Ancient DNA in archaeology: From archaeological excavation to laboratory analyses

Sa razvojem tehnologije, analize drevne DNK postale su sastavni deo istraživanja u arheologiji, doprinoseći saznanjima vezanim za istoriju populacija. Međutim, analize drevne DNK predstavljaju izazov iz nekoliko razloga. Destruktivne su i skupe, a osim toga, u arheološkom uzorku je prisutna veoma mala količina degradovane i hemijski izmenjene endogene DNK. Nepovoljni sredinski uslovi u kojima je materijal pronađen i skladišten (poput visokih temperatura i vlage) podstiču dalju degradaciju organskog materijala. Dodatni problem je i moguća kontaminacija savremenom DNK, do koje može doći u bilo kom koraku analiza. Optimalno i pažljivije rukovanje uzorcima prilikom iskopavanja i skladištenja istog, dovodi do povećanja količine endogene DNK u uzorku i smanjivanja cene sekvenciranja. Pokazano je da se najveći prinos drevne DNK postiže izdvajanjem iz kalcifikovanog materijala, a posebno unutrašnjeg dela petrozne kosti. Pretvaranje kostiju u prah obavlja se u laboratorijama specijalno posvećenim ovim analizama, u kojima su preduzete određene mere i protokoli kako bi se kontaminacija sprečila. U zavisnosti od istraživačkog pitanja i nakon provere očuvanosti DNK, donosi se odluka da li će se sekvencirati ceo genom i na kojoj dubini ili samo delovi genoma od interesa. U slučaju nedostupnosti jedarne DNK, mitohondrijalna DNK kao i polimorfni genetički markeri na Y hromozomu mogu biti informativni pri određivanju materalnih, tj. pateralnih linija, ali za razliku od jedarne DNK ne mogu pružiti potpunu informaciju o demografskim promenama i populacionoj dinamici. Međutim, kako bismo stvarno razumeli prošlost, neophodna je dobra komunikacija i razmena informacija između arheologa, antropologa, genetičara i lingvista.With the development of molecular methods, especially Next Generation Sequencing platforms, ancient DNA analyses have been integrated into archaeological analyses so as to help answer the questions about past populations. However, the extraction of ancient DNA (aDNA) from archaeological samples is a very challenging process as the amount of endogenous DNA is often very low due to several reasons. Firstly, aDNA is very badly preserved in samples, especially in warm and humid climates. Even in optimal conditions, such as caves and cold and dry environments, DNA is degraded to shorter fragments and chemically modified. Secondly, besides the DNA of interest, there is a huge amount of exogenous and microbial DNA in samples. And finally, contamination with modern DNA represents a constant threat to the authenticity of the analysis and could be introduced at any step of the analyses, either during excavation, museum storage or laboratory work. Since these methods are destructive and expensive, it is a duty of archaeologists, museum curators as well as biologists, to take certain steps to prevent further degradation of the material and possible contamination. One of the most essential steps is the use of gloves while handling samples. Moreover, samples should not be washed or chemically treated and they should be properly stored in a dry place, without fluctuations in temperature and moisture. The optimal storage and handling could greatly affect the amount of endogenous DNA in samples, which dictates further possibilities of analyses as well as the cost. Researches so far have showed that the inner part of petrous bones yields the highest amount of endogenous DNA, as it is the densest bone in mammals and therefore more resistant to contamination. In the absence of petrous bones, other material such as teeth or hard, but not porous bones could be used as well. Conversion of bones into powder for DNA extraction has to be carried out in special facilities dedicated only to aDNA analysis to prevent contamination. With the development of NGS, it became possible to successfully determine very short and degraded sequences (characteristic for aDNA) even from very small amounts of old and badly preserved samples and to significantly reduce the time of sequencing, as well as its cost. Whether the research includes sequencing of the whole genome at different sequencing depth or capturing specific sequences of the genome depends greatly on DNA preservation, research question and the cost. In addition, mitochondrial DNA and genetic markers on Y chromosome could be very informative of maternal and paternal genetic ancestry, respectively. However, regardless of the research question, it is of the outermost importance that genetic data is observed in context, together with anthropological and archaeological data. It is only in this way that we can obtain reliable information about the past

Ancient genomes provide insights into family structure and the heredity of social status in the early Bronze Age of southeastern Europe

Twenty-four palaeogenomes from Mokrin, a major Early Bronze Age necropolis in southeastern Europe, were sequenced to analyse kinship between individuals and to better understand prehistoric social organization. 15 investigated individuals were involved in genetic relationships of varying degrees. The Mokrin sample resembles a genetically unstructured population, suggesting that the community's social hierarchies were not accompanied by strict marriage barriers. We find evidence for female exogamy but no indications for strict patrilocality. Individual status differences at Mokrin, as indicated by grave goods, support the inference that females could inherit status, but could not transmit status to all their sons. We further show that sons had the possibility to acquire status during their lifetimes, but not necessarily to inherit it. Taken together, these findings suggest that Southeastern Europe in the Early Bronze Age had a significantly different family and social structure than Late Neolithic and Early Bronze Age societies of Central Europe

Ancient DNA analyses of individuals of the Mokrin necropolis (2100 - 1800 BC): the reconstruction of kinship and social organization of Early Bronze Age society

Analiza biološkog srodstva u arheologiji pruža uvid u problematiku rođačkih odnosa u kontekstu organizovanja strukture društva i prenosa statusa i materijalnog bogatstva sa generacije na generaciju. Razvoj paleogenetike, kroz analizu drevne DNK i u određivanje srodstva, doprineo je razumevanju socijalne organizacije i socijalnih promena u ranim egalitarnim i hijerarhijski organizovanim društvima. Dvadeset četiri petrozne kosti sa rano-bronzanodopske nekropole u Mokrinu analizirane su novom generacijom sekvenciranja kako bi se uvidilo postoji li vertikalna i horizontalna socijalna diferencijacija na ovoj nekropoli i na koji način su se materijalni i socijalni status nasleđivali. Analizom srodstva detektovano je 9 porodičnih veza koje uključuju 15 individua. Sveukupni podaci sugerišu da su bogatstvo i status vezani za pol individue: dok sinovi nisu nasleđivali socijalni status od bioloških majki, ali su imali priliku da ga zasluže tokom života, žene su mogle naslediti status od majki ili ga postići putem socijalnih veza. Genetički podaci takođe sugerišu da se ne može prihvatiti hipoteza o podeljenosti nekropole na južni i severni deo na osnovu porodičnih veza. Veliki diverzitet uniparentalnih genetičkih markera sugeriše da društvo u Mokrinu nije bilo matrilokalno, ali i da nije bilo strogo patrilokalno, dok procenjeni koeficijent inbridinga ukazuje na odsustvo genetičke strukturiranosti. Osnovne populacione analize na populaciji sa Mokrina ne pokazuju nikakvo odstupanje od očekivanih proporcija porekla karakterističnog za bronzano doba, niti uzorci izlaze iz opsega genetičke varijabilnosti savremenih populacija u Evropi. Na istim uzorcima procenjena je učestalost alela funkcionalnih markera za depigmentaciju kože i toleranciju na mleko. Izvedeni aleli povezani sa svetlom kožom i plavim očima prisutni su u populaciji sa Mokrina, dok je učestalost alela za toleranciju laktoze kod 18 uzoraka sa Mokrina niska.Detecting biological relatedness is significant in archeology as it is believed that kinship ties were important in organization of societies and that they were the main mechanism of transgenerational transmission of status and wealth. With development of paleogenetics field, ancient DNA became efficient tool in determining kinship in order to better understand social organization and social changes in early egalitarian and hierarchical societies. Twenty-four petrous bones from Early Bronze Age Mokrin necropolis were sequenced with NGS method to address the hypotheses regarding vertical and horizontal stratification of the necropolis and to obtain information whether the wealth and status were hereditary. Nine kin relationships were identified among 15 individuals. The observed results indicate that the wealth and status were depended on the individuals’ sex: men could not inherit status from their biological mothers, but they probably could achieve it during life, while women could inherit the status or accomplish it through their links with prestigious individuals. aDNA analyses could not confirm hypothesis regarding the division of necropolis into Northern and Southern parts in which each community was integrated through kinship ties. The great variability of uniparental genetic markers indicates that Mokrin society was not matriliear, but it was also not a strictly patrilocal, while estimated inbreeding coefficient indicates that the population was genetically unstructured. The basic population analyses do not show any unexpected results of ancestry proportions, and all Mokrin samples could be placed within genetic variation of modern Europeans. Frequency of alleles for skin depigmentation markers and lactose tolerance were also estimated in Mokrin population. Derived alleles associated with light hair and blue eyes are detected, while the frequency of allele for lactose tolerance in 18 Mokrin samples is low

Low Prevalence of Lactase Persistence in Bronze Age Europe Indicates Ongoing Strong Selection over the Last 3,000 Years

Lactase persistence (LP), the continued expression of lactase into adulthood, is the most strongly selected single gene trait over the last 10,000 years in multiple human populations. It has been posited that the primary allele causing LP among Eurasians, rs4988235-A [1], only rose to appreciable frequencies during the Bronze and Iron Ages [2, 3], long after humans started consuming milk from domesticated animals. This rapid rise has been attributed to an influx of people from the Pontic-Caspian steppe that began around 5,000 years ago [4, 5]. We investigate the spatiotemporal spread of LP through an analysis of 14 warriors from the Tollense Bronze Age battlefield in northern Germany (similar to 3,200 before present, BP), the oldest large-scale conflict site north of the Alps. Genetic data indicate that these individuals represent a single unstructured Central/Northern European population. We complemented these data with genotypes of 18 individuals from the Bronze Age site Mokrin in Serbia (similar to 4,100 to similar to 3,700 BP) and 37 individuals from Eastern Europe and the Pontic-Caspian Steppe region, predating both Bronze Age sites (similar to 5,980 to similar to 3,980BP). We infer low LP in all three regions, i.e., in northern Germany and South-eastern and Eastern Europe, suggesting that the surge of rs4988235 in Central and Northern Europe was unlikely caused by Steppe expansions. We estimate a selection coefficient of 0.06 and conclude that the selection was ongoing in various parts of Europe over the last 3,000 years

Low prevalence of lactase persistence in bronze age europe indicates ongoing strong selection over the last 3,000 years

Lactase persistence (LP), the continued expression of lactase into adulthood, is the most strongly selected single gene trait over the last 10,000 years in multiple human populations. It has been posited that the primary allele causing LP among Eurasians, rs4988235-A [1], only rose to appreciable frequencies during the Bronze and Iron Ages [2, 3], long after humans started consuming milk from domesticated animals. This rapid rise has been attributed to an influx of people from the Pontic-Caspian steppe that began around 5,000 years ago [4, 5]. We investigate the spatiotemporal spread of LP through an analysis of 14 warriors from the Tollense Bronze Age battlefield in northern Germany (∼3,200 before present, BP), the oldest large-scale conflict site north of the Alps. Genetic data indicate that these individuals represent a single unstructured Central/Northern European population. We complemented these data with genotypes of 18 individuals from the Bronze Age site Mokrin in Serbia (∼4,100 to ∼3,700 BP) and 37 individuals from Eastern Europe and the Pontic- Caspian Steppe region, predating both Bronze Age sites (∼5,980 to ∼3,980 BP). We infer low LP in all three regions, i.e., in northern Germany and South-eastern and Eastern Europe, suggesting that the surge of rs4988235 in Central and Northern Europe was unlikely caused by Steppe expansions. We estimate a selection coefficient of 0.06 and conclude that the selection was ongoing in various parts of Europe over the last 3,000 years