovered have shown there was a discontinuity between late hunter- in Neolithic Anatolian farmers (Mathieson et al., 2015). In gatherers and early farmers, and later extant European the analysis by Mathieson et al. (2018), the individual with populations, in most locations throughout Europe. An ex- U8b1b showed little evidence that their genome-wide varia- ception has been in SE Europe and the Baltic where cultural tion was more similar to earlier Neolithic or Mesolithic diffusion may have played a larger role (Jones et al., 2017). groups, displaying a mixture of mostly Balkans Neolithic The VC haplotype distribution indicates common haplo- with some contribution from western hunter-gatherers, types among Eurasian populations (Richards et al., 2002). Ukraine Neolithic, and steppe Yamnaya. In fact, the Trypil- These include haplogroups H, T, K, and W. The majority of lians in Mathieson et al. (2018) had up to 80% Neolithic Vol. 128, 2020 ANCIENT DNA ANALYSIS ON HUMAN REMAINS FROM VERTEBA CAVE 7

Anatolian ancestry. Based on these data, combined with our As the population increases and resources reach an upper preliminary results, it appears the Trypillians were very limit, groups would begin to split and settle into new loca- much a distinct people with ancestral roots tied to early Ne- tions in close geographic proximity. Sampling individuals olithic groups from Anatolia. from each of these new sites, we would expect to see the Haplogroup W was also observed in several specimens maintenance of population stability since the groups, though deriving from Site G3 (Table 3). Although we are unsure if genetically related, are spread out and thus would maintain all of these haplogroups come from a single or multiple indi- population equilibrium with bidirectional migration. If, at viduals, this observation is interesting in that it is relatively some point in time, these sites again become aggregated be- rare and isolated among Neolithic samples. It has, however, cause of increased population size, and we were to sample been found in samples dating to the Bronze Age (Wilde et from this new, larger archaeological site, we would again al., 2014). Wilde et al. (2014) found haplogroup W present witness demographic expansion simply because the overall in two samples from the Early Bronze Age associated with population size has increased. Therefore, farmer populations the Yamnaya and Usatovo cultures. The Usatovo culture (c. as a whole (over the course of the Neolithic) generally see a 3500–2500 BC) was found in Romania, Moldova, and trend for increased population expansion as local villages southern Ukraine. It was a conglomeration of Tripolye and turn into larger settlements (as could be the case for peak North Pontic steppe cultures. Therefore, this individual occupation at VC, where nearby settlements tended to be could link the Trypillian peoples to the Usatovo peoples and large). However, if we sample from each of those localities perhaps to the greater Yamnaya steppe migrations during the over time, as perhaps we are seeing with our results when we Bronze Age that lead to the Corded Ware culture (Kristiansen include samples from all sites (chambers), then we do not et al., 2017). see demographic expansion, but rather maintenance of pop- VC contains archaeological evidence for the Tripolye ulation size over time. cultural complex, including implements for agrarian cultiva- Archaeologically, it has been documented that Tripolye tion, including grain processing. Based on the material cul- settlements began to disappear at the beginning of the ture, the immensity of certain settlements (some housing up Bronze Age. The reasons for this vary, but could be influ- to 10000 people; Müller et al., 2016), as well as the deterio- enced by their interaction with steppe groups from the east. ration in biological health resulting from grain consumption, One of the possibilities for settlement abandonment is war- it is clear the subsistence economy of the Trypillians was fare. It has been well documented at VC that interpersonal based on agriculture. Previous studies report that modern violence was a common phenomena (Anthony, 2007; hunter-gatherers do not indicate a signal of demographic Madden et al., 2018). Madden et al. (2018) found a high expansion in mismatch distribution and/or Tajima’s D test, degree of trauma-related cranial injuries among Trypillian but farmers tend to show expansion based on increasing burials. It is believed these individuals were killed by an numbers of individuals living in sedentary conditions outside raiding group and were later buried by members of (Watson et al., 1996; Oota et al., 2002). To investigate the Tripolye culture. overall demography in VC, we examined population genetic The Trypillians were one of the last of the ‘Old Europe’ statistics. There was no evidence that the Trypillian people cultures that lived along the shores of the Danube River. By (at least their maternal lineages) experienced any demo- 5800 calBP, many of these Neolithic Danubian cultures were graphic expansion, at least during the Late Neolithic wiped out after the arrival of pastoralists from the steppe (Table 4); although we find a negative value for Tajima’s D (Anthony, 2007). It is believed that by 5300 calBP, the Tryp- when analyzing all of the sampled individuals (n = 22), it is illians were in conflict with members of the Usatovo culture not significant. to the south, no longer benefiting from trade relationships However, we are unable to treat our sample population as across the forest–steppe boundaries. being deposited at or around the same time period. Although Another explanation for the sudden collapse of Tripolye most individuals included in this study come from Site 7, culture may be an early form of plague, recently document- which has been firmly established to date to c. 5500 calBP ed, that was widespread from Siberia to the Baltic at c. 5000 based on ceramics and radiocarbon dating (Nikitin et al., YBP (Rasmussen et al., 2015; Andrades Valtueña et al., 2010; Ledogar et al., 2019), other chambers in the cave are 2017; Rascovan et al., 2019). Neolithic communities con- not as confidently dated. For example, Chamber G3 had very tracting this early form of the plague would have devastated few artifacts from the Tripolye culture. Given this caveat, we Tripolye mega-sites, thus creating a demographic collapse analyzed separately individuals buried in Site 7 (n = 15) and that we are only glimpsing in our population genetic analy- found a negative Tajima’s D that was not significant ses. To get a better understanding of the demographic col- (–1.34944; P = 0.085). lapse in Tripolye society, we will need to obtain genome- An explanation for our observed population size stability wide data to further explore how these early agropastoralists as seen in Tajima’s D might be due to sampling strategy and eventually declined or were replaced by steppe nomads from the temporal component of ancient DNA sites. If a popula- the east. tion migrates in low numbers into a new environment, such as the case with early migrating farmers from Anatolia, we Conclusions would slowly see an increase in the population as they become increasingly sedentary over time. If we were to sample In this study, we have shown that mtDNA diversity during from this site and test for demographic expansion, we would the Eneolithic at the VC site is closely related to early Euro- most likely see that reflected in a statistic such as Tajima’sD . pean farmers and that the represented haplogroups are qual- 8 R.W. SCHMIDT ET AL. Anthropological Science itatively different from the mtDNA haplotypes found during ensure the quality of sequencing data of mitochondrial DNA the Mesolithic and Early Neolithic at other Ukrainian sites. in highly degraded samples. Legal Medicine, 16: 52–55. 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