Reconstructing Denisovan Anatomy Using DNA Methylation Maps.

Denisovans are an extinct group of humans whose morphology remains unknown. Here, we present a method for reconstructing skeletal morphology using DNA methylation patterns. Our method is based on linking unidirectional methylation changes to loss-of-function phenotypes. We tested performance by reconstructing Neanderthal and chimpanzee skeletal morphologies and obtained >85% precision in identifying divergent traits. We then applied this method to the Denisovan and offer a putative morphological profile. We suggest that Denisovans likely shared with Neanderthals traits such as an elongated face and a wide pelvis. We also identify Denisovan-derived changes, such as an increased dental arch and lateral cranial expansion. Our predictions match the only morphologically informative Denisovan bone to date, as well as the Xuchang skull, which was suggested by some to be a Denisovan. We conclude that DNA methylation can be used to reconstruct anatomical features, including some that do not survive in the fossil record.

Ancient Human Genomes and Environmental DNA from the Cement Attaching 2,000-Year-Old Head Lice Nits.

Over the past few decades, there has been a growing demand for genome analysis of ancient human remains. Destructive sampling is increasingly difficult to obtain for ethical reasons, and standard methods of breaking the skull to access the petrous bone or sampling remaining teeth are often forbidden for curatorial reasons. However, most ancient humans carried head lice and their eggs abound in historical hair specimens. Here we show that host DNA is protected by the cement that glues head lice nits to the hair of ancient Argentinian mummies, 1,500-2,000 years old. The genetic affinities deciphered from genome-wide analyses of this DNA inform that this population migrated from north-west Amazonia to the Andes of central-west Argentina; a result confirmed using the mitochondria of the host lice. The cement preserves ancient environmental DNA of the skin, including the earliest recorded case of Merkel cell polyomavirus. We found that the percentage of human DNA obtained from nit cement equals human DNA obtained from the tooth, yield 2-fold compared with a petrous bone, and 4-fold to a bloodmeal of adult lice a millennium younger. In metric studies of sheaths, the length of the cement negatively correlates with the age of the specimens, whereas hair linear distance between nit and scalp informs about the environmental conditions at the time before death. Ectoparasitic lice sheaths can offer an alternative, nondestructive source of high-quality ancient DNA from a variety of host taxa where bones and teeth are not available and reveal complementary details of their history.

Analysis of oral microbiome from fossil human remains revealed the significant differences in virulence factors of modern and ancient Tannerella forsythia.

BACKGROUND: Recent advances in the next-generation sequencing (NGS) allowed the metagenomic analyses of DNA from many different environments and sources, including thousands of years old skeletal remains. It has been shown that most of the DNA extracted from ancient samples is microbial. There are several reports demonstrating that the considerable fraction of extracted DNA belonged to the bacteria accompanying the studied individuals before their death. RESULTS: In this study we scanned 344 microbiomes from 1000- and 2000- year-old human teeth. The datasets originated from our previous studies on human ancient DNA (aDNA) and on microbial DNA accompanying human remains. We previously noticed that in many samples infection-related species have been identified, among them Tannerella forsythia, one of the most prevalent oral human pathogens. Samples containing sufficient amount of T. forsythia aDNA for a complete genome assembly were selected for thorough analyses. We confirmed that the T. forsythia-containing samples have higher amounts of the periodontitis-associated species than the control samples. Despites, other pathogens-derived aDNA was found in the tested samples it was too fragmented and damaged to allow any reasonable reconstruction of these bacteria genomes. The anthropological examination of ancient skulls from which the T. forsythia-containing samples were obtained revealed the pathogenic alveolar bone loss in tooth areas characteristic for advanced periodontitis. Finally, we analyzed the genetic material of ancient T. forsythia strains. As a result, we assembled four ancient T. forsythia genomes - one 2000- and three 1000- year-old. Their comparison with contemporary T. forsythia genomes revealed a lower genetic diversity within the four ancient strains than within contemporary strains. We also investigated the genes of T. forsythia virulence factors and found that several of them (KLIKK protease and bspA genes) differ significantly between ancient and modern bacteria. CONCLUSIONS: In summary, we showed that NGS screening of the ancient human microbiome is a valid approach for the identification of disease-associated microbes. Following this protocol, we provided a new set of information on the emergence, evolution and virulence factors of T. forsythia, the member of the oral dysbiotic microbiome.

Ancient DNA reveals the chronology of walrus ivory trade from Norse Greenland.

The importance of the Atlantic walrus ivory trade for the colonization, peak, and collapse of the medieval Norse colonies on Greenland has been extensively debated. Nevertheless, no studies have directly traced medieval European ivory back to distinct Arctic populations of walrus. Analysing the entire mitogenomes of 37 archaeological specimens from Europe, Svalbard, and Greenland, we here discover that Atlantic walrus comprises two monophyletic mitochondrial (MT) clades, which diverged between 23 400 and 251 120 years ago. Our improved genomic resolution allows us to reinterpret the geographical distribution of partial MT data from 306 modern and nineteenth-century specimens, finding that one of these clades was exclusively accessible to Greenlanders. With this discovery, we ascertain the biological origin of 23 archaeological specimens from Europe (most dated between 900 and 1400 CE). These results reveal a significant shift in trade from an early, predominantly eastern source towards a near exclusive representation of Greenland ivory. Our study provides empirical evidence for how this remote Arctic resource was progressively integrated into a medieval pan-European trade network, contributing to both the resilience and vulnerability of Norse Greenland society.

Bioarchaeological investigation of individuals with suspected multibacillary leprosy from the mediaeval leprosarium of St Mary Magdalen, Winchester, Hampshire, UK.

Introduction. We have examined four burials from the St Mary Magdalen mediaeval leprosarium cemetery in Winchester, Hampshire, UK. One (Sk.8) was a male child, two (Sk.45 and Sk.52) were adolescent females and the fourth (Sk.512) was an adult male. The cemetery was in use between the 10th and 12th centuries. All showed skeletal lesions of leprosy. Additionally, one of the two females (Sk.45) had lesions suggestive of multi-cystic tuberculosis and the second (Sk.52) of leprogenic odontodysplasia (LO), a rare malformation of the roots of the permanent maxillary incisors.Gap statement. Relatively little is known of the manifestations of lepromatous leprosy (LL) in younger individuals from the archaeological record.Aims and Methodology. To address this, we have used ancient DNA testing and osteological examination of the individuals, supplemented with X-ray and microcomputed tomography (micro-CT) scan as necessary to assess the disease status.Results and Conclusions. The presence of Mycobacterium leprae DNA was confirmed in both females, and genotyping showed SNP type 3I-1 strains but with a clear genotypic variation. We could not confirm Mycobacterium tuberculosis complex DNA in the female individual SK.45. High levels of M. leprae DNA were found within the pulp cavities of four maxillary teeth from the male child (Sk.8) with LO, consistent with the theory that the replication of M. leprae in alveolar bone may interfere with root formation at key stages of development. We report our biomolecular findings in these individuals and review the evidence this site has contributed to our knowledge of mediaeval leprosy.

Ancient human dental calculus metadata collection and sampling strategies: Recommendations for best practices.

OBJECTIVES: Ancient human dental calculus is a unique, nonrenewable biological resource encapsulating key information about the diets, lifestyles, and health conditions of past individuals and populations. With compounding calls its destructive analysis, it is imperative to refine the ways in which the scientific community documents, samples, and analyzes dental calculus so as to maximize its utility to the public and scientific community. MATERIALS AND METHODS: Our research team conducted an IRB-approved survey of dental calculus researchers with diverse academic backgrounds, research foci, and analytical specializations. RESULTS: This survey reveals variation in how metadata is collected and utilized across different subdisciplines and highlights how these differences have profound implications for dental calculus research. Moreover, the survey suggests the need for more communication between those who excavate, curate, and analyze biomolecular data from dental calculus. DISCUSSION: Challenges in cross-disciplinary communication limit researchers' ability to effectively utilize samples in rigorous and reproducible ways. Specifically, the lack of standardized skeletal and dental metadata recording and contamination avoidance procedures hinder downstream anthropological applications, as well as the pursuit of broader paleodemographic and paleoepidemiological inquiries that rely on more complete information about the individuals sampled. To provide a path forward toward more ethical and standardized dental calculus sampling and documentation approaches, we review the current methods by which skeletal and dental metadata are recorded. We also describe trends in sampling and contamination-control approaches. Finally, we use that information to suggest new guidelines for ancient dental calculus documentation and sampling strategies that will improve research practices in the future.

Bioanthropological analysis of human remains from the archaic and classic period discovered in Puyil cave, Mexico.

OBJECTIVES: Determine the geographic place of origin and maternal lineage of prehistoric human skeletal remains discovered in Puyil Cave, Tabasco State, Mexico, located in a region currently populated by Olmec, Zoque and Maya populations. MATERIALS AND METHODS: All specimens were radiocarbon (14C) dated (beta analytic), had dental modifications classified, and had an analysis of 13 homologous reference points conducted to evaluate artificial cranial deformation (ACD). Following DNA purification, hypervariable region I (HVR-1) of the mitogenome was amplified and Sanger sequenced. Finally, Next Generation Sequencing (NGS) was performed for total DNA. Mitochondrial DNA (mtDNA) variants and haplogroups were determined using BioEdit 7.2 and IGV software and confirmed with MITOMASTER and WebHome softwares. RESULTS: Radiocarbon dating (14C) demonstrated that the inhabitants of Puyil Cave lived during the Archaic and Classic Periods and displayed tabular oblique and tabular mimetic ACD. These pre-Hispanic remains exhibited five mtDNA lineages: A, A2, C1, C1c and D4. Network analysis revealed a close genetic affinity between pre-Hispanic Puyil Cave inhabitants and contemporary Maya subpopulations from Mexico and Guatemala, as well as individuals from Bolivia, Brazil, the Dominican Republic, and China. CONCLUSIONS: Our results elucidate the dispersal of pre-Hispanic Olmec and Maya ancestors and suggest that ACD practices are closely related to Olmec and Maya practices. Additionally, we conclude that ACD has likely been practiced in the region since the Middle-Archaic Period.

Using loop-primer mediated PCR to enhance the detection of poorly preserved DNA.

Ancient DNA is vitally important in evolutionary research, and obtaining authentic ancient DNA sequences is critical for a proper analysis. However, it is difficult to acquire amplicons accurately and efficiently from ancient DNA templates using current techniques. Here, we established a loop-primer-mediated amplification method (L-PCR) to obtain target ancient DNA sequences with high accuracy and efficiency. The method was tested using 66 ancient samples (including 27 pig bones or teeth and 39 chicken bones) and serially diluted modern animal DNA templates. Compared to nested PCR, L-PCR was proven to be more efficient and accurate and could obtain more amplicons from both ancient pig samples and chicken bones and detect as low as 10-3 ng/µl modern pig template DNA. The efficiency was at least 100-fold that of the nested PCR. The results suggest that L-PCR is advantageous for obtaining authentic DNA sequences from poorly preserved or recalcitrant ancient specimens.

The elusive parasite: comparing macroscopic, immunological, and genomic approaches to identifying malaria in human skeletal remains from Sayala, Egypt (third to sixth centuries AD).

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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12520-021-01350-z.

PyDamage: automated ancient damage identification and estimation for contigs in ancient DNA de novo assembly.

DNA de novo assembly can be used to reconstruct longer stretches of DNA (contigs), including genes and even genomes, from short DNA sequencing reads. Applying this technique to metagenomic data derived from archaeological remains, such as paleofeces and dental calculus, we can investigate past microbiome functional diversity that may be absent or underrepresented in the modern microbiome gene catalogue. However, compared to modern samples, ancient samples are often burdened with environmental contamination, resulting in metagenomic datasets that represent mixtures of ancient and modern DNA. The ability to rapidly and reliably establish the authenticity and integrity of ancient samples is essential for ancient DNA studies, and the ability to distinguish between ancient and modern sequences is particularly important for ancient microbiome studies. Characteristic patterns of ancient DNA damage, namely DNA fragmentation and cytosine deamination (observed as C-to-T transitions) are typically used to authenticate ancient samples and sequences, but existing tools for inspecting and filtering aDNA damage either compute it at the read level, which leads to high data loss and lower quality when used in combination with de novo assembly, or require manual inspection, which is impractical for ancient assemblies that typically contain tens to hundreds of thousands of contigs. To address these challenges, we designed PyDamage, a robust, automated approach for aDNA damage estimation and authentication of de novo assembled aDNA. PyDamage uses a likelihood ratio based approach to discriminate between truly ancient contigs and contigs originating from modern contamination. We test PyDamage on both on simulated aDNA data and archaeological paleofeces, and we demonstrate its ability to reliably and automatically identify contigs bearing DNA damage characteristic of aDNA. Coupled with aDNA de novo assembly, Pydamage opens up new doors to explore functional diversity in ancient metagenomic datasets.

Tuberculosis infection in a late-medieval Hungarian population.

The AD 16-17(th) century skeletal series from Bácsalmás-Óalmás (southern Hungary) has already been the subject of previous paleopathological studies concerning TB-related bone lesions. Due to recent development of macroscopic and molecular diagnostic methods in paleopathology and paleomicrobiology, a five-year international research program was recently started in order to re-evaluate the TB-related lesions in the complete series, comprising 481 skeletons. The skeletal material of these individuals was examined using macromorphological methods focusing on both classical/advanced stage skeletal TB alterations and atypical/early-stage TB lesions. Paleomicrobial analysis was used to study the presence of Mycobacterium tuberculosis complex (MTBC) DNA both in morphologically positive and negative cases. Samples were tested for the repetitive element IS6110 and further characterized by spoligotyping. In the whole series, 283 possible cases of TB infections were identified based on morphological alterations. Skeletal samples of eighteen individuals, morphologically positive as well as negative cases, were selected for further biomolecular examinations. Among them, seven individuals were PCR positive for the repetitive IS6110 sequence of the MTBC genome. Compared to the few cases of TB from the Bácsalmás-Óalmás series previously described, a much higher prevalence of MTBC infected skeletons was revealed in this study. The atypical/early stage skeletal lesions occurred significantly more frequently than the so-called classical alterations. Paleomicrobial analysis confirmed a prevalence of MTBC infection nearing 40% among the selected sample. Preliminary results also indicated better preservation of bacterial DNA in the compact layer of long bones and teeth, while spoligotyping suggested infection by different MTBC pathogens.