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Reconstruction of ancient epigenomes by DNA methylation (DNAm) can shed light into the composition of cell types, disease states, and age at death. However, such analysis is hampered by impaired DNA quality and little is known how decomposition affects DNAm. In this study, we determined if EPIC Illumina BeadChip technology is applicable for specimens from mummies of the eighteenth century CE. Overall, the signal intensity on the microarray was extremely low, but for one of two samples we were able to detect characteristic DNAm signals in a subset of CG dinucleotides (CpGs), which were selected with a stringent processing pipeline. Using only these CpGs we could train epigenetic signatures with reference DNAm profiles of multiple tissues and our predictions matched the fact that the specimen was lung tissue from a 28-year-old woman. Thus, we provide proof of principle that Illumina BeadChips are applicable for DNAm profiling in ancient samples.

The perpetual burden of tuberculosis (TB) keeps drawing the focus of research on this disease. Among other risk factors (e.g., poor living conditions, malnutrition, smoking, HIV infection, etc.), being in close contact with a TB infected person requires special attention. For a better understanding of the disease, paleopathological investigations concerning TB have been carried out with various techniques for a long a time; nevertheless, analysis of incidence among family members is hardly possible in past populations. An exceptional group of naturally mummified individuals, the collection of the Vác mummies (Hungary, 18th century CE), is known about the large TB incidence rate, which has been revealed by aDNA analysis. Besides the high rate of TB infection, another interesting aspect of the collection is that in some cases, the family connections could be reconstructed. In this paper, we present the mycocerosic acid profiles gained by HPLC-HESI-MS measurements of two Vác mummies, who were mother and daughter according to the personal records. Earlier metagenomic analysis already revealed mixed M. tuberculosis infection with the same bacterial strains in both individuals; moreover, the same bacterial strains were recorded in both cases.

Herrmann Klaatsch eignete sich 1905 unter dubiosen Umständen eine australische Räuchermumie an und übergab sie der Berliner Gesellschaft für Anthropologie, Ethnologie und Urgeschichte (BGAEU). Aus historischen Quellen ist bekannt, dass es sich hierbei um die sterblichen Überreste von „Ng:tja“ handelt, auch unter dem Namen Barry Clarke bekannt. Der Herkunftsort der Mumie befindet sich im nordöstlichen Queensland. 1914 übergab die BGAEU diese Mumie dann als Dauerleihgabe an das Ethnologische Museum Berlin, wo sie bis zur Restitution an die Nachfahren von Ng:tja im Jahr 2017 blieb. Mit den Nachfahren von Ng:tja wurde eine Vereinbarung über die anthropologische Untersuchung und Beprobung getroffen. Die genetischen Analysen haben ergeben, dass die Mumie aus dem australischen Raum kommt und zur indigenen Bevölkerung Australiens gehört. Die mitochondriale DNA weist zudem auf das Herkunftsgebiet Queensland hin und das Geschlecht konnte, in Übereinstimmung mit der anthropologischen Untersuchung, genetisch als männlich bestimmt werden.

SummaryHuns, Avars and conquering Hungarians were Migration Period nomadic groups which arrived in three successive waves in the Carpathian Basin between the 5th and 9th centuries. Based on historical data each of these groups are thought to have arrived from Asia, although their exact origin and relation to other ancient and modern populations has been debated. In this study we have sequenced 9 Hun, 143 Avar and 113 Hungarian conquest period samples, and identified three core populations, representing immigrants from each period, with no recent European ancestry. Our results suggest that this “immigrant core” of both Huns and Avars originated in present day Mongolia, and their origin can be traced back to Xiongnus. On the other hand, the “immigrant core” of the conquering Hungarians derived from an earlier admixture of Mansis, early Sarmatians and descendants of late Xiongnus. In addition, we detected shared Hun-related ancestry in numerous Avar and Hungarian conquest period genetic outliers indicating a genetic link between these successive nomadic groups. Aside from the immigrant core groups we identified that the majority of the individuals from each period were local residents, harboring “native European” ancestry.

Huns, Avars, and conquering Hungarians were migration-period nomadic tribal confederations that arrived in three successive waves in the Carpathian Basin between the 5

Nucleic acid extraction from complex environmental and ancient tissue material is prone to co-extract inhibitory substances that make further molecular analysis difficult or impossible. This co-extraction occurs in both solid-phase and liquid-phase/organic nucleic acid extraction protocols. Currently, the widely used method to overcome inhibition includes the addition of bovine serum albumin (BSA) to the downstream enzymatic reactions or the dilution of the nucleic acid extracts. BSA, however, seems to reduce the inhibitory effect of certain compounds only, and excessive dilution may change the original DNA composition. In this study, we introduce an innovative new method using linear polyacrylamide (LPA) to efficiently precipitate and purify nucleic acids extracted from complex environmental and ancient tissue samples in one working step. The LPA method replaces the precipitation step in classic liquid-phase/organic extraction protocols or can be easily applied as an additional post-extraction step on impure DNA extracts. As a proof of concept, we experimented with this method on different ancient human mummy samples (bones, soft tissues and gut contents) from different time periods (5000 BC-1800 AD), as well as on complex environmental samples (e.g. soil, activated sludge and animal faeces) known to contain inhibitory compounds. We demonstrated that LPA precipitates nucleic acids, even in an aqueous ethanol solution without the addition of chaotropic salts, resulting in the recovery of highly pure DNA from all tested samples that displayed inhibition with previously published extraction protocols. Compared to the current, most widely used silica-based extraction method for ancient and sedimentary DNA, our LPA method resulted in comparable DNA qualities and overall DNA compositions (human endogenous content and microbial diversity). In conclusion, our LPA method with its high purifying capacity provides an important alternative to the commonly used DNA extraction protocols in the environmental and ancient DNA (aDNA) fields.

The Vác Mummy Collection comprises 265 well documented mummified individuals from the late 16th to the early 18th century that were discovered in 1994 inside a crypt in Vác, Hungary. This collection offers a unique opportunity to study the relationship between humans and pathogens in the pre-antibiotic era, as previous studies have shown a high proportion of tuberculosis (TB) infections among the individuals. In this study, we recovered ancient DNA with shotgun sequencing from a rib bone sample of a 18th century midwife. This individual is part of the collection and shows clear skeletal changes that are associated with tuberculosis and syphilis. To provide molecular proof, we applied a metagenomic approach to screen for ancient pathogen DNA. While we were unsuccessful to recover any ancient Treponema pallidum DNA, we retrieved high coverage ancient TB DNA and identified a mixed infection with two distinct TB strains by detailed single-nucleotide polymorphism and phylogenetic analysis. Thereby, we have obtained comprehensive results demonstrating the long-time prevalence of mixed infections with the sublineages L4.1.2.1/Haarlem and L4.10/PGG3 within the local community in preindustrial Hungary and put them in context of sociohistorical factors.

The identity of the mummified Lady from the Barfusser Church in Basel, Switzerland has been unsolved for decades, despite the prominent location of the burial place in front of the choir screen. A recent multidisciplinary research approach came up with a possible candidate, Anna Catharina Bischoff who died in Basel in 1787 with an age of 69 years (1719–1787). To verify the identity of the mummy, genealogists of the Citizen Science Basel discovered three living individuals of the maternal lineage of two different family branches, separated from Anna Catharina Bischoff by up to 22 generations. In this study we compare the ancient mitochondrial DNA of the mummy recovered from a premolar to the mitochondrial DNA of these three candidates. Initially the mitochondrial hypervariable regions I and II of the living individuals were screened using the Sanger sequencing method. This was followed by a mitochondrial capture approach and next generation sequencing to enrich for the whole mitochondrial genome of the mummy and one living person. A full mitochondrial genome has been recovered of both individuals sharing an identical haplotype. The sequence was assigned to the mitochondrial haplogroup U5a1+!16192 including two private mutations 10006G and 16293C. Only by using an interdisciplinary approach combining ancient DNA analysis and genealogy a maternal lineage of a non-noble family spanning 22 generations could be confirmed.

The bacterium Helicobacter pylori (H. pylori) infects the stomachs of approximately 50% of all humans. With its universal occurrence, high infectivity and virulence properties it is considered as one of the most severe global burdens of modern humankind. It has accompanied humans for many thousands of years, and due to its high genetic variability and vertical transmission, its population genetics reflects the history of human migrations. However, especially complex demographic events such as the colonisation of Europe cannot be resolved with population genetic analysis of modern H. pylori strains alone. This is best exemplified with the reconstruction of the 5300-year-old H. pylori genome of the Iceman, a European Copper Age mummy. Our analysis provided precious insights into the ancestry and evolution of the pathogen and underlined the high complexity of ancient European population history. In this review we will provide an overview on the molecular analysis of H. pylori in mummified human remains that were done so far and we will outline methodological advancements in the field of ancient DNA research that support the reconstruction and authentication of ancient H. pylori genome sequences.

Although malaria is one of the oldest and most widely distributed diseases affecting humans, identifying and characterizing its presence in ancient human remains continue to challenge researchers. We attempted to establish a reliable approach to detecting malaria in human skeletons using multiple avenues of analysis: macroscopic observations, rapid diagnostic tests, and shotgun-capture sequencing techniques, to identify pathological changes, Plasmodium antigens, and Plasmodium DNA, respectively. Bone and tooth samples from ten individuals who displayed skeletal lesions associated with anaemia, from a site in southern Egypt (third to sixth centuries AD), were selected. Plasmodium antigens were detected in five of the ten bone samples, and traces of Plasmodium aDNA were detected in six of the twenty bone and tooth samples. There was relatively good synchronicity between the biomolecular findings, despite not being able to authenticate the results. This study highlights the complexity and limitations in the conclusive identification of the Plasmodium parasite in ancient human skeletons. Limitations regarding antigen and aDNA preservation and the importance of sample selection are at the forefront of the search for malaria in the past. We confirm that, currently, palaeopathological changes such as cribra orbitalia are not enough to be certain of the presence of malaria. While biomolecular methods are likely the best chance for conclusive identification, we were unable to obtain results which correspond to the current authentication criteria of biomolecules. This study represents an important contribution in the refinement of biomolecular techniques used; also, it raises new insight regarding the consistency of combining several approaches in the identification of malaria in past populations.

Summary We subjected human paleofeces dating from the Bronze Age to the Baroque period (18th century AD) to in-depth microscopic, metagenomic, and proteomic analyses. The paleofeces were preserved in the underground salt mines of the UNESCO World Heritage site of Hallstatt in Austria. This allowed us to reconstruct the diet of the former population and gain insights into their ancient gut microbiome composition. Our dietary survey identified bran and glumes of different cereals as some of the most prevalent plant fragments. This highly fibrous, carbohydrate-rich diet was supplemented with proteins from broad beans and occasionally with fruits, nuts, or animal food products. Due to these traditional dietary habits, all ancient miners up to the Baroque period have gut microbiome structures akin to modern non-Westernized individuals whose diets are also mainly composed of unprocessed foods and fresh fruits and vegetables. This may indicate a shift in the gut community composition of modern Westernized populations due to quite recent dietary and lifestyle changes. When we extended our microbial survey to fungi present in the paleofeces, in one of the Iron Age samples, we observed a high abundance of Penicillium roqueforti and Saccharomyces cerevisiae DNA. Genome-wide analysis indicates that both fungi were involved in food fermentation and provides the first molecular evidence for blue cheese and beer consumption in Iron Age Europe., Graphical abstract, Highlights • Gut microbiome and diet of European salt miners determined using paleofeces • Until the Baroque, the microbiome resembled that of modern non-Westernized people • Food-fermenting fungi in Iron Age feces indicates blue cheese and beer consumption, Maixner et al. describe the gut microbiome and diet of European salt miners using paleofeces dating from the Bronze Age to the Baroque period. This analysis provides evidence for recent changes in the gut microbiome due to industrialization and for the consumption of fermented food and beverages in Iron Age Europe.

Loss of gut microbial diversity1–6 in industrial populations is associated with chronic diseases7, underscoring the importance of studying our ancestral gut microbiome. However, relatively little is known about the composition of pre-industrial gut microbiomes. Here we performed a large-scale de novo assembly of microbial genomes from palaeofaeces. From eight authenticated human palaeofaeces samples (1,000–2,000 years old) with well-preserved DNA from southwestern USA and Mexico, we reconstructed 498 medium- and high-quality microbial genomes. Among the 181 genomes with the strongest evidence of being ancient and of human gut origin, 39% represent previously undescribed species-level genome bins. Tip dating suggests an approximate diversification timeline for the key human symbiont Methanobrevibacter smithii. In comparison to 789 present-day human gut microbiome samples from eight countries, the palaeofaeces samples are more similar to non-industrialized than industrialized human gut microbiomes. Functional profiling of the palaeofaeces samples reveals a markedly lower abundance of antibiotic-resistance and mucin-degrading genes, as well as enrichment of mobile genetic elements relative to industrial gut microbiomes. This study facilitates the discovery and characterization of previously undescribed gut microorganisms from ancient microbiomes and the investigation of the evolutionary history of the human gut microbiota through genome reconstruction from palaeofaeces., Ancient microbiomes from palaeofaeces are more similar to non-industrialized than industrialized human gut microbiomes regardless of geography, but 39% of their de novo reconstructed genomes represent previously undescribed microbial species.

Objective Review of the current advancements in the field of paleogenetics that provide new opportunities in studying the evolution of rare genetic bone diseases. Material and Methods Based on cases from the literature, the genetics of rare bone diseases will be introduced and the main methodological issues will be addressed, focusing on the opportunities presented by the application of aDNA analyses in the field of paleopathology. Results Medical literature provides large datasets on the genes responsible for rare bone disorders. These genes, subdivided in functional categories, display important future targets when analyzing rare genetic bone disorders in ancient human remains. Conclusions Knowledge on both phenotype and genotype is required to study rare diseases in ancient human remains. Significance The proposed interdisciplinary research will provide new insight into the occurrence and spread of genetic risk factors in the past and will help in the diagnostics of these rare and often neglected diseases. Limitations The current limitations in ancient DNA research and targeting the disease-causing specific mutations (e.g., somatic or germline). Suggestions for Further Research Methodological advancements and candidate gene lists provide the optimal basis for future interdisciplinary studies of rare genetic bone disorders in ancient human remains.

Although much progress has been made in the study of cell wall biosynthetic genes in the model filamentous fungus Aspergillus nidulans, there are still targets awaiting characterization. An example is the gene celA (ANIA_08444) encoding a putative mixed linkage glucan synthase. To characterize the role of celA, we deleted it in A. nidulans, analyzed the phenotype of the mycelium and performed RNA-Seq. The strain shows a very strong phenotype, namely “balloons” along the hyphae and aberrant conidiophores, as well as an altered susceptibility to cell wall drugs. These data suggest a potential role of the gene in cell wall-related processes. The Gene Ontology term Enrichment analysis shows increased expression of secondary metabolite biosynthetic genes (sterigmatocystin in particular) in the deleted strain. Our results show that the deletion of celA triggers a strong phenotype reminiscent of cell wall-related aberrations and the upregulation of some secondary metabolite gene clusters in A. nidulans.

The Museo Egizio in Turin holds a unique collection of 116 mummified or skeletal human bodies or body parts. This collection is currently under study by the "Mummy Conservation Project", a collaborative venture of the Museo Egizio, the Institute for Mummy Studies of Eurac Research, the Soprintendenza Archeologia del Piemonte, and the Horus Group, whose aim is to improve mummy conservation techniques. In the context of this project, an assessment of the state of preservation of the mummified human remains was carried out by monitoring basic physical parameters (temperature, relative humidity and water activity) and by performing a fungal survey. The latter revealed the presence on the mummified material of fungal spores and mycelia that could possibly pose a biodegradative threat. However, all the current physical parameters show that the mummies are stored under optimal environmental conditions, which will suppress any microbial up-growth. ملخص البحث: يحتوي المتحف المصري في تورينو على مجموعة فريدة تضمّ 116 قطعة ما بين مومياوات بشرية محنطة أو هياكل عظمية أو أجزاء بشرية محنطة. حالياً هذه المجموعة قيد الدراسة من قبل "مشروع الحفاظ على المومياوات"، هو مشروع تعاوني ما بين المتحف المصري ومعهد دراسات المومياء لأبحاث يوراك ومكتب الحفاظ على التراث الأثري في مقاطعة بييمونتي ومجموعة حورس، التي تهدف إلى تحسين تقنيات الحفاظ على المومياوات. في نطاق هذا المشروع، تم تقييم حالة الحفظ للبقايا البشرية المحنطة من خلال مراقبة العوامل الفيزيائية الأساسية (درجة الحرارة والرطوبة النسبية ونشاط الجزيئات المائية) وإجراء مسح للفطور. كشفت هذه الأخيرة عن وجود فطور وبكتريات فطرية على المواد المحنطة والتي يمكن أن تشكل تهديداً بالتحلل الحيوي. ومع ذلك تظهر جميع المقاييس الفيزيائية الحالية أن المومياوات يتم تخزينها في ظل ظروف بيئية مثالية، والتي سوف تثبط أي نمو للميكروبات.

The Tyrolean Iceman, commonly known as Ötzi, is the world’s oldest glacier mummy and one of the best investigated ancient human remains in the world. Since the discovery of the 5,300-year-old Copper Age individual in 1991, in a glacier in the Eastern Italian Alps, a variety of morphological, biochemical, and molecular analyses have been performed that revealed important insights into his origin, his life habits, and the circumstances surrounding his demise. In more recent research, the mummy was subjected to cutting-edge modern research methodologies currently focusing on high-throughput sequence analysis of ancient biomolecules (DNA, proteins, lipids) that are still preserved in the mummified tissues. This application of innovative “-omics” technologies revealed novel insights on the ancestry, disease predisposition, diet, and the presence of pathogens in the glacier mummy. In this review, the most important and actual results of the molecular studies will be highlighted.

Prevotella copri is a common inhabitant of the human gut. Interest in P. copri has gathered pace due to conflicting reports on whether it is beneficial or detrimental to health. In a cross-continent meta-analysis exploiting >6,500 available metagenomes supported by new isolate sequencing and recovery of high-quality genomes from metagenomes, we obtained >1,000 P. copri genomes. This 100-fold increase over existing isolate genomes allowed the genetic and global population structure of P. copri to be explored at an unprecedented depth. We demonstrate P. copri is not a monotypic species, but encompasses four distinct clades (>10% inter-clade vs. P. copri complex, comprising clades A, B, C and D. We show the complex is near ubiquitous in non-Westernised populations (95.4% versus 29.6% in Westernised populations), where all four clades are typically co-present within an individual (61.6% of the cases), in contrast to Westernised populations (4.6%). Genomic analysis of the complex reveals substantial and complementary functional diversity, including the potential for utilisation of complex carbohydrates, suggestive that multi-generational dietary modifications may be a driver for the reduced P. copri prevalence in Westernised populations. Analysis of ancient stool microbiomes highlights a similar pattern of P. copri presence consistent with modern non-Westernised populations, allowing us to estimate the time of clade delineation to pre-date human migratory waves out of Africa. Our analysis reveals P. copri to be far more diverse than previously appreciated and this diversity appears to be underrepresented in Western-lifestyle populations.

We applied ancient DNA methods to shed light on the origin of ancient Hungarians and their relation to modern populations. Hungarians moved into the Carpathian Basin from the Eurasian Pontic steppes in the year 895 AD as a confederation of seven tribes, but their further origin remains obscure. Here, we present 17 mtDNA haplotypes and four Y-chromosome haplogroups, which portray the genetic composition of an entire small cemetery of the first generation Hungarians. Using novel algorithms to compare these mitochondrial DNA haplogroups with other ancient and modern Eurasian data, we revealed that a significant portion of the Hungarians probably originated from a long ago consolidated gene pool in Central Asia-South Siberia, which still persists in modern Hungarians. Another genetic layer of the early Hungarians was obtained during their westward migrations by admixing with various populations of European origin, and an important component of these was derived from the Caucasus region. Most of the modern populations, which are genetically closest relatives of ancient Hungarians, today speak non-Indo-European languages. Our results contribute to our understanding of the peopling of Europe by providing ancient DNA data from a still genetically poorly studied period of medieval human migrations.

Tuberculosis (TB) was a large burden of infections that peaked during the 19th century in Europe. Mummies from the 18th century CE, discovered in the crypt of a church at Vac, Hungary, had high TB prevalence, as revealed by amplification of key fragments of TB DNA and genome-wide TB analysis. Complementary methods are needed to confirm these diagnoses and one approach uses the identification of specific lipid biomarkers, such as TB mycocerosic acids (MCs). Previously, MC derivatives were profiled by specialised gas chromatography-mass spectrometry (GC-MS), so an alternative more direct approach has been developed. Underivatized MCs are extracted and analysed by high-performance liquid chromatography linked to a mass spectrometer, in heated electrospray ionisation mode (HPLC-HESI-MS). The method was validated using representatives of the Mycobacterium tuberculosis complex and other mycobacteria and tested on six Vac mummy cases, previously considered positive for TB infection. Analysing both rib and soft tissue samples, four out of six cases gave profiles of main C32 and major C29 and C39 mycocerosates correlating well with those of M. tuberculosis. Multidisciplinary methods are needed in the diagnosis of ancient tuberculosis; this new protocol accesses important confirmatory evidence, as demonstrated by the confirmation of TB in the Vac mummies.

Summary The history of humankind is marked by the constant adoption of new dietary habits affecting human physiology, metabolism, and even the development of nutrition-related disorders. Despite clear archaeological evidence for the shift from hunter-gatherer lifestyle to agriculture in Neolithic Europe [1], very little information exists on the daily dietary habits of our ancestors. By undertaking a complementary -omics approach combined with microscopy, we analyzed the stomach content of the Iceman, a 5,300-year-old European glacier mummy [2, 3]. He seems to have had a remarkably high proportion of fat in his diet, supplemented with fresh or dried wild meat, cereals, and traces of toxic bracken. Our multipronged approach provides unprecedented analytical depth, deciphering the nutritional habit, meal composition, and food-processing methods of this Copper Age individual., Highlights • The last meal of the Iceman, a European Copper Age mummy, was reconstructed • Our multipronged approach deciphers the meal composition and food processing • His high-fat diet was supplemented with wild meat and cereals, Maixner et al. report the dietary reconstruction of the Iceman’s last meal using a combined multi-omics approach. The stomach content analysis of the 5,300-year-old glacier mummy shows that the Iceman’s diet preceding his death was a mix of carbohydrates, proteins, and lipids, well adjusted to the energetic requirements of his high-altitude trekking.

The paleopathological analysis of a well-preserved young adult female skeleton from the AD 7-8th century (Avar Age) in Hungary revealed multiple lytic lesions in all of the thoracic and lumbar vertebral bodies. The lesions were characterized by smooth marginal zones and space-occupying mass appearance. The considerable loss of spongy bone in the thoracolumbar vertebrae resulted in angular deformity and fusion, characteristic of the healing stage of TB. Osteolytic lesions were also observed on the vertebral processes, ribs and sternum. On the endocranial surface, abnormal blood vessel impressions were revealed, indicating some kind of meningitis. The X-ray and CT analysis of the affected bones detected abnormal structures and cystic zones of destruction. The lesions were however not always bordered by areas of increased density, which is typical in cystic TB. Vertebral remains were also subjected to biomolecular analysis in two different laboratories, which attested the presence of Mycobacterium tuberculosis complex (MTBC) DNA and supported the paleopathological diagnosis of TB. Spoligotyping analysis confirmed the presence of MTBC DNA and more specifically an infection caused by bacteria belonging to the M. tuberculosis lineage. This case study provides new data for the paleoepidemiology of TB in this geographical area and historical period, and draws attention to the great variability of TB lesions in the human skeleton.

Reconstruction of relatedness and ancestry from ancient DNA from Medieval burial provides new insights into kinship behavior., From historical and archeological records, it is posited that the European medieval household was a combination of close relatives and recruits. However, this kinship structure has not yet been directly tested at a genomic level on medieval burials. The early 7th century CE burial at Niederstotzingen, discovered in 1962, is the most complete and richest example of Alemannic funerary practice in Germany. Excavations found 13 individuals who were buried with an array of inscribed bridle gear, jewelry, armor, and swords. These artifacts support the view that the individuals had contact with France, northern Italy, and Byzantium. This study analyzed genome-wide sequences recovered from the remains, in tandem with analysis of the archeological context, to reconstruct kinship and the extent of outside contact. Eleven individuals had sufficient DNA preservation to genetically sex them as male and identify nine unique mitochondrial haplotypes and two distinct Y chromosome lineages. Genome-wide analyses were performed on eight individuals to estimate genetic affiliation to modern west Eurasians and genetic kinship at the burial. Five individuals were direct relatives. Three other individuals were not detectably related; two of these showed genomic affinity to southern Europeans. The genetic makeup of the individuals shares no observable pattern with their orientation in the burial or the cultural association of their grave goods, with the five related individuals buried with grave goods associated with three diverse cultural origins. These findings support the idea that not only were kinship and fellowship held in equal regard: Diverse cultural appropriation was practiced among closely related individuals as well.

Paleogenetics offers a unique opportunity to study human evolution, population dynamics, and disease evolution in situ. Although histologic and computed x-ray tomographic investigations of ancient mummies have clearly shown that atherosclerosis has been present in humans for more than 5,000 years, limited data are available on the presence of genetic predisposition for cardiovascular disease in ancient human populations. In a previous whole-genome study of the Tyrolean Iceman, a 5,300-year-old glacier mummy from the Alps, an increased risk for coronary heart disease was detected. The Iceman's genome revealed several single nucleotide polymorphisms that are linked with cardiovascular disease in genome-wide association studies. Future genetic studies of ancient humans from various geographic origins and time periods have the potential to provide more insights into the presence and possible changes of genetic risk factors in our ancestors. The study of ancient humans and a better understanding of the interaction between environmental and genetic influences on the development of heart diseases may lead to a more effective prevention and treatment of the most common cause of death in the modern world.HighlightsOldest evidence on the presence of genetic predisposition for cardiovascular disease in the 5,300-year-old Tyrolean Iceman.Future genetic studies of ancient humans have the potential to provide more insights into the presence and possible changes of genetic risk factors in our ancestors.The study of ancient humans and a better understanding of the interaction between environmental and genetic influences on the development of heart diseases may lead to more effective prevention and treatment.

Summary Prevotella copri is a common human gut microbe that has been both positively and negatively associated with host health. In a cross-continent meta-analysis exploiting >6,500 metagenomes, we obtained >1,000 genomes and explored the genetic and population structure of P. copri. P. copri encompasses four distinct clades (>10% inter-clade genetic divergence) that we propose constitute the P. copri complex, and all clades were confirmed by isolate sequencing. These clades are nearly ubiquitous and co-present in non-Westernized populations. Genomic analysis showed substantial functional diversity in the complex with notable differences in carbohydrate metabolism, suggesting that multi-generational dietary modifications may be driving reduced prevalence in Westernized populations. Analysis of ancient metagenomes highlighted patterns of P. copri presence consistent with modern non-Westernized populations and a clade delineation time pre-dating human migratory waves out of Africa. These findings reveal that P. copri exhibits a high diversity that is underrepresented in Western-lifestyle populations., Graphical Abstract, Highlights • P. copri is not a monotypic species but composed of four distinct clades • The P. copri complex is more prevalent in populations with non-Westernized lifestyles • P. copri clades are frequently co-present within non-Westernized individuals • Ancient stool samples suggest Westernization leads to P. copri underrepresentation, Tett et al. find that the intestinal microbe Prevotella copri encompasses four distinct clades constituting the P. copri complex. The complex is prevalent in non-Westernized populations where co-presence of all clades is commonly observed within individuals. Analysis of ancient stool samples supports Westernization as contributing to reduced P. copri prevalence.

Summary Nitrospira are the most widespread and diverse known nitrite-oxidizing bacteria and key nitrifiers in natural and engineered ecosystems. Nevertheless, their ecophysiology and environmental distribution are understudied because of the recalcitrance of Nitrospira to cultivation and the lack of a molecular functional marker, which would allow the detection of Nitrospira in the environment. Here we introduce nxrB, the gene encoding subunit beta of nitrite oxidoreductase, as a functional and phylogenetic marker for Nitrospira. Phylogenetic trees based on nxrB of Nitrospira were largely congruent to 16S ribosomal RNA-based phylogenies. By using new nxrB-selective polymerase chain reaction primers, we obtained almost full-length nxrB sequences from Nitrospira cultures, two activated sludge samples, and several geographically and climatically distinct soils. Amplicon pyrosequencing of nxrB fragments from 16 soils revealed a previously unrecognized diversity of terrestrial Nitrospira with 1801 detected species-level operational taxonomic units (OTUs) (using an inferred species threshold of 95% nxrB identity). Richness estimates ranged from 10 to 946 coexisting Nitrospira species per soil. Comparison with an archaeal amoA dataset obtained from the same soils [Environ. Microbiol. 14: 525–539 (2012)] uncovered that ammonia-oxidizing archaea and Nitrospira communities were highly correlated across the soil samples, possibly indicating shared habitat preferences or specific biological interactions among members of these nitrifier groups.

The Tyrolean Iceman, a Copper-age ice mummy, is one of the best-studied human individuals. While the genome of the Iceman has largely been decoded, tissue-specific proteomes have not yet been investigated. We studied the proteome of two distinct brain samples using gel-based and liquid chromatography-mass spectrometry-based proteomics technologies together with a multiple-databases and -search algorithms-driven data-analysis approach. Thereby, we identified a total of 502 different proteins. Of these, 41 proteins are known to be highly abundant in brain tissue and 9 are even specifically expressed in the brain. Furthermore, we found 10 proteins related to blood and coagulation. An enrichment analysis revealed a significant accumulation of proteins related to stress response and wound healing. Together with atomic force microscope scans, indicating clustered blood cells, our data reopens former discussions about a possible injury of the Iceman's head near the site where the tissue samples have been extracted.

Sommersdorf Castle (Bavaria, Germany) is a medieval castle complex which has been inhabited by the aristocratic family von Crailsheim. The deceased were entombed in a crypt located in the parapets underneath the castle's church, resulting in mummification of the bodies. Based on the family chronicle and oral history, identities have been ascribed to the mummies. The aim of the study is therefore to test the accuracy of the historical records in comparison to archaeological, anthropological and genetic data. Today, the crypt houses eleven wooden coffins from the 17th to 19th century AD. In ten of these, mummified and scattered human remains were found. Archive records were studied in order to identify names, ancestry, titles, occupation, date of birth and death, and place of interment of the individuals. The coffins were visually inspected and dated by typo-chronology, and the mummified and scattered skeletal remains were subjected to a physical anthropological examination. In total, the crypt contains the remains of a minimum number of nine individuals, among them three adult males, five adult females and one infant. A detailed scientific examination, including prior conservation, ancient DNA analyses, and computed tomography (CT), was performed on five mummies. By means of the CT data age at death, sex, body height, pathologies, and anatomical variants were investigated. CT analysis further showed that the bodies were naturally mummified. Mitochondrial DNA analyses revealed that the tested individuals are not maternally related. In addition, health, living conditions and circumstances of death of the entombed individuals could be highlighted. Being confronted with the strengths, weaknesses and limitations of each methodological approach, probable identification was achieved in two cases.

The analysis of nucleic acids in ancient samples is largely limited to DNA. Small noncoding RNAs (microRNAs) are known to be evolutionary conserved and stable. To gain knowledge on miRNAs measured from ancient samples, we profiled microRNAs in cryoconserved mummies. First, we established the approach on a World War One warrior, the "Kaiserjager", which has been preserved for almost one century. Then, we profiled seven ancient tissue specimens including skeletal muscle, stomach mucosa, stomach content and two corpus organ tissues of the 5,300-year-old copper age mummy Iceman and compared these profiles to the presence of organ-specific miRNAs in modern tissues. Our analyses suggest the presence of specific miRNAs in the different Iceman's tissues. Of 1,066 analyzed human miRNAs, 31 were discovered across all biopsies and 87 miRNAs were detected only in a single sample. To check for potential microbiological contaminations, all miRNAs detected in Iceman samples and not present in ancient samples were mapped to 14,582 bacterial and viral genomes. We detected few hits (3.9% of miRNAs compared with 3.6% of miRNAs). Interestingly, the miRNAs with higher abundance across all ancient tissues were significantly enriched for Guanine (P value of 10-13) and Cytosine (P value of 10-7). The same pattern was observed for modern tissues. Comparing miRNAs measured from ancient organs to modern tissue patterns highlighted significant similarities, e.g., for miRNAs present in the muscle. Our first comprehensive analysis of microRNAs in ancient human tissues indicates that these stable molecules can be detected in tissue specimens after 5,300 years.

The attire of the Tyrolean Iceman, a 5,300-year-old natural mummy from the Ötzal Italian Alps, provides a surviving example of ancient manufacturing technologies. Research into his garments has however, been limited by ambiguity surrounding their source species. Here we present a targeted enrichment and sequencing of full mitochondrial genomes sampled from his clothes and quiver, which elucidates the species of production for nine fragments. Results indicate that the majority of the samples originate from domestic ungulate species (cattle, sheep and goat), whose recovered haplogroups are now at high frequency in today’s domestic populations. Intriguingly, the hat and quiver samples were produced from wild species, brown bear and roe deer respectively. Combined, these results suggest that Copper Age populations made considered choices of clothing material from both the wild and domestic populations available to them. Moreover, these results show the potential for the recovery of complete mitochondrial genomes from degraded prehistoric artefacts.

Modern next generation sequencing technologies produce vast amounts of data in the context of large-scale metagenomic studies, in which complex microbial communities can be reconstructed to an unprecedented level of detail. Most prominent examples are human microbiome studies that correlate the bacterial taxonomic profile with specific physiological conditions or diseases.In order to perform these analyses high-throughput computational tools are needed that are able to process these data within a short time while preserving a high level of sensitivity and specificity.Here we present MALT (MEGAN ALignment Tool) a program for the ultrafast alignment and analysis of metagenomic DNA sequencing data. MALT processes hundreds of millions of sequencing reads within only a few hours. In addition to the alignment procedure MALT implements a taxonomic binning algorithm that is able to specifically assign reads to bacterial species. Its tight integration with the interactive metagenomic analysis software MEGAN allows for visualization and further analyses of results.We demonstrate MALT by its application to the metagenomic analysis of two ancient microbiomes from oral cavity and lung samples of the 5,300-year-old Tyrolean Iceman. Despite the strong environmental background, MALT is able to pick up the weak signal of the original microbiomes and identifies multiple species that are typical representatives of the respective host environment.