DNA for Romania and adjacent areas
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About us

The Romania Dual Y-DNA & mtDNA Project welcomes all who have a direct paternal line and/or direct maternal line with Romanian ancestry. This includes the National Genographic participants, or those who tested with other companies and wish to transfer their results to FTDNA. Note: In addition to the confines of Romania, all surnames with Y-DNA/mtDNA Romanian heritage are welcome, irrespective of the location for the most distant known ancestor. We are also accepting those who have an ancestral connection with Romanian lands, in spite of border changes during the years. As such, we welcome not only those from Romania, but also from Moldova, Ukraine, Hungary, Serbia (Serbian Banat and Timok), Bulgaria, Israel and diaspora in general, who have Romanian ancestry (via name, family history or spoken language). Vlachs/Aromanians/Macedo-Romanians/Istro-Romanians are also welcome to join our group. The same applies for those with known Hungarian, German, Ashkenazim Jewish (, Ashkenazim Levites, Serbian etc. roots. Please note that subgroups are now created based on haplogroups for both Y-DNA and mtDNA. Initially I grouped members based on "perceived ethnicity" but the DNA results prompted me to switch to haplogroups instead.

PUT YOURSELF ON THE MAP! We would like to encourage everyone to put yourself on the map! More about DNA testing at If you have any questions, please e-mail the administrator and co-administrators. For those of you who do not speak English well enough, our administrator will be more than happy to assist you and communicate with you directly in Romanian.

Family Search has made available the Moldova Church Books, 1811-1936 at 

Published papers:

Ancient DNA from South-East Europe Reveals Different Events during Early and Middle Neolithic Influencing the European Genetic Heritage - June 8, 2015

Montserrat Hervella, Mihai Rotea, Neskuts Izagirre, Mihai Constantinescu, Santos Alonso, Mihai Ioana, Cătălin Lazăr, Florin Ridiche, Andrei Dorian Soficaru, Mihai G. Netea, Concepcion de-la-Rua

The importance of the process of Neolithization for the genetic make-up of European populations has been hotly debated, with shifting hypotheses from a demic diffusion (DD) to a cultural diffusion (CD) model. In this regard, ancient DNA data from the Balkan Peninsula, which is an important source of information to assess the process of Neolithization in Europe, is however missing. In the present study we show genetic information on ancient populations of the South-East of Europe. We assessed mtDNA from ten sites from the current territory of Romania, spanning a time-period from the Early Neolithic to the Late Bronze Age. mtDNA data from Early Neolithic farmers of the Starčevo Criş culture in Romania (Cârcea, Gura Baciului and Negrileşti sites), confirm their genetic relationship with those of the LBK culture (Linienbandkeramik Kultur) in Central Europe, and they show little genetic continuity with modern European populations. On the other hand, populations of the MiddleLate Neolithic (Boian, Zau and Gumelniţa cultures), supposedly a second wave of Neolithic migration from Anatolia, had a much stronger effect on the genetic heritage of the European populations. In contrast, we find a smaller contribution of Late Bronze Age migrations to the genetic composition of Europeans. Based on these findings, we propose that permeation of mtDNA lineages from a second wave of Middle-Late Neolithic migration from North-West Anatolia into the Balkan Peninsula and Central Europe represent an important contribution to the genetic shift between Early and Late Neolithic populations in Europe, and consequently to the genetic make-up of modern European populations.

Y-Chromosome Analysis in Individuals Bearing the Basarab Name of the First Dynasty of Wallachian Kings
Begoña Martinez-Cruz et al., 2012

Vlad III The Impaler, also known as Dracula, descended from the dynasty of Basarab, the first rulers of independent Wallachia, in present Romania. Whether this dynasty is of Cuman (an admixed Turkic people that reached Wallachia from the East in the 11th century) or of local Romanian (Vlach) origin is debated among historians. Earlier studies have demonstrated the value of investigating the Y chromosome of men bearing a historical name, in order to identify their genetic origin. We sampled 29 Romanian men carrying the surname Basarab, in addition to four Romanian populations (from counties Dolj, N = 38; Mehedinti, N = 11; Cluj, N = 50; and Brasov, N = 50), and compared the data with the surrounding populations. We typed 131 SNPs and 19 STRs in the non-recombinant part of the Y-chromosome in all the individuals. We computed a PCA to situate the Basarab individuals in the context of Romania and its neighboring populations. Different Y-chromosome haplogroups were found within the individuals bearing the Basarab name. All haplogroups are common in Romania and other Central and Eastern European populations. In a PCA, the Basarab group clusters within other Romanian populations. We found several clusters of Basarab individuals having a common ancestor within the period of the last 600 years. The diversity of haplogroups found shows that not all individuals carrying the surname Basarab can be direct biological descendants of the Basarab dynasty. The absence of Eastern Asian lineages in the Basarab men can be interpreted as a lack of evidence for a Cuman origin of the Basarab dynasty, although it cannot be positively ruled out. It can be therefore concluded that the Basarab dynasty was successful in spreading its name beyond the spread of its genes.

Population History of the Dniester-Carpathians: Evidence from Alu Insertion and Y-Chromosome Polymorphisms
Dissertation for the Faculty of Biology Ludwig-Maximilians-Universität at München, Germany, Alexandru Varzari, Republic of Moldova, 27 July 2006

Paleo-mtDNA analysis and population genetic aspects of old Thracian populations from South-East of Romania
Authors: Cardos G., Stoian V., Miritoiu N., Comsa A., Kroll A., Voss S., Rodewald A. © 2004 Romanian Society of Legal Medicine

Y-Chromosome Diversity in Modern Bulgarians: New Clues about Their Ancestry

Authors: Karachanak S, Grugni V, Fornarino S, Nesheva D, Al-Zahery N, et al. (2013). PLoS ONE 8(3): e56779. doi:10.1371/journal.pone.0056779

Investigation of the fine structure of European populations with applications to disease association studies
European Journal of Human Genetics (2008) 16, 1413 – 1429, Macmillan Publishers Limited 2008

A substantial prehistoric European ancestry among Ashkenazi maternal lineages
Nature Communications, 8 Oct 2013

Indian Signatures in the Westernmost Edge of the European Romani Diaspora: New Insight from Mitogenomes
PLOS ONE, October 2013
Authors: Alberto Gomez-Carballa, Jacobo Pardo-Seco, Laura Fachal, Ana Vega, Miriam Cebey, Nazareth Martinon-Torres, Federico Martinon-Torres, Antonio Salas 

ABSTRACT: In agreement with historical documentation, several genetic studies have revealed ancestral links between the European Romani and India. The entire mitochondrial DNA (mtDNA) of 27 Spanish Romani was sequenced in order to shed further light on the origins of this population. The data were analyzed together with a large published dataset (mainly hypervariable region I [HVS-I] haplotypes) of Romani (N = 1,353) and non-Romani worldwide populations (N.150,000). Analysis of mitogenomes allowed the characterization of various Romani-specific clades. M5a1b1a1 is the most distinctive European Romani haplogroup; it is present in all Romani groups at variable frequencies (with only sporadic findings in nonRomani) and represents 18% of their mtDNA pool. Its phylogeographic features indicate that M5a1b1a1 originated 1.5 thousand years ago (kya; 95% CI: 1.3–1.8) in a proto-Romani population living in Northwest India. U3 represents the most characteristic Romani haplogroup of European/Near Eastern origin (12.4%); it appears at dissimilar frequencies across the continent (Iberia: ,31%; Eastern/Central Europe: ,13%). All U3 mitogenomes of our Iberian Romani sample fall within a new sub-clade, U3b1c, which can be dated to 0.5 kya (95% CI: 0.3–0.7); therefore, signaling a lower bound for the founder event that followed admixture in Europe/Near East. Other minor European/Near Eastern haplogroups (e.g. H24, H88a) were also assimilated into the Romani by introgression with neighboring populations during their diaspora into Europe; yet some show a differentiation from the phylogenetically closest non-Romani counterpart. The phylogeny of Romani mitogenomes shows clear signatures of low effective population sizes and founder effects. Overall, these results are in good agreement with historical documentation, suggesting that cultural identity and relative isolation have allowed the Romani to preserve a distinctive mtDNA heritage, with some features linking them unequivocally to their ancestral Indian homeland.

Origins and Divergence of the Roma (Gypsies)
Authors: Gresham et al. (AJHG, 2001)

Genetic Structure of the Paternal Lineage of the Roma People
Authors: Horolma Pamjav, Andrea Zalan, Judit Beres, Melinda Nagy and Yuet Meng Chang, American Journal of Physical Anthropology 000:000–000 (2011)

ABSTRACT: According to written sources, Roma (Romanies, Gypsies) arrived in the Balkans around 1,000 years ago from India and have subsequently spread through several parts of Europe. Genetic data, particularly from the Y chromosome, have supported this model, and can potentially refine it. We now provide an analysis of Y-chromosomal markers from five Roma and two non-Roma populations (N=787) in order to investigate the genetic relatedness of the Roma population groups to one another, and to gain further understanding of their likely Indian origins, the genetic contribution of non-Roma males to the Roma populations, and the early history of their splits and migrations in Europe. The two main sources of the Roma paternal gene pool were identified as South Asian and European. The reduced diversity and expansion of H1a-M82 lineages in all Roma groups imply shared descent from a single paternal ancestor in the Indian subcontinent. The Roma paternal gene pool also contains a specific subset of E1b1b1a-M78 and J2a2-M67 lineages, implying admixture during early settlement in the Balkans and the subsequent influx into the Carpathian Basin. Additional admixture, evident in the low and moderate frequencies of typical European haplogroups I1-M253, I2a-P37.2, I2b-M223, R1b1-P25, and R1a1-M198, has occurred in a more population-specific manner.

Population data for Y-chromosome haplotypes defined by 17 STRs in South-East Romania
Authors: Florin Stanciu, Veronica Cutar, Sorina Pirlea, Veronica Stoian, Ionel Marius Stoian, Olivia Sevastre, Oana Raluca Popescu, Legal Medicine 12 (2010) 259-264
Forensic Science Institute, Bucharest, Romania, Department of Genetics, Faculty of Biology, University of Bucharest, Bucharest, Romania

The team of scientists analyzed 122 unrelated males from nine counties of the Romania South-East region: Arges (2 samples), Calarasi (52), Constanta (5), Dâmbovita (6), Ialomita (2), Olt (19), Prahova (4), Teleorman (4) and Vrancea (28). The region of the Romanian capital city, Bucharest, was excluded from the study. Results for the Romanian male population (for 9 and 12 analyzed Y-STR loci) indicate higher genetical closeness to southern Slavic populations (Macedonian, Serbian, Bosnia–Herzegovinian and Croatia), followed by northern Slavic populations (Russian and Polish), as well as Lithuanian, Hungarian and Greek. Regarding the genetic similarity between these peoples, two main assumptions were formulated: (a) the historical recent event with the greatest influence on the current population genetic structure was the Slavic migration from the North Eastern Europe towards the Balkan Peninsula or (b) the genetic similarity between these peoples reflects the distribution of a Paleo–Balkan population, such as Thracians. These findings corroborate with a previous population study regarding South-Eastern Romania and the surrounding countries, which had been made using autosomal STR loci and integrates the South-Eastern Romania population among southern Slavic populations in the big picture of Slavic peoples described by the Rebała et al. [Rebała K, Mikulich AI, Tsybovsky IS, Siváková D, Dzˇupinková Z, Szczerkowska-Dobosz A, et al. Y-STR variation among Slavs: evidence for the Slavic homeland in the middle Dnieper basin. J Hum Genet 2007;52(5):406–14.]



"The modern kingdom of Rumania consists of the provinces of Moldavia, Wallachia, Dobruja, Bessarabia, Transylvania, part of the Banat, and the Bukovina. The last four, while the majority of their inhabitants are Rumanians, have been Rumanian territory only since the World War. Moldavia is bounded on the west by the crest of the Carpathians, on the east by the Pruth River; Wallachia is bounded on the north by the Transylvanian Alps, and on the south by the Danube. Dobruja is the plain lying between the northward curve of the Danube and the Black Sea; it includes the important seaport of Constanza. In Moldavia and Wallachia the great majority of the population is Rumanian; the same is true to a large extent of Bessarabia, but in Transylvania there are large populations of Germans and of Magyars, already discussed in previous sections. In the Banat again there are many Hungarians, and a number of Serbs, while in the Dobruja lives one of the most scrambled populations of Europe. Here Bulgars, Ottoman Turks, Tatars, Gaguz, who claim to be descendants of the Kumans, Armenians, Kurds, Caucasic peoples, and a few of almost all the other peoples of Eastern Europe and western Asia are to be found. The Dobruja is as varied as the contents of an ethnological museum, and like a museum, each group clings tenaciously to everything that is its own. The inhabitants of Dobruja include, of course, both Gypsies and Jews, and Rumania is one of the greatest concentration points for both in Europe. The Jews form 5 per cent of the population of the pre-war section of the kingdom, and are especially numerous in northern Moldavia and the Bukovina, where their zone of concentration forms an extension of that in Polish Galicia. The Moldavian Jews, who are mainly of Polish or Russian antecedents, speak their own language, wear a separate costume, and mix little if at all with the Rumanian population.

In classical times Transylvania, Wallachia, and Moldavia formed what known as Dacia, and the Dacians were considered to be a branch of the Thracians. The Dacians included an upper class, distinguished by the practice of wearing brimless felt hats, Scythian style, and a peasantry, among whom the men went bare-headed, with their hair long, as do the older and more conservative of the present-day Rumanian peasants. Between 105 and 107 A.D. Trajan conquered Dacia, and made it a Roman province; the warlike inhabitants, who had long resisted the Romans, fled in great numbers, while their villages were being plundered; later, many are said to have returned. The Romans placed many colonists in Dacia, and for its defense established there the permanent headquarters of the thirteenth legion. In 256 A.D. the Goths arrived, and the Romans began a hasty departure; it is likely that many of the inhabitants of the country left with them. During the century and a half of Roman rule, the language of Dacia became Latin, and modern Rumanian is without doubt a descendant of that colonial speech. During the maximum extension of the empire, Latin and its derivatives were spoken in a wide zone peripheral to Rome, including the Iberian peninsula, Gaul, Switzerland, the Tyrol, and much of the territory lying between the head of the Adriatic and the Black Sea. Albanian, with its strong Latin infusion, must be considered a partial product of this extension; elsewhere Ladin, Romansch, and Rumanian must be considered survivals in the face of the barbarian invasions which converted most of southeastern Europe to Germanic, Slavic, Uralic, and Altaic speech.

Foreigners designate Rumanians and Rumanian speakers by the term Vlach; the Vlachs are the Rumanian speakers to be found throughout southeastern Europe, whether living in Rumania, Bulgaria, Greece, Albania, Yugoslavia, or elsewhere. The word Vlach, which is a derivative from the Gothic, by way of Slavic, means “foreigner”; it is a cognate of our own word “Welsh,” used by the Anglo-Saxons to designate Kymric-speaking Britons, and of “Walloon.” The modern Vlach language, while basically Latin, shares with Albanian certain structural peculiarities which it must derive from Thracian or Illyrian, and at the same time contains a large number of Slavic roots. The use of a Romance language in Rumania today is not a simple case of a Romanized Dacian survival; the history of Rumania is too complicated to permit this explanation alone. After the departure of the Romans, Dacia was overrun by Goths, by Slavs, by Bulgars, by many kinds of Tatars, and by Ottoman Turks. It is very likely that the Vlach survival in these lands was only partial until the late Middle Ages, when the peasants who had resisted the inroads of these conquerors were joined by their kinsmen returning from Bulgaria and Macedonia, and from beyond the Carpathians. Since then the expansion of the Vlachs in what is now Rumania has been constant and, east of the Carpathians, nearly complete. The Vlachs have always been far wanderers; many of them are shepherds, and the pastoral life has been as important to them, until modern times, as agriculture.

In Macedonia and northern Greece, and in Southern Albania, Vlach colonists are nomads living in black tents like those of Arabs, and like those which one may suppose the Scythians used before them. In Dalmatia they were during the Middle Ages an important people. Dubrovnik (Ragusa) was originally a Vlach town. In the peninsula of Istria, now inhabited mostly by Slovenes and Italians, a small group of Vlach speakers, the Cici, has resisted assimilation to this day. These Istrian Vlachs, early invaders of Illyrian territory, are the remnants of a former link in the continuity of the Roman Empire between the Atlantic and the Black Sea. In view of the complex ethnic history of Rumania, the living Rumanians may be expected to show evidence of a multiplicity of racial origin. To native Dacian elements, which must have included a blend of indigenous Neolithic peoples with Satem-speaking Nordics, have been added whatever population the Romans brought and which did not run away, and a multitude of early Slavs whom the Vlachs absorbed. Other elements, Ugric, Tatar, and Gothic, were probably of lesser importance. The Rumanians, as a whole, in the early part of the present century, had a mean stature of roughly 167 cm, which is probably nearly representative today.

There is little regional variation; what there is indicates that the mountaineers of the northern Rumanian Carpathians may be taller than the rest, since the villagers of Fundul Moldovii, studied by Rainer, have a mean of nearly 170 cm; those living on the Bessarabian plain amongst the Ukrainians seem to be the shortest, with a mean as low as 165 cm. A greater variation is found in the cephalic index; on the plains of Moldavia and Wallachia, and in the Dobruja, the Rumanians are as a rule mesocephals or sub-brachycephals, with means of 80 to 81; they are are nearly as long-headed as the Bulgarians. In the mountains, however, they are fully Dinaric or Alpine in their brachycephaly, with a mean of 85.4 in Fundul Moldovii in the Bukovinian highlands, and of 86 in Bukovina in general, where they equal the brachycephaly of the Huzuls.

Within the curve of the Carpathians, they are also completely brachycephalic; means from Rumanians in Transylvania and in the Banat lie between 84 and 85, although in the village of Dragus, an old and completely Rumanian settlement lying just inside the bend in the mountain crest where the Transylvanian Alps become the eastern Carpathians, and not far from the Saxon city of Kronstadt, the mean is 86.6. We are dealing, therefore, with two kinds of Rumanians; the mesocephalic ones of the eastern plains, and the brachycephalic ones of the Carpathians and the lands to the west. The Carpathians form a sharp boundary delimiting the eastward and northeastward extension of Alpine brachycephaly in Europe. This boundary shows little regard for language or for ethnic tradition.

The Rumanians of the plains show a general metrical similarity to the Neo-Danubians of the Slavic countries to the north, and at the same time a relationship to the longer-headed Bulgarians. The village of Nerejul Mare, some eighty miles north of Bucharest on the southeastern slope of the Carpathians, will serve as an example of the plains population, although the mean cephalic index of its inhabitants, 81.5, is higher than in some districts. The mean stature is 166.8 cm, the relative sitting height 52.7. Eighty-eight per cent of the men have black or dark brown to brown hair, the rest light brown or blond. Pure dark eyes are found among 54 per cent, light eyes among 11 per cent, with the rest mixed, mostly dark-mixed. Thus the population is prevailingly brunet, as well as moderately tall, intermediate in body build, and sub-brachycephalic. The mean head length of 186 mm, and breadth of 151 mm show a moderately small head size; the auricular height of 125 mm is relatively high. The face is of moderate size, with a height of 121 mm, and breadths of 102 mm for the minimum frontal, 140 mm. for the bizygomatic, and 106 mm. for the bigonial. The nose is small, with a height of 53.2 mm and a breadth of 34.2 mm. The face is mesoprosopic, with a facial index of 86, and leptorrhine, with a nasal index of 65. While these cranial and facial indices place the inhabitants of Nerejul Mare definitely in the same class with the peasantry of most of Russia, the intensity of hair and eye pigmentation, and the narrowness of the forehead and nose, as contrasted to the breadth of the jaw, suggest the brunet long-headed element in Bulgaria and Greece. Rainer finds these moderately tall Mediterraneans among his villagers, as well as individuals of Neo-Danubian, Slavic-looking type; Alpines and Dinarics are partly responsible for the elevation of the cephalic index, and Norics are present as a Nordic by-product. In Moldavia as a whole, however, the Neo-Danubian and Black Sea Mediterranean forms are the two elements of greatest importance, and the same is true of Wallachia.

The mountaineers of Fundul Moldovii, in the Bukovina, are taller than the villagers just studied, with a mean stature, quoted above, of 169.5 cm; their cephalic index mean is 85.4, while their nasal index reaches the low mean of 60. They are somewhat lighter eyed than the plainsmen, and darker haired. Their heads are broader, with a mean width of 157 mm, rather than shorter, and hence larger. Their faces are longer (124 mm) and broader (144 mm), while both foreheads and jaws also exceed those of the Moldavian villagers in breadth, and their nasal lengths (56.4 mm) are considerably greater. Fifteen per cent have flattened occiputs Although only 20 per cent have convex nasal profiles, in the great majority the forward jut of the nose, accompanied by a straight or wavy profile, is great. The Fundul Moldovii people are in great majority Dinarics; a few appear Alpine, and a few others Noric. By and large, if the inhabitants of this village were transported to northern Albania and given a change of costume, few anthropologists would be able to tell the difference between the newcomers and the native tribesmen. The inhabitants of Dragus, farther south and on the Transylvanian side, and no farther from Bucharest than Nerejul Mare, are just as Dinaric metrically as the Bukovinian villagers; their heads are, in fact, shorter, with a mean length of 182 mm., as are their faces; they resemble to a certain extent the Dinaric form common among Serbs. Leaving the political boundaries of Rumania, we find two groups of Vlachs who have been the subjects of special study; those of Macedonia and of Istria.

The Vlachs of Macedonia are the tallest of the many varied ethnic groups which compose that region, with a mean stature of 168 cm., and have the greatest absolute head length (188 mm). They are low brachycephals, with a mean cephalic index of 83, are predominantly dark-haired and dark-eyed, and straight-nosed. They show some Dinaric influences, as do all the peoples of Macedonia; on the whole, however, their closest affiliation is with the brunet mesocephals and dolichocephals of the eastern Balkan area. There are, nevertheless, a few blonds among them, and these are usually Nordic. The Istrian Vlachs, on the other hand, are complete Dinarics with a mean stature of 169 cm., a cephalic index of 86, and head and facial dimensions which cannot be distinguished from those of most Dinarics. In their high brachycephaly, however, and in their facial and nasal lengths, as well as in a predominant brunet tendency, they are much closer to the Tyrolese, and especially to the Ladin-speakers, than to the Slovenes among whom they live. They are also very similar to their distant linguistic relatives in the Carpathians.

The Vlachs, a widespread and numerous people in southeastern Europe, are the descendants of Romanized aborigines, and of other peoples whom these latter have absorbed. They have no racial homogeneity, but vary regionally according to the races long seated in the regions where they live. In the northeast, where the Moldavian plain forms a continuation of the Black Earth region of southern Russia, the Neo-Danubian type of the Black Earth region is predominant; in the southeast, where a local Atlanto-Mediterranean type is concentrated, the Vlachs tend to assume that form; west of the Carpathians, and near the crest of that range, they are Dinarics of the first rank, comparable to that other group of mountain-dwelling speakers of Neo-Latin, the Ladiner.

In studying the racial composition of southern Russia, there was evidence of a moderately tall, long-headed, brunet Mediterranean form, which is concentrated along the northern shore of the Black Sea, but which also appears sporadically in the entire Russian population. To western Europeans and Americans, it is better known than its frequency would warrant, for it is exemplified by several world famous ballerinas and opera singers. This is the Mediterranean racial division which the Russian anthropologists call Pontic and which the Poles recognize as a very minor element in their own population. It is with little doubt of Neolithic date in southern Russia, Rumania, Bulgaria, and the Hellespont region, and probably in Greece and the Aegean. In most of Thrace it seems more basic than the Danubian, or at least more common. What its relationship may be to the introduction of the Neolithic economy into Europe by land or by sea, cannot be determined without more data."

From The Races of Europe, by Carleton Stevens Coon, written before WWII Source:
Download the entire volume as a ZIP file (about 17 Mb) at:

Map Map of Macedonia, Thracia, Illyria, Moesia and Dacia - after 271 AD (printed in 1849)
Map Post-Glacial Population expansions (Early Holocene) Post-Glacial Population expansions (Early Holocene)

T Cardos G, Paleo-mtDNA analysis of old Thracian populations from S-E Romania

Rom J Leg Med 12 (4) 239 – 246 (2004)© 2004 Romanian Society of Legal Medicine
Stoian V., Miritoiu N., Department of Genetics-University of Bucharest, Bucharest, Romania
Comsa A., Institute of Anthropology-Romanian Academy of Science, Bucharest, Romania
Kroll A., Romanian National Institute of Archaeology/Thracology -Romanian Academy of Science, Bucharest, Romania
Voss S., Rodewald A.
Corresponding author: Prof. Dr. Dr. Director of Institute of Human Biology, University of  Hamburg, Allende-Platz 2, 20146 Hamburg, Germany; tel: 0049-40-428382271, fax: 428383174, e-mail: Department of Human Biology-University of Hamburg, Germany

Note: Certain technical aspects have been omitted from the article, for space purposes

ABSTRACT: Paleo-mtDNA analysis and population genetic aspects of old Thracian populations from South-East of Romania.

We have performed a study of mtDNA polymorphisms (HVR I and HVR II sequences) on the skeletal remains of some old Thracian populations from SE of Romania, dating from the Bronze and Iron Age in order to show their contribution to the foundation of the modern Romanian genetic pool and the degree of their genetic kinships with other old and modern human European populations. For this purpose we have applied and adapted three DNA extraction methods: the phenol/chloroform, the guanidine isotiocianat and silica particles and thirdly the Invisorb Forensic Kit (Invitek)-based DNA extraction method. We amplified by PCR short fragments of HVR I and HVR II and sequenced them by the Sanger method.So far, we have obtained mtDNA from 13 Thracian individuals, which we have compared with several modern mtDNA sequences from 5 European present-day populations.Our results reflect an evident genetic similarity between the old Thracian individuals and the modern populations from SE of Europe.

The scientific research in the field of ancient and degraded DNA has opened new possibilities and prospects for generally studying the evolution of life and studying the evolution of humankind in particular and also in forensic sciences. The major sources of biological material to examine the past are skeletons and rarely some soft tissues (especially in case of mummies). The mitochondrial DNA (mtDNA) markers are often used in such studies because of some particular traits of mt genome, such as: its presence in multiple copies per cell; its maternal inheritance; lack of recombination; and its mutation rate about 10 times faster than the average nuclear genes. From the whole mt genome, the most used in population or phylogenetic studies is the D-loop region or the control region, an uncoding sequence of about900 bp in the human mt genome. This mtDNA region shows the highest sequence polymorphism from the whole mt genome and mainly consists of HVR I and HVR II, which are the highly valuable DNA markers for such kind of studies. In the human mt genome, the HVR I consists of 341 bp between the nucleotide position (np) 16,024 bp to np 16,365 and the HVR II consists of 267 bp from np 73 to np 340.The two HVR mt regions reveal about 3 % variability between two unrelated individuals. In this context our study has focused on the mtDNA analysis (HVR I and HVR II region polymorphisms) on the skeletal remains of individuals from some old Thracian populations found in SE of Romania, dating from the Bronze and Iron Age, in order to show their genetic kinship with other old and modern European populations and their contribution to the foundation of the modern Romanian genetic pool.

Historical context

From archaeological and anthropological sources it is known that the Thracian people were formed during along historical lapse of time by an admixture of aboriginal and new arrived human groups. By a progressive development, these people had constituted in their basic elements during the 3rd - 2nd millennium B.C. The Thracian people lived on the land between the Carpathian Mountains (towards the N) and the Aegean Sea (towards the S), yet some Thracian elements were also found northwest of Little Asia and Slovakia. There is also a lot of archaeological material ascribed to the Thracian populations discovered in SE and E of Romania.

Material and methods

The biological material from our study has been represented by bones and teeth belonging to some individuals from the old Thracian populations from SE of Romania, which have either been well or less preserved, depending on the environmental factors from the archaeological site. The human fossil bones of 20 individuals dating about 3200-4100 years, from the Bronze Age, belonging to some cultures such as Tei, Monteoru and Noua, were found in graves from some necropoles in SE of Romania, namely in Zimnicea, Smeeni, Candesti,Cioinagi-Balintesti, Gradistea-Coslogeni and Sultana-Malu Rosu. The human fossil bones and teeth of 27 individuals from the early Iron Age, dating from the 10th - 7th century B.C. from the Hallstatt Era (the Babadag Culture), were found extremely SE of Romania near the Black Sea coast, in some settlements from Dobrogea, namely: Jurilovca, Satu Nou, Babadag, Niculitel and Enisala-Palanca. They had fossilized in some common pits, most of them on stone surfaces, covered by loess and an ash layer. Most of the fossil teeth were much better preserved than many human old bones probably due to the enamel layer which protect teeth against damaging induced by diagenetic factors. All human fossils from our study have been preserved at room temperature since their discovery between the years 1997-2003.

Sample preparation and DNA extraction

First and foremost, to prevent any contamination, sterile protective equipment,instruments, reagents and three different rooms for ancient DNA (aDNA) extraction, PCR amplification and cleaning-up the PCR products were used. To remove any previous contamination, the skeletal samples were treated by firstly removing the outer layer, secondly exposing them to UV light for 10 minutes on each side, thirdly washing them with absolute EtOH, EtOH 70%, distilled water, and lastly letting them dry at 30°C in the oven over night. Once again, the skeletal samples were exposed to UV light for 10 minutes on each side and then ground to a fine powder. For aDNA extraction we used the three following methods:
  • the phenol-based DNA extraction method as described by Humme and modified it by removing the decalcification step and prolonging the cell lysis step to 15-18 hours;
  • the guanidine tiocianat and silica-based method described by Hoss and Pääbo and modified it by prolonging the extraction step to 15-18 hours and removing the final elution step. The DNA extracts with silica suspension were used for the DNA amplification byPCR reactions;
  • the Invisorb Forensic Kit (Invitek)-based DNA extraction method modified it by using silica particles instead of carrier suspension. To detect any contamination of the reagents, a negative control (without sample) was used for each aDNA extraction.
Results and discussion

The four DNA sequences have been amplified from extracts obtained by all three DNA extraction methods described above. Up to now we have obtained aDNA from 6 individuals from the Bronze Age and from 7 individuals from the Iron Age. The other samples still require further extractions and/or amplifications in order to confirm the results. Only the HVR I polymorphisms are presented below because we have so far only obtained a few and short Thracian HVR II sequences, without scientific relevance for a sequence analysis.

The comparison of HVR I sequences (the two fragments) of old Thracian populations and 5 European present populations: Thracian, Romanian, Albanian, Bulgarian, Greek, Italian; CRS = Cambridge Reference Sequence; N = unknown nucleotide
First, we have done the alignment of the Thracian mtDNA sequences separately with each sample of 20 sequences from the five modern European populations by using BIOEDIT programme and we noticed that in the mtDNA sequences analysed there are 12 nucleotide positions which gave us some information about the genetic kinship of the five modern European populations with the old Thracian populations (alignments not shown). Then, from all modern European mtDNA sequences we have chosen only the most informative HVR I sequences, which contain at least one of the 12 informative nucleotide positions, to align them with the mtDNA sequences of the old Thracian populations to show their genetic kinship more relevant. The 12 nucleotide positions in which there are common point mutations in comparison with the European mitochondrial sequence CRS, in the sixth populations analysed here are as follows:

1) 16126 np with T→C transition is shown by 10 Italian, 1 Greek, 2 Albanian, and 2 Romanian individuals,
2) 16129 np with G→A transition shown by 1 Thracian, 1 Italian, 3 Greek, 1 Bulgarian and 3 Albanian individuals,
3) 16145 np with G→A transition shown by 1 Thracian and 1 Italian individuals,
4) 16186 np with C→T transition shown by 1 Thracian, 3 Italian, 1 Greek, 1 Romanian individuals,
5) 16190 np with T→C transition shown by 1 Thracian, 2 Romanian, 8 Italian, 4 Greek,3 Bulgarian and 2 Albanian individuals,
6) 16193 np with C→T transition shown by 1 Thracian, 2 Greek and 1 Albanian individuals,
7) 16223 np with C→T transition shown by 1Thracian, 1 Romanian, 1 Greek, 2 Albanian and 2 Bulgarian individuals,
8) 16283 np with A→C transversion shown by 1 Thracian and 1 Romanian individuals,
9) 16294 np with C→T transition shown by 1 Romanian, 1 Italian and 1 Albanian individuals,
10) 16311 np with T→C transition shown by 1 Thracian, 1 Romanian, 3 Greek, 4 Italian and 5 Albanian individuals,
11) 16356 np with T→C transition shown by 2 Romanian and 1 Bulgarian individuals and
12) 16362 np with T→C transition shown by 2 Thracian, 4 Romanian, 4 Bulgarian and 2 Italian individuals.
As we can notice in the latter alignment, the Thracian individuals have shown informative point mutations in 7 np, the Romanian, Greek and Albanian individuals in 8 np, the Italian individuals in 7 np and the Bulgarian individuals in only 5 np out of the 12 most informative nucleotide positions presented above. As concerns the frequency of point mutations in the 12 nucleotide positions we have realized that the Italian individuals show the highest mutation frequency with 12.5%, followed by the Thracian individuals with 8.3%, the Albanian individuals with 7.5%, the Romanian and Greek individuals with 6.25% and the Bulgarian individuals with only 4.6%. Computing the frequency of common point mutations of the present-day European population with the Thracian population has resulted that the Italian (7.9%), the Albanian (6.3%) and the Greek (5.8%) have shown a bias of closer genetic kinship with the Thracian individuals than the Romanian and Bulgarian individuals (only 4.2%).

Conclusions and prospects

In conclusion we believe that our results reflect an evident genetic similarity between the old Thracian individuals analysed up to now in our study and the modern populations from SE of Europe. So far we can just suppose, that the old Thracian populations would have been able to contribute to the foundation of the Romanian modern genetic pool. More mtDNA sequences from Thracian individuals are needed in order to perform an complex objective statistical analysis.


We would like to thank Dan Botezatu and Georgeta Miu from the Institute of Thracology-Iasi and Andrei Soficaru from the Institute of Anthropology-Bucharest for their help in giving us some skeletal samples from the Bronze Age and Hermann Müller from the Department of Human Biology-University of Hamburg forhis graphical contribution.

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