Reply to Barton et al: signatures of natural selection during the Black Death.

Barton et al.1 raise several statistical concerns regarding our original analyses2 that highlight the challenge of inferring natural selection using ancient genomic data. We show here that these concerns have limited impact on our original conclusions. Specifically, we recover the same signature of enrichment for high FST values at the immune loci relative to putatively neutral sites after switching the allele frequency estimation method to a maximum likelihood approach, filtering to only consider known human variants, and down-sampling our data to the same mean coverage across sites. Furthermore, using permutations, we show that the rs2549794 variant near ERAP2 continues to emerge as the strongest candidate for selection (p = 1.2×10-5), falling below the Bonferroni-corrected significance threshold recommended by Barton et al. Importantly, the evidence for selection on ERAP2 is further supported by functional data demonstrating the impact of the ERAP2 genotype on the immune response to Y. pestis and by epidemiological data from an independent group showing that the putatively selected allele during the Black Death protects against severe respiratory infection in contemporary populations.

Mapping bone microstructure trace element composition: Sr, Ba, Cu, and Pb in an early modern Danish skeleton.

OBJECTIVES: A Geographical Information System (GIS) approach enhances the acquisition, management, and analysis of trace element data from cortical bone. A high-resolution spatial dimension expands the research potential of Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) data from cortical bone cross-sections. The chemical characterization of hundreds of osteons, notably sequences of superimposed osteons, permits more exacting studies of individual life histories than is possible with analyses of bulk bone samples. METHODS: A GIS procedure was used to estimate Sr, Ba, Pb, and Cu concentrations, originally generated through LA-ICP-MS, for bone microstructural features, notably fragmentary and intact osteons, in a human femoral cross-section. The skeleton is from Ribe, Denmark, and dates to the early modern period. RESULTS: Postmortem chemical alteration was limited to the bone's outer and inner margins. Two dietary indicators, Sr and Ba, and two socioeconomic indicators, Pb and Cu, measured for individual osteons were correlated with one another. Osteon sequences indicate concentrations of all four elements increased late in life for this individual. CONCLUSIONS: The application of GIS procedures expedites fine-grained analyses of variation in the distribution of trace elements in bone microstructure identifiable in cortical bone cross-sections. It provides an efficient means of extracting the most information possible from LA-ICP-MS data about the lives of people in the past. Combining the two procedures makes it easier to track exposure to elements such as Pb across the part of an individual's life represented by osteon sequences.

Emergence, continuity, and evolution of Yersinia pestis throughout medieval and early modern Denmark.

The historical epidemiology of plague is controversial due to the scarcity and ambiguity of available data.1,2 A common source of debate is the extent and pattern of plague re-emergence and local continuity in Europe during the 14th-18th century CE.3 Despite having a uniquely long history of plague (∼5,000 years), Scandinavia is relatively underrepresented in the historical archives.4,5 To better understand the historical epidemiology and evolutionary history of plague in this region, we performed in-depth (n = 298) longitudinal screening (800 years) for the plague bacterium Yersinia pestis (Y. pestis) across 13 archaeological sites in Denmark from 1000 to 1800 CE. Our genomic and phylogenetic data captured the emergence, continuity, and evolution of Y. pestis in this region over a period of 300 years (14th-17th century CE), for which the plague-positivity rate was 8.3% (3.3%-14.3% by site). Our phylogenetic analysis revealed that the Danish Y. pestis sequences were interspersed with those from other European countries, rather than forming a single cluster, indicative of the generation, spread, and replacement of bacterial variants through communities rather than their long-term local persistence. These results provide an epidemiological link between Y. pestis and the unknown pestilence that afflicted medieval and early modern Europe. They also demonstrate how population-scale genomic evidence can be used to test hypotheses on disease mortality and epidemiology and help pave the way for the next generation of historical disease research.

Using SXRF and LA-ICP-TOFMS to Explore Evidence of Treatment and Physiological Responses to Leprosy in Medieval Denmark.

Leprosy can lead to blood depletion in Zn, Ca, Mg, and Fe and blood enrichment in Cu. In late medieval Europe, minerals were used to treat leprosy. Here, physiological responses to leprosy and possible evidence of treatment are investigated in enamel, dentine, and cementum of leprosy sufferers from medieval Denmark (n = 12) and early 20th century Romania (n = 2). Using SXRF and LA-ICP-TOFMS, 12 elements were mapped in 15 tooth thin sections, and the statistical covariation of paired elements was computed to assess their biological relevance. The results show marked covariations in the Zn, Ca, and Mg distributions, which are compatible with clinical studies but cannot be directly attributed to leprosy. Minerals used historically as a treatment for leprosy show no detectable intake (As, Hg) or a diffuse distribution (Pb) related to daily ingestion. Intense Pb enrichments indicate acute incorporations of Pb, potentially through the administration of Pb-enriched medication or the mobilization of Pb from bone stores to the bloodstream during intense physiological stress related to leprosy. However, comparisons with a healthy control group are needed to ascertain these interpretations. The positive correlations and the patterns observed between Pb and essential elements may indicate underlying pathophysiological conditions, demonstrating the potential of SXRF and LA-ICP-TOFMS for paleopathological investigations.

Population trends and the transition to agriculture: Global processes as seen from North America.

Agriculture-specifically an intensification of the production of readily stored food and its distribution-has supported an increase in the global human population throughout the Holocene. Today, with greatly accelerated of growth during recent centuries, we have reached about 8 billion people. Human skeletal and archaeobotanical remains clarify what occurred over several millennia of profound societal and population change in small-scale societies once distributed across the North American midcontinent. Stepwise, not gradual, changes in the move toward an agriculturally based life, as indicated by plant remains, left a demographic signal reflecting age-independent ([Formula: see text]) mortality as estimated from skeletons. Designated the age-independent component of the Siler model, it is tracked through the juvenility index (JI), which is increasingly being used in studies of archaeological skeletons. Usually interpreted as a fertility indicator, the JI is more responsive to age-independent mortality in societies that dominated most of human existence. In the midcontinent, the JI increased as people transitioned to a more intensive form of food production that prominently featured maize. Several centuries later, the JI declined, along with a reversion to a somewhat more diverse diet and a reduction in overall population size. Changes in age-independent mortality coincided with previously recognized increases in intergroup conflict, group movement, and pathogen exposure. Similar rises and falls in JI values have been reported for other parts of the world during the emergence of agricultural systems.

Evolution of immune genes is associated with the Black Death.

Infectious diseases are among the strongest selective pressures driving human evolution1,2. This includes the single greatest mortality event in recorded history, the first outbreak of the second pandemic of plague, commonly called the Black Death, which was caused by the bacterium Yersinia pestis3. This pandemic devastated Afro-Eurasia, killing up to 30-50% of the population4. To identify loci that may have been under selection during the Black Death, we characterized genetic variation around immune-related genes from 206 ancient DNA extracts, stemming from two different European populations before, during and after the Black Death. Immune loci are strongly enriched for highly differentiated sites relative to a set of non-immune loci, suggesting positive selection. We identify 245 variants that are highly differentiated within the London dataset, four of which were replicated in an independent cohort from Denmark, and represent the strongest candidates for positive selection. The selected allele for one of these variants, rs2549794, is associated with the production of a full-length (versus truncated) ERAP2 transcript, variation in cytokine response to Y. pestis and increased ability to control intracellular Y. pestis in macrophages. Finally, we show that protective variants overlap with alleles that are today associated with increased susceptibility to autoimmune diseases, providing empirical evidence for the role played by past pandemics in shaping present-day susceptibility to disease.

Paleodemography: From archaeology and skeletal age estimation to life in the past.

Much of paleodemography, an interdisciplinary field with strong ties to archaeology, among other disciplines, is oriented toward clarifying the life experiences of past people and why they changed over time. We focus on how human skeletons contribute to our understanding of preindustrial demographic regimes, including when changes took place that led to the world as we know it today. Problems with existing paleodemographic practices are highlighted, as are promising directions for future work. The latter requires both better age estimates and innovative methods to handle data appropriately. Age-at-death estimates for adult skeletons are a particular problem, especially for adults over 50 years that undoubtedly are mistakenly underrepresented in published studies of archaeological skeletons. Better age estimates for the entirety of the lifespan are essential to generate realistic distributions of age at death. There are currently encouraging signs that after about a half-century of intensive, and sometimes contentious, research, paleodemography is poised to contribute much to understandings of evolutionary processes, the structure of past populations, and human-disease interaction, among other topics.

Leprosy in medieval Denmark: Exploring life histories through a multi-tissue and multi-isotopic approach.

OBJECTIVES: By focusing on two Danish leprosaria (Naestved and Odense; 13th-16th c. CE) and using diet and origin as proxies, we follow a multi-isotopic approach to reconstruct life histories of patients and investigate how leprosy affected both institutionalized individuals and the medieval Danish community as a whole. MATERIALS AND METHODS: We combine archaeology, historical sources, biological anthropology, isotopic analyses (δ13 C, δ15 N, δ34 S, 87 Sr/86 Sr) and radiocarbon dating, and further analyze bones with different turnover rates (ribs and long bones). RESULTS: The δ13 C, δ15 N and δ34 S results indicate a C3 terrestrial diet with small contributions of marine protein for leprosy patients and individuals from other medieval Danish sites. A similar diet is seen through time, between males and females, and patients with and without changes on facial bones. The isotopic comparison between ribs and long bones reveals no significant dietary change. The δ34 S and 87 Sr/86 Sr results suggest that patients were local to the regions of the leprosaria. Moreover, the radiocarbon dates show a mere 50% agreement with the arm position dating method used in Denmark. CONCLUSIONS: A local origin for the leprosy patients is in line with historical evidence, unlike the small dietary contribution of marine protein. Although only 10% of the analyzed individuals have rib/long bone offsets that undoubtedly show a dietary shift, the data appear to reveal a pattern for 25 individuals (out of 50), with elevated δ13 C and/or δ15 N values in the ribs compared to the long bones, which points toward a communal type of diet and reveals organizational aspects of the institution.

Transition analysis applied to third molar development in a Danish population.

INTRODUCTION: Age assessment based on dental development is often requested in order to assess whether an individual is older or younger than 18 years of age. There are several statistical approaches to estimate age based upon third molar development. The aim of this study was to apply the principles of transition analysis (TA) to a Danish reference material and to evaluate whether it was indicated to include a model that allows for logistic non-linearity as opposed to applying a model only allowing for logistic linearity. For this we chose to use the generalized additive model (gam) and the generalized linear model (glm), respectively. MATERIAL AND METHOD: A cross-sectional sample comprising 1302 panoramic radiographs of Danish subjects in the chronological age range of 13-25 years was included. All present third molars had been scored according to the 10-stage method of Gleiser and Hunt. Each transition from one stage to the subsequent stage was analyzed according to the statistical approach of TA and fitted with both the generalized linear model (glm) and the generalized additive model (gam). In order to assess whether gam or glm was more parsimonious for each transition individually, the Akaikon information criterion (AIC) was applied. RESULTS: The results emphasized the importance of applying a statistical model that sufficiently captures the spread of the age estimate. The AIC values showed that some transitions were sufficiently described by glm whereas for others the gam curves fitted significantly better. CONCLUSION: We recommend that for an age assessment tool based on TA, both a fitting allowing for non-linearity and one allowing only for linearity should be included.

Genetic resiliency and the Black Death: No apparent loss of mitogenomic diversity due to the Black Death in medieval London and Denmark.

OBJECTIVES: In the 14th century AD, medieval Europe was severely affected by the Great European Famine as well as repeated bouts of disease, including the Black Death, causing major demographic shifts. This high volatility led to increased mobility and migration due to new labor and economic opportunities, as evidenced by documentary and stable isotope data. This study uses ancient DNA (aDNA) isolated from skeletal remains to examine whether evidence for large-scale population movement can be gleaned from the complete mitochondrial genomes of 264 medieval individuals from England (London) and Denmark. MATERIALS AND METHODS: Using a novel library-conserving approach to targeted capture, we recovered 264 full mitochondrial genomes from the petrous portion of the temporal bones and teeth and compared genetic diversity across the medieval period within and between English (London) and Danish populations and with contemporary populations through population pairwise ΦST analysis. RESULTS: We find no evidence of significant differences in genetic diversity spatially or temporally in our dataset, yet there is a high degree of haplotype diversity in our medieval samples with little exact sequence sharing. DISCUSSION: The mitochondrial genomes of both medieval Londoners and medieval Danes suggest high mitochondrial diversity before, during and after the Black Death. While our mitochondrial genomic data lack geographically correlated signals, these data could be the result of high, continual female migration before and after the Black Death or may simply indicate a large female effective population size unaffected by the upheaval of the medieval period. Either scenario suggests a genetic resiliency in areas of northwestern medieval Europe.

Meeting Report: The Role of Beliefs and Perception on Body Size. Proceedings of the 26th Aschauer Soiree, Held at Aschauhof, Altenhof, Germany, May 26th, 2018.

Thirty-one scientists met at Aschauhof, Germany to discuss the role of beliefs and self-perception on body size. In view of apparent growth stimulatory effects of dominance within the social group that is observed in social mammals, they discussed various aspects of competitive growth strategies and growth adjustments. Presentations included new data from Indonesia, a cohort-based prospective study from Merida, Yucatan, and evidence from recent meta-analyses and patterns of growth in the socially deprived. The effects of stress experienced during pregnancy and adverse childhood events were discussed, as well as obesity in school children, with emphasis on problems when using z-scores in extremely obese children. Aspects were presented on body image in African-American women, and body perception and the disappointments of menopause in view of feelings of attractiveness in different populations. Secular trends in height were presented, including short views on so called 'racial types' vs bio-plasticity, and historic data on early-life nutritional status and later-life socioeconomic outcomes during the Dutch potato famine. New tools for describing body proportions in patients with variable degrees of phocomelia were presented along with electronic growth charts. Bio-statisticians discussed the influence of randomness, community and network structures, and presented novel tools and methods for analyzing social network data.

Selective mortality in middle-aged American women with Diffuse Idiopathic Skeletal Hyperostosis (DISH).

OBJECTIVE: A mortality sample of white American male and female skeletons was examined to illustrate a simple means of identifying skeletal conditions associated with an increased risk of dying relatively early in adulthood and to determine if males and females with Diffuse Idiopathic Skeletal Hyperostosis (DISH) displayed the same general age-specific pattern of mortality. METHODS: Age-specific probability distributions for DISH were generated from 416 white Americans who died from the 1980s to the present, and whose remains were donated to the University of Tennessee Forensic Anthropology Center. The age-specific frequency of DISH is analyzed using an empirical smoothing algorithm. Doing so allows for the identification of deviations (i.e., local maxima) from monotonically increasing age-specific probabilities. RESULTS: In females (N = 199), there is a peak in the frequency of individuals with DISH around 60 years of age where 37.0% of the individuals have DISH. It is matched only by the frequency (38.7%) in the oldest females, those over 85 years old. In contrast, DISH frequencies for males (N = 217) increase monotonically with advancing age, reaching 62.5% in the ≥86 years age group. There was an association between DISH and high body weight in women, particularly those who died before they reached the age of 75. CONCLUSIONS: Early-onset DISH in white American women is associated with an increased risk of dying indicated by a local maximum in the probability curve. Should this finding be replicated in additional mortality samples and the reason DISH is associated with early death is established, beyond being heavy, this radiologically visible ossification of the spine could be a potential component of health-monitoring programs for middle-aged women.

Ancient DNA study reveals HLA susceptibility locus for leprosy in medieval Europeans.

Leprosy, a chronic infectious disease caused by Mycobacterium leprae (M. leprae), was very common in Europe till the 16th century. Here, we perform an ancient DNA study on medieval skeletons from Denmark that show lesions specific for lepromatous leprosy (LL). First, we test the remains for M. leprae DNA to confirm the infection status of the individuals and to assess the bacterial diversity. We assemble 10 complete M. leprae genomes that all differ from each other. Second, we evaluate whether the human leukocyte antigen allele DRB1*15:01, a strong LL susceptibility factor in modern populations, also predisposed medieval Europeans to the disease. The comparison of genotype data from 69 M. leprae DNA-positive LL cases with those from contemporary and medieval controls reveals a statistically significant association in both instances. In addition, we observe that DRB1*15:01 co-occurs with DQB1*06:02 on a haplotype that is a strong risk factor for inflammatory diseases today.

Ancient genomes reveal a high diversity of Mycobacterium leprae in medieval Europe.

Studying ancient DNA allows us to retrace the evolutionary history of human pathogens, such as Mycobacterium leprae, the main causative agent of leprosy. Leprosy is one of the oldest recorded and most stigmatizing diseases in human history. The disease was prevalent in Europe until the 16th century and is still endemic in many countries with over 200,000 new cases reported annually. Previous worldwide studies on modern and European medieval M. leprae genomes revealed that they cluster into several distinct branches of which two were present in medieval Northwestern Europe. In this study, we analyzed 10 new medieval M. leprae genomes including the so far oldest M. leprae genome from one of the earliest known cases of leprosy in the United Kingdom-a skeleton from the Great Chesterford cemetery with a calibrated age of 415-545 C.E. This dataset provides a genetic time transect of M. leprae diversity in Europe over the past 1500 years. We find M. leprae strains from four distinct branches to be present in the Early Medieval Period, and strains from three different branches were detected within a single cemetery from the High Medieval Period. Altogether these findings suggest a higher genetic diversity of M. leprae strains in medieval Europe at various time points than previously assumed. The resulting more complex picture of the past phylogeography of leprosy in Europe impacts current phylogeographical models of M. leprae dissemination. It suggests alternative models for the past spread of leprosy such as a wide spread prevalence of strains from different branches in Eurasia already in Antiquity or maybe even an origin in Western Eurasia. Furthermore, these results highlight how studying ancient M. leprae strains improves understanding the history of leprosy worldwide.

Life not death: Epidemiology from skeletons.

Analytically sophisticated paleoepidemiology is a relatively new development in the characterization of past life experiences. It is based on sound paleopathological observations, accurate age-at-death estimates, an explicit engagement with the nature of mortality samples, and analytical procedures that owe much to epidemiology. Of foremost importance is an emphasis on people, not skeletons. Transforming information gleaned from the dead, a biased sample of individuals who were once alive at each age, into a form that is informative about past life experiences has been a major challenge for bioarchaeologists, but recent work shows it can be done. The further development of paleoepidemiology includes essential contributions from paleopathology, archaeology or history (as appropriate), and epidemiology.

Stressing out in medieval Denmark: An investigation of dental enamel defects and age at death in two medieval Danish cemeteries.

The influence of early life stress on later life experiences has become a major focus of research in medicine and more recently in bioarchaeology. Dental enamel, which preserves a record of childhood stress events, represents an important resource for this investigation when paired with the information from adult skeletal remains, such as age at death. The purpose of this research was to use a life history approach to the exploration of sex differences in the relationship between childhood stress and adult longevity by examining accentuated striae of Retzius (AS). A medieval Danish sample (n=70) drawn from the rural cemetery of Sejet and the urban cemetery of Ole Wormsgade was considered for AS and age at death. The results suggest sex differences in survivorship, with more stress being associated with reduced survivorship in males and increased survivorship in females. A consideration of AS formation time also suggests a difference in the impact of developmental timing between males and females. These results are interpreted in terms of differential frailty and selective mortality, drawing in both biomedical and cultural perspectives.

Sex estimation from modern American humeri and femora, accounting for sample variance structure.

OBJECTIVES: A new procedure for skeletal sex estimation based on humeral and femoral dimensions is presented, based on skeletons from the United States. The approach specifically addresses the problem that arises from a lack of variance homogeneity between the sexes, taking into account prior information about the sample's sex ratio, if known. MATERIAL AND METHODS: Three measurements useful for estimating the sex of adult skeletons, the humeral and femoral head diameters and the humeral epicondylar breadth, were collected from 258 Americans born between 1893 and 1980 who died within the past several decades. RESULTS: For measurements individually and collectively, the probabilities of being one sex or the other were generated for samples with an equal distribution of males and females, taking into account the variance structure of the original measurements. The combination providing the best estimates correctly classifies 88.3% of the skeletons, with 10.8% considered unknown and 0.9% assigned to the wrong sex. DISCUSSION: Probabilities of correct assignments are a better means of categorizing individuals as male or female than the sectioning points commonly used in skeletal studies. That is because it is possible to estimate the observer's certainty that the individual represented by measured bones was one sex or the other. A computer program is available that simultaneously considers samples of unequal sex composition. It is useful when there is contextual information available about the nature of skeletal samples (e.g., a mass burial from a battle or genocide).

Cranial vault trauma and selective mortality in medieval to early modern Denmark.

To date, no estimates of the long-term effect of cranial vault fractures on the risk of dying have been generated from historical or prehistoric skeletons. Excess mortality provides a perspective on the efficacy of modern treatment, as well as the human cost of cranial injuries largely related to interpersonal violence in past populations. Three medieval to early modern Danish skeletal samples are used to estimate the effect of selective mortality on males with cranial vault injuries who survived long enough for bones to heal. The risk of dying for these men was 6.2 times higher than it was for their uninjured counterparts, estimated through a simulation study based on skeletal observations. That is about twice the increased risk of dying experienced by modern people with traumatic brain injuries. The mortality data indicate the initial trauma was probably often accompanied by brain injury. Although the latter cannot be directly observed in skeletal remains, it can be inferred through the relative risks of dying. The ability to identify the effects of selective mortality in this skeletal sample indicates it must be taken into account in paleopathological research. The problem is analogous to extrapolating from death register data to modern communities, so epidemiological studies based on mortality data have the same inherent possibility of biases as analyses of ancient skeletons.

Genome-wide comparison of medieval and modern Mycobacterium leprae.

Leprosy was endemic in Europe until the Middle Ages. Using DNA array capture, we have obtained genome sequences of Mycobacterium leprae from skeletons of five medieval leprosy cases from the United Kingdom, Sweden, and Denmark. In one case, the DNA was so well preserved that full de novo assembly of the ancient bacterial genome could be achieved through shotgun sequencing alone. The ancient M. leprae sequences were compared with those of 11 modern strains, representing diverse genotypes and geographic origins. The comparisons revealed remarkable genomic conservation during the past 1000 years, a European origin for leprosy in the Americas, and the presence of an M. leprae genotype in medieval Europe now commonly associated with the Middle East. The exceptional preservation of M. leprae biomarkers, both DNA and mycolic acids, in ancient skeletons has major implications for palaeomicrobiology and human pathogen evolution.

Schleswig: medieval leprosy on the boundary between Germany and Denmark.

Leprosy was a well-recognized and dreaded disease in medieval Europe. The disease is reported to have reached Germany with the Roman invasion and it was present in Scandinavia in the first centuries AD. This paper estimates and analyzes the frequency of leprosy among adult people buried in one of five medieval cemeteries in the city of Schleswig. Seven different dichotomous osteological lesions indicative of leprosy were analyzed, and it was possible to score at least one of these conditions on 350 adult skeletons (aged 15 or older). The scores were transformed to a statistic indicating the likelihood that the person to whom the skeleton belonged suffered from leprosy. It was found that the frequency of leprosy in the five cemeteries varied between 9 and 44%. Four of the five cemeteries showed frequencies ranging from 35 and 44% and with no statistically significant differences among them. The fifth cemetery showed a significantly lower frequency of leprosy (9%). The distribution of female age at death does not appear to be affected by leprosy status. This means that females experienced a considerably elevated risk of dying once they had contracted leprosy as the disease usually has a mid-adulthood age of onset. In four of the five cemeteries males with leprosy died in higher ages than men without leprosy--in two of the cemeteries the difference was statistically significant. This indicates that leprosy usually added less to the risk of dying among men than among women in medieval Schleswig.