The Great Hungarian Plain was a crossroads of cultural transformations that have shaped European prehistory. Here we analyse a 5,000-year transect of human genomes, sampled from petrous bones giving consistently excellent endogenous DNA yields, from 13 Hungarian Neolithic, Copper, Bronze and Iron Age burials including two to high (~22 × ) and seven to ~1 × coverage, to investigate the impact of these on Europe’s genetic landscape. These data suggest genomic shifts with the advent of the Neolithic, Bronze and Iron Ages, with interleaved periods of genome stability. The earliest Neolithic context genome shows a European hunter-gatherer genetic signature and a restricted ancestral population size, suggesting direct contact between cultures after the arrival of the first farmers into Europe. The latest, Iron Age, sample reveals an eastern genomic influence concordant with introduced Steppe burial rites. We observe transition towards lighter pigmentation and surprisingly, no Neolithic presence of lactase persistence.

Ancient DNA (aDNA) research involves invasive and destructive sampling procedures that are often incompatible with anthropological, anatomical, and bioarcheological analyses requiring intact skeletal remains. The osseous labyrinth inside the petrous bone has been shown to yield higher amounts of endogenous DNA than any other skeletal element; however, accessing this labyrinth in cases of a complete or reconstructed skull involves causing major structural damage to the cranial vault or base. Here, we describe a novel cranial base drilling method (CBDM) for accessing the osseous labyrinth from the cranial base that prevents damaging the surrounding cranial features, making it highly complementary to morphological analyses. We assessed this method by comparing the aDNA results from one petrous bone processed using our novel method to its pair, which was processed using established protocols for sampling disarticulated petrous bones. We show a decrease in endogenous DNA and molecular copy numbers when the drilling method is used; however, we also show that this method produces more endogenous DNA and higher copy numbers than any postcranial bone. Our results demonstrate that this minimally-invasive method reduces the loss of genetic data associated with the use of other skeletal elements and enables the combined craniometric and genetic study of individuals with archeological, cultural, and evolutionary value.

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1The distribution, antiquity and epidemiology of tuberculosis (TB) have previously been studied in 2 osteoarchaeological material in the eastern part of Hungary, mainly on the Great Plain. The 3 purpose of this study is to map the occurrence of skeletal TB in different centuries in the Western 4 part of Hungary, Transdanubia, and to present new cases we have found. Paleopathological 5 analysis was carried out using gross observation supported by radiographic and molecular 6 methods. A large human osteoarchaeological sample (n=5684) from Transdanubian 7 archaeological sites ranging from the 2nd to the 18th centuries served as a source of material. 8Spinal TB was observed in seven individuals (in three specimens with Pott's disease two of 9 which also had cold abscess) and hip TB was assumed in one case. The results of DNA for 10Mycobacterium tuberculosis were positive in seven of the eight cases identified by 11 paleopathology, and negative in the assumed case of hip TB. However, the molecular results are 12 consistent with highly fragmented DNA, which limited further analysis. Based on the present 13 study and previously published cases, osteotuberculosis was found in Transdanubia mainly 14 during the 9th-13th centuries. However, there are no signs of TB in many other 9th-13th 15 centuries, even in those that lie geographically close to those where osteotuberculous cases were 16found. This may be due to a true absence of TB caused by the different living conditions, way of 17 life, or origin of these populations An alternative explanation is that TB was present in some 18 individuals with no typical paleopathology, but that death occurred before skeletal morphological 19 features could develop. 20 21 3

Study Design: Case report. A C C E P T E D Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.Objective: To characterise the paleopathology presented in the skeleton of a 45-50-year-old male indicative of tuberculous spondylitis and to confirm by the detection of ancient DNA.Summary of Background Data: Tuberculosis (TB) is an infectious disease prevalent in both present and ancient human populations. The disease is primarily located within the lungs, so although characacteristic bone lesions can lead to a clear diagnosis, skeletal TB occurs in only 5-6% of TB infections, even in historical cases. In addition, the visual appearance of human skeletal remains may be influenced by the environmental conditions at the burial site.However, it is important to recognise ancient skeletal TB, because this can provide important data on the history of Mycobacterium tuberculosis and gives an unique opportunity for physicians to observe the natural outcome of the infection from the pre-antibiotic era.Methods: Paleopathological analysis was carried out using careful visual observation supported by ancient DNA analysis. Approximately 60 mg of bone powder from rib fragments was examined and DNA from the M. tuberculosis complex was detected by PCR targeting specific genetic loci of the IS6110 and IS1081 regions. Results:The skeleton is part of a human osteoarchaeological collection (n=274) from the 12th-13th century Transylvanian archaeological site of Peteni, in modern-day Romania. The individual, a 45-50-year-old male, showed gross pathology typical of tuberculous spondylitis.The paleopathological diagnosis was supported by analysis for M. tuberculosis complex ancient DNA.Conclusions: This case demonstrates that TB was present in Transylvania (Romania) during the 12-13th century and adds to the growing body of knowledge on the history of this disease.