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To investigate variation in ancient DNA recovery of Brucella melitensis, the causative agent of brucellosis, from multiple tissues belonging to one individual MATERIALS: 14 samples were analyzed from the mummified remains of the Blessed Sante, a 14Shotgun sequencing data from was examined to determine the presence of Brucella DNA.Three of the 14 samples contained authentic ancient DNA, identified as belonging to B. melitensis. A genome (23.81X depth coverage, 0.98 breadth coverage) was recovered from a kidney stone. Nine of the samples contained reads classified as B. melitensis (7-169), but for many the data quality was insufficient to withstand our identification and authentication criteria.We identified significant variation in the preservation and abundance of B. melitensis DNA present across multiple tissues, with calcified nodules yielding the highest number of authenticated reads. This shows how greatly sample selection can impact pathogen identification.Our results demonstrate variation in the preservation and recovery of pathogen DNA across tissues. This study highlights the importance of sample selection in the reconstruction of infectious disease burden and highlights the importance of a holistic approach to identifying disease.Study focuses on pathogen recovery in a single individual.Further analysis of how sampling impacts aDNA recovery will improve pathogen aDNA recovery and advance our understanding of disease in past peoples.

International audience; 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.

Bartonet al.1raise several statistical concerns regarding our original analyses2that 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 FSTvalues 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 nearERAP2continues to emerge as the strongest candidate for selection (p = 1.2×10−5), falling below the Bonferroni-corrected significance threshold recommended by Bartonet al. Importantly, the evidence for selection onERAP2is further supported by functional data demonstrating the impact of theERAP2genotype on the immune response toY. pestisand 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.

The recent influx of genomic data has provided greater insights into the molecular basis for regressive evolution, or vestigialization, through gene loss and pseudogenization. As such, the analysis of gene degradation patterns has the potential to provide insights into the evolutionary history of regressed anatomical traits. We specifically applied these principles to the xenarthran radiation (anteaters, sloths, armadillos), which is characterized by taxa with a gradation in regressed dental phenotypes. Whether the pattern among extant xenarthrans is due to an ancient and gradual decay of dental morphology or occurred repeatedly in parallel is unknown. We tested these competing hypotheses by examining 11 core dental genes in most living species of Xenarthra, characterizing shared inactivating mutations and patterns of relaxed selection during their radiation. Here we report evidence of independent and distinct events of dental gene loss in the major xenarthran subclades. First, we found strong evidence of complete enamel loss in the common ancestor of sloths and anteaters, suggested by the inactivation of four enamel-associated genes (AMELX, AMTN, MMP20, ENAM). Next, whereas sloths halted their dental regression, presumably a critical event that ultimately permitted adaptation to an herbivorous lifestyle, anteaters continued losing genes on the path towards complete tooth loss. Echoes of this event are recorded in the genomes of all living anteaters, being marked by a 2-bp deletion in a gene critical for dentinogenesis (DSPP) and a putative shared 1-bp insertion in a gene linked to tooth retention (ODAPH). By contrast, in the two major armadillo clades, genes pertaining to the gingival-tooth junction appear to have been independently inactivated prior to losing all or most enamel. These genomic data provide evidence for a complex evolutionary history with multiple pathways and rates of anatomical regression, and underscore the utility of using pseudogenes to reconstruct evolutionary history when fossils are sparse.

Sedimentary ancient DNA (sedaDNA) has been established as a viable biomolecular proxy for tracking taxon presence through time in a local environment, even in the total absence of surviving tissues. SedaDNA is thought to survive through mineral binding, facilitating long-term biomolecular preservation, but also challenging DNA isolation. Two common limitations in sedaDNA extraction are the carryover of other substances that inhibit enzymatic reactions, and the loss of authentic sedaDNA when attempting to reduce inhibitor co-elution. Here, we present a sedaDNA extraction procedure paired with targeted enrichment intended to maximize DNA recovery. Our procedure exhibits a 7.7–19.3x increase in on-target plant and animal sedaDNA compared to a commercial soil extraction kit, and a 1.2–59.9x increase compared to a metabarcoding approach. To illustrate the effectiveness of our cold spin extraction and PalaeoChip capture enrichment approach, we present results for the diachronic presence of plants and animals from Yukon permafrost samples dating to the Pleistocene-Holocene transition, and discuss new potential evidence for the late survival (~9700 years ago) of mammoth (Mammuthus sp.) and horse (Equus sp.) in the Klondike region of Yukon, Canada. This enrichment approach translates to a more taxonomically diverse dataset and improved on-target sequencing.

Pleistocene glacial-interglacial cycles are correlated with dramatic temperature oscillations. Examining how species responded to these natural fluctuations can provide valuable insights into the impacts of present-day anthropogenic climate change. Here we present a phylogeographic study of the extinct American mastodon (Mammut americanum), based on 35 complete mitochondrial genomes. These data reveal the presence of multiple lineages within this species, including two distinct clades from eastern Beringia. Our molecular date estimates suggest that these clades arose at different times, supporting a pattern of repeated northern expansion and local extirpation in response to glacial cycling. Consistent with this hypothesis, we also note lower levels of genetic diversity among northern mastodons than in endemic clades south of the continental ice sheets. The results of our study highlight the complex relationships between population dispersals and climate change, and can provide testable hypotheses for extant species expected to experience substantial biogeographic impacts from rising temperatures., Pleistocene population dynamics can inform the consequences of current climate change. This phylogeography of 35 complete American mastodon mitochondrial genomes suggests distinct lineages in this species repeatedly expanded northwards and then went locally extinct in response to glacial cycles.

The discovery of mass burial sites is rare in Europe, particularly in rural areas. Recent excavations at Thornton Abbey in Lincolnshire have revealed a previously unknown catastrophic mass grave containing the remains of at least 48 men, women and children, with radiocarbon dating placing the event in the fourteenth century AD. The positive identification of Yersinia pestis in sampled skeletal remains suggests that the burial population died from the Black Death. This site represents the first Black Death mass grave found in Britain in a non-urban context, and provides unique evidence for the devastating impact of this epidemic on a small rural community.

Significance The Justinianic Plague (circa 541 to 750 CE) has recently featured prominently in scholarly and popular discussions. Current consensus accepts that it resulted in the deaths of between a quarter and half of the population of the Mediterranean, playing a key role in the fall of the Roman Empire. Our contribution argues that earlier estimates are founded on a small subset of textual evidence and are not supported by many other independent types of evidence (e.g., papyri, coins, inscriptions, and pollen archaeology). We therefore conclude that earlier analyses of the mortality and social effects of the plague are exaggerated, and that the nontextual evidence suggests plague did not play a significant role in the transformation of the Mediterranean world or Europe., Existing mortality estimates assert that the Justinianic Plague (circa 541 to 750 CE) caused tens of millions of deaths throughout the Mediterranean world and Europe, helping to end antiquity and start the Middle Ages. In this article, we argue that this paradigm does not fit the evidence. We examine a series of independent quantitative and qualitative datasets that are directly or indirectly linked to demographic and economic trends during this two-century period: Written sources, legislation, coinage, papyri, inscriptions, pollen, ancient DNA, and mortuary archaeology. Individually or together, they fail to support the maximalist paradigm: None has a clear independent link to plague outbreaks and none supports maximalist reconstructions of late antique plague. Instead of large-scale, disruptive mortality, when contextualized and examined together, the datasets suggest continuity across the plague period. Although demographic, economic, and political changes continued between the 6th and 8th centuries, the evidence does not support the now commonplace claim that the Justinianic Plague was a primary causal factor of them.

Traditionally, paleontologists have relied on the morphological features of bones and teeth to reconstruct the evolutionary relationships of extinct animals.

The COVID-19 pandemic has the potential to affect the human microbiome in infected and uninfected individuals, having a substantial impact on human health over the long term. This pandemic intersects with a decades-long decline in microbial diversity and ancestral microbes due to hygiene, antibiotics, and urban living (the hygiene hypothesis). High-risk groups succumbing to COVID-19 include those with preexisting conditions, such as diabetes and obesity, which are also associated with microbiome abnormalities. Current pandemic control measures and practices will have broad, uneven, and potentially long-term effects for the human microbiome across the planet, given the implementation of physical separation, extensive hygiene, travel barriers, and other measures that influence overall microbial loss and inability for reinoculation. Although much remains uncertain or unknown about the virus and its consequences, implementing pandemic control practices could significantly affect the microbiome. In this Perspective, we explore many facets of COVID-19−induced societal changes and their possible effects on the microbiome, and discuss current and future challenges regarding the interplay between this pandemic and the microbiome. Recent recognition of the microbiome’s influence on human health makes it critical to consider both how the microbiome, shaped by biosocial processes, affects susceptibility to the coronavirus and, conversely, how COVID-19 disease and prevention measures may affect the microbiome. This knowledge may prove key in prevention and treatment, and long-term biological and social outcomes of this pandemic.

The multitude of factors alleged to have contributed to the late Quaternary mass extinction of some two-thirds of Earth’s megafauna is complicated by the coarse record of buried macro-fossils. In response, micro-methods such as ancient DNA have been increasingly able to augment discontinuous palaeontological records to investigate the relative timings of vegetation turnover versus megafaunal extirpations—all in the absence of biological tissues. Here, we present sedimentary ancient DNA data retrieved using the PalaeoChip Arctic-1.0 bait-set diachronically identifying fauna and flora from permafrost cores recovered from the Klondike region of central Yukon, Canada dating between 30,000–6000 calendar years BP. We observe a substantial turnover in ecosystem composition between 13,000–10,000 BP with the rise of woody shrubs and the disappearance of mammoth-steppe vegetation. We also identify a lingering signal of Equus sp. (North American horse) and Mammuthus primigenius (woolly mammoth) from multiple samples thousands of years after their last dated macro-fossils, possibly as late as the mid-Holocene.

International audience; The COVID-19 pandemic has the potential to affect the human microbiome in infected and uninfected individuals, having a substantial impact on human health over the long term. This pandemic intersects with a decades-long decline in microbial diversity and ancestral microbes due to hygiene, antibiotics, and urban living (the hygiene hypothesis). High-risk groups succumbing to COVID-19 include those with preexisting conditions, such as diabetes and obesity, which are also associated with microbiome abnormalities. Current pandemic control measures and practices will have broad, uneven, and potentially long-term effects for the human microbiome across the planet, given the implementation of physical separation, extensive hygiene, travel barriers, and other measures that influence overall microbial loss and inability for reinoculation. Although much remains uncertain or unknown about the virus and its consequences, implementing pandemic control practices could significantly affect the microbiome. In this Perspective, we explore many facets of COVID-19−induced societal changes and their possible effects on the microbiome, and discuss current and future challenges regarding the interplay between this pandemic and the microbiome. Recent recognition of the microbiome’s influence on human health makes it critical to consider both how the microbiome, shaped by biosocial processes, affects susceptibility to the coronavirus and, conversely, how COVID-19 disease and prevention measures may affect the microbiome. This knowledge may prove key in prevention and treatment, and long-term biological and social outcomes of this pandemic.

Summary The compounding challenges of low signal, high background, and uncertain targets plague many metagenomic sequencing efforts. One solution has been DNA capture, wherein probes are designed to hybridize with target sequences, enriching them in relation to their background. However, balancing probe depth with breadth of capture is challenging for diverse targets. To find this balance, we have developed the HUBDesign pipeline, which makes use of sequence homology to design probes at multiple taxonomic levels. This creates an efficient probe set capable of simultaneously and specifically capturing known and related sequences. We validated HUBDesign by generating probe sets targeting the breadth of coronavirus diversity, as well as a suite of bacterial pathogens often underlying sepsis. In separate experiments demonstrating significant, simultaneous enrichment, we captured SARS-CoV-2 and HCoV-NL63 in a human RNA background and seven bacterial strains in human blood. HUBDesign ( https://github.com/zacherydickson/HUBDesign ) has broad applicability wherever there are multiple organisms of interest.

According to a recent article published in Genome Biology, Duggan and coworkers sequenced and partially assembled five genomes of smallpox vaccines from the nineteenth century. No information regarding the ends of genomes was presented, and they are important to understand the evolutionary relationship of the different smallpox vaccine genomes during the centuries. We re-assembled the genomes, which include the largest genomes in the vaccinia lineage and one true horsepox strain. Moreover, the assemblies reveal a diverse genetic structure in the genome ends. Our data emphasize the concurrent use of horsepox and horsepox-related viruses as the smallpox vaccine in the nineteenth century.

The ability to sequence genomes from ancient biological material has provided a rich source of information for evolutionary biology and engaged considerable public interest. Although most studies of ancient genomes have focused on vertebrates, particularly archaic humans, newer technologies allow the capture of microbial pathogens and microbiomes from ancient and historical human and non-human remains. This coming of age has been made possible by techniques that allow the preferential capture and amplification of discrete genomes from a background of predominantly host and environmental DNA. There are now near-complete ancient genome sequences for three pathogens of considerable historical interest — pre-modern bubonic plague (Yersinia pestis), smallpox (Variola virus) and cholera (Vibrio cholerae) — and for three equally important endemic human disease agents — Mycobacterium tuberculosis (tuberculosis), Mycobacterium leprae (leprosy) and Treponema pallidum pallidum (syphilis). Genomic data from these pathogens have extended earlier work by paleopathologists. There have been efforts to sequence the genomes of additional ancient pathogens, with the potential to broaden our understanding of the infectious disease burden common to past populations from the Bronze Age to the early 20th century. In this review we describe the state-of-the-art of this rapidly developing field, highlight the contributions of ancient pathogen genomics to multidisciplinary endeavors and describe some of the limitations in resolving questions about the emergence and long-term evolution of pathogens., Sebastián Duchêne and colleagues highlight the impact of advances in ancient DNA techniques on our understanding of the emergence and persistence of pathogens from a historical and epidemiological perspective.

Vaccination has transformed public health, most notably including the eradication of smallpox. Despite its profound historical importance, little is known of the origins and diversity of the viruses used in smallpox vaccination. Prior to the twentieth century, the method, source and origin of smallpox vaccinations remained unstandardised and opaque. We reconstruct and analyse viral vaccine genomes associated with smallpox vaccination from historical artefacts. Significantly, we recover viral molecules through non-destructive sampling of historical materials lacking signs of biological residues. We use the authenticated ancient genomes to reveal the evolutionary relationships of smallpox vaccination viruses within the poxviruses as a whole.

SARS-CoV-2 is a novel betacoronavirus and the aetiological agent of the current COVID-19 outbreak that originated in Hubei Province, China. While polymerase chain reaction is the front-line tool for SARS-CoV-2 surveillance, application of amplification-free and culture-free methods for isolation of SARS-CoV-2 RNA, partnered with next-generation sequencing, would provide a useful tool for both surveillance and research of SARS-CoV-2. We here release into the public domain a set of bait capture hybridization probe sequences for enrichment of SARS-CoV-2 RNA from complex biological samples. These probe sequences have been designed using rigorous bioinformatics methods to provide sensitivity, accuracy, and minimal off-target hybridization. Probe design was based on existing, validated approaches for detecting antimicrobial resistance genes in complex samples and it is our hope that this SARS-CoV-2 bait capture platform, once validated by those with samples in hand, will be of aid in combating the current outbreak.

Rome initiated several campaigns to expand, conquer, and enslave local Italic populations following the establishment of the republic in 504 BCE. However, the cultural and biological changes resulting from Roman subjugation across Italy remain a topic of intense historical debate. Although important, historic and archaeological lines of evidence fail to track the impact of forced enslavement and enculturation at individual and broader genetic scales and, more generally, offer fewer clues regarding the potential affinities of Roman period Italians to European, Near Eastern, western Asian and North African populations at this time. In this paper, we present the whole mitochondrial (mtDNA) genomes of 30 Roman period (1st–4th centuries CE) individuals buried in the Vagnari necropolis in southern Italy. We integrate the mtDNA data with previously published bioarchaeological and isotope (δ18O and 87Sr/86Sr) data for the Vagnari assemblage and compare Roman haplogroup composition to 15 newly sequenced mitochondrial genomes obtained from a pre-Roman Iron Age skeletal assemblage, located in close proximity to Vagnari. Additionally, we contrast our South Italian dataset with a further 332 complete ancient mtDNA genomes from the pan-Mediterranean region, Europe, western Asia and North African regions. Population pairwise ΦST values suggest that Roman Italians share closer genetic similarity to Neolithic, Bronze Age, and Armenian Iron Age populations from western and central Eurasia than with Iron Age Italians, Ptolemaic, and Roman period Egyptians. Vagnari individuals with δ18O, 87Sr/86Sr, and mtDNA data suggest a predominantly local demographic was employed at the site. However, two individuals belong to eastern Eurasian haplogroup D4b1c, indicating that the maternal ancestors of these two individuals migrated to South Italy prior to the 1st century CE. Additionally, we provide the first genetic evidence for possible maternal relatedness in a Roman period skeletal assemblage. Our research highlights the significance of integrating multiple lines of bioarchaeological data to inform interpretations about Roman colonial expansion and its impact on population structure.

Plasmodium falciparum is a significant human pathogen, particularly in the historical context of the ancient Mediterranean region. The causative species of malaria are “invisible” in the historical record, while malaria as a disease entity is indirectly supported by evidence from literary works (e.g., the Hippocratic Corpus, Celsus’ De Medicina) and non-specific skeletal pathological responses. Although ancient DNA may demonstrate the presence of a pathogen, there remain theoretical and methodological challenges in contextualizing such molecular evidence. Here we present a framework to explore the biosocial context of malaria in 1st–4th c. CE central-southern Italy using genomic, literary, epidemiological, and archaeological evidence to highlight relationships between the Plasmodium parasite, human hosts, Anopheles vector, and environment. By systematically integrating these evidentiary sources, our approach highlights the importance of disease ecology (e.g., climate and landscape) and human-environment interactions (e.g., land use patterns, such as agriculture or infrastructure activities) that differentially impact the potential scope of malaria in the past.

Recent advances in ancient DNA methodologies have enabled the retrieval of highly degraded DNA from contexts with poor preservation conditions. While paleofeces have previously been shown to contain endogenous DNA of the defecator, the preserved DNA is composed of a mixture of diverse microbial, floral and fungal constituents, with limited DNA from the host. However, in situations where skeletal remains are unavailable, paleofeces can serve as an important alternative genetic source, allowing for the molecular identification of the target species and diet. Here, we describe the extraction of ancient DNA from a paleofecal sample found within Bechan Cave (southeastern Utah, USA). Previous work in the cave has suggested that these remains likely stem from Mammuthus. We used a comprehensive proboscidean bait set which was used to enrich a nearly complete mitochondrial genome (81.6%) at an average coverage depth of 8.1×. Phylogenetic analysis of the derived consensus sequence revealed that the Bechan Cave bolus does indeed derive from Mammuthus, and its sequence falls within Clade 1 (haplogroups F or C), most similar to specimens identified as Mammuthus columbi.

The Museum of London selected four individuals for multidisciplinary scientific analyses in order to establish their ancestry, aspects of their personal appearance and health. We also reinterpreted their burial context in order to better understand how identity was constructed and expressed in this unique Roman settlement. Our study discovered the presence of people with Black and White European ancestry, some of whom had migrated from the southern Mediterranean. The most surprising result was that Harper Road woman's chromosomes were male. Overall, our experience of undertaking a multidisciplinary study served to further underline the need for these different techniques to be used in combination when investigating past identities. The mtDNA results were very broad and required the mobility isotopes to better understand their significance, while the aDNA evidence confirmed the osteological analysis. In terms of public engagement at the Museum of London, the ability to determine hair and eye colour had a significant impact.

SummaryAncient environmental DNA has been established as a viable biomolecular proxy for tracking taxonomic presence through time in a local environment, even in the total absence of primary tissues. It is thought that sedimentary ancient DNA (sedaDNA) survives through mineral binding. And while these organo-mineral complexes likely facilitate long-term preservation, they also challenge our ability to release and isolate target molecules. Two limitations in sedaDNA extraction impede many palaeoenvironmental reconstructions: the post-extraction carryover of enzymatic inhibitors, and sedaDNA loss when attempting to reduce inhibitor co-elution. Here, we present an optimised eDNA targeted enrichment approach for reconstructing past environments. Our new extraction protocol with targeted enrichment averages a 14.6-fold increase in on-target plant and animal DNA compared to a commercial soil extraction kit, and a 22.6-fold increase compared to a PCR metabarcoding approach. To illustrate the effectiveness of the PalaeoChip Arctic1.0 protocol, we present results of plant and animal presence from permafrost samples and discuss new potential evidence for the late survival (ca. 9685 BP) of mammoth (Mammuthus sp.) and horse (Equus sp.) in the Klondike Region of Yukon, Canada. This approach translates to a more diverse and sensitive dataset with increased sequencing efficiency of ecologically informative sedaDNA.

Identification of the nucleotide sequences encoding antibiotic resistance elements and determination of their association with antibiotic resistance are critical to improve surveillance and monitor trends in antibiotic resistance. Current methods to study antibiotic resistance in various environments rely on extensive deep sequencing or laborious culturing of fastidious organisms, both of which are heavily time-consuming operations., Identification of the nucleotide sequences encoding antibiotic resistance elements and determination of their association with antibiotic resistance are critical to improve surveillance and monitor trends in antibiotic resistance. Current methods to study antibiotic resistance in various environments rely on extensive deep sequencing or laborious culturing of fastidious organisms, both of which are heavily time-consuming operations. An accurate and sensitive method to identify both rare and common resistance elements in complex metagenomic samples is needed. Referencing the sequences in the Comprehensive Antibiotic Resistance Database, we designed a set of 37,826 probes to specifically target over 2,000 nucleotide sequences associated with antibiotic resistance in clinically relevant bacteria. Testing of this probe set on DNA libraries generated from multidrug-resistant bacteria to selectively capture resistance genes reproducibly produced higher numbers of reads on target at a greater length of coverage than shotgun sequencing. We also identified additional resistance gene sequences from human gut microbiome samples that sequencing alone was not able to detect. Our method to capture the resistome enables a sensitive means of gene detection in diverse environments where genes encoding antibiotic resistance represent less than 0.1% of the metagenome.

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 Φ ST 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.

Living sloths represent two distinct lineages of small-sized mammals that independently evolved arboreality from terrestrial ancestors. The six extant species are the survivors of an evolutionary radiation marked by the extinction of large terrestrial forms at the end of the Quaternary. Until now, sloth evolutionary history has mainly been reconstructed from phylogenetic analyses of morphological characters. Here, we used ancient DNA methods to successfully sequence 10 extinct sloth mitogenomes encompassing all major lineages. This includes the iconic continental ground sloths Megatherium, Megalonyx, Mylodon, and Nothrotheriops and the smaller endemic Caribbean sloths Parocnus and Acratocnus. Phylogenetic analyses identify eight distinct lineages grouped in three well-supported clades, whose interrelationships are markedly incongruent with the currently accepted morphological topology. We show that recently extinct Caribbean sloths have a single origin but comprise two highly divergent lineages that are not directly related to living two-fingered sloths, which instead group with Mylodon. Moreover, living three-fingered sloths do not represent the sister group to all other sloths but are nested within a clade of extinct ground sloths including Megatherium, Megalonyx, and Nothrotheriops. Molecular dating also reveals that the eight newly recognized sloth families all originated between 36 and 28 million years ago (mya). The early divergence of recently extinct Caribbean sloths around 35 mya is consistent with the debated GAARlandia hypothesis postulating the existence at that time of a biogeographic connection between northern South America and the Greater Antilles. This new molecular phylogeny has major implications for reinterpreting sloth morphological evolution, biogeography, and diversification history. Extant sloths are the survivors of an evolutionary radiation marked by the extinction of large terrestrial forms of the Ice Age. By sequencing ten mitogenomes from extinct sloths, Delsuc et al. present a new molecular phylogeny revealing widespread morphological convergence with major implications for reinterpreting sloth evolutionary history. Fil: Delsuc, Frédéric. Université Montpellier II; Francia Fil: Kuch, Melanie. Mcmaster Ancient Dna Centre; Canadá Fil: Gibb, Gillian C.. Massey University; Nueva Zelanda. Université Montpellier II; Francia Fil: Karpinski, Emil. Mcmaster Ancient Dna Centre; Canadá. Mcmaster University; Canadá Fil: Hackenberger, Dirk. Mcmaster Ancient Dna Centre; Canadá Fil: Szpak, Paul. Trent University; Canadá Fil: Martinez, Jorge Gabriel. Universidad Nacional de Tucumán; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Estudios Sociales. Universidad Nacional de Tucumán. Instituto Superior de Estudios Sociales; Argentina Fil: Mead, Jim I.. East Tennessee State University; Estados Unidos Fil: McDonald, H. Gregory. Bureau of Land Management; Estados Unidos Fil: Macphee, Ross Douglas Earle. American Museum of Natural History; Estados Unidos Fil: Billet, Guillaume. Université Pierre et Marie Curie; Francia Fil: Hautier, Lionel. Natural History Museum; Reino Unido. Université Montpellier II; Francia Fil: Poinar, Hendrik N.. Mcmaster Ancient Dna Centre; Canadá

Human evolution has been punctuated by climate anomalies, structuring environments, deadly infections, and altering landscapes. How well humans adapted to these new circumstances had direct effects on fitness and survival. Here, how the gut microbiome could have contributed to human evolutionary success through contributions to host nutritional buffering and infectious disease resistance is reviewed. How changes in human genetics, diet, disease exposure, and social environments almost certainly altered microbial community composition is also explored. Emerging research points to the microbiome as a key player in host responses to environmental change. Therefore, the reciprocal interactions between humans and their microbes are likely to have shaped human patterns of local adaptation throughout our shared evolutionary history. Recent alterations in human lifestyle, however, are altering human microbiomes in unprecedented ways. The consequences of interrupted host-microbe relationships for human adaptive potential in the future are unknown.

Xenarthra (armadillos, sloths, and anteaters) constitutes one of the four major clades of placental mammals. Despite their phylogenetic distinctiveness in mammals, a reference phylogeny is still lacking for the 31 described species. Here we used Illumina shotgun sequencing to assemble 33 new complete mitochondrial genomes, establishing Xenarthra as the first major placental clade to be fully sequenced at the species level for mitogenomes. The resulting data set allowed the reconstruction of a robust phylogenetic framework and timescale that are consistent with previous studies conducted at the genus level using nuclear genes. Incorporating the full species diversity of extant xenarthrans points to a number of inconsistencies in xenarthran systematics and species definition. We propose to split armadillos in two distinct families Dasypodidae (dasypodines) and Chlamyphoridae (euphractines, chlamyphorines, and tolypeutines) to better reflect their ancient divergence, estimated around 42 million years ago. Species delimitation within long-nosed armadillos (genus Dasypus) appeared more complex than anticipated, with the discovery of a divergent lineage in French Guiana. Diversification analyses showed Xenarthra to be an ancient clade with a constant diversification rate through time with a species turnover driven by high but constant extinction. We also detected a significant negative correlation between speciation rate and past temperature fluctuations with an increase in speciation rate corresponding to the general cooling observed during the last 15 million years. Biogeographic reconstructions identified the tropical rainforest biome of Amazonia and the Guianan shield as the cradle of xenarthran evolutionary history with subsequent dispersions into more open and dry habitats. Fil: Gibb, Gillian C.. Universite de Montpellier; Francia. Massey Universit; Nueva Zelanda Fil: Condamine, Fabien L.. University of Gothenburg; Suecia. Universite de Montpellier; Francia. University of Alberta; Canadá Fil: Kuch, Melanie. McMaster University; Canadá Fil: Enk, Jacob. McMaster University; Canadá Fil: Moraes Barros, Nadia. Universidade Do Porto; Portugal. Universidade de Sao Paulo; Brasil Fil: Superina, Mariella. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Poinar, Hendrik N.. McMaster University; Canadá Fil: Delsuc, Frederic. Universite de Montpellier; Francia

Summary The historical record attests to the devastation malaria exacted on ancient civilizations, particularly the Roman Empire [1]. However, evidence for the presence of malaria during the Imperial period in Italy (1st–5th century CE) is based on indirect sources, such as historical, epigraphic, or skeletal evidence. Although these sources are crucial for revealing the context of this disease, they cannot establish the causative species of Plasmodium . Importantly, definitive evidence for the presence of malaria is now possible through the implementation of ancient DNA technology. As malaria is presumed to have been at its zenith during the Imperial period [1], we selected first or second molars from 58 adults from three cemeteries from this time: Isola Sacra (associated with Portus Romae, 1st–3rd century CE), Velia (1st–2nd century CE), and Vagnari (1st–4th century CE). We performed hybridization capture using baits designed from the mitochondrial (mtDNA) genomes of Plasmodium spp. on a prioritized subset of 11 adults (informed by metagenomic sequencing). The mtDNA sequences generated provided compelling phylogenetic evidence for the presence of P. falciparum in two individuals. This is the first genomic data directly implicating P. falciparum in Imperial period southern Italy in adults.

Elephantids are the world's most iconic megafaunal family, yet there is no comprehensive genomic assessment of their relationships. We report a total of 14 genomes, including 2 from the American mastodon, which is an extinct elephantid relative, and 12 spanning all three extant and three extinct elephantid species including an ∼120,000-y-old straight-tusked elephant, a Columbian mammoth, and woolly mammoths. Earlier genetic studies modeled elephantid evolution via simple bifurcating trees, but here we show that interspecies hybridization has been a recurrent feature of elephantid evolution. We found that the genetic makeup of the straight-tusked elephant, previously placed as a sister group to African forest elephants based on lower coverage data, in fact comprises three major components. Most of the straight-tusked elephant's ancestry derives from a lineage related to the ancestor of African elephants while its remaining ancestry consists of a large contribution from a lineage related to forest elephants and another related to mammoths. Columbian and woolly mammoths also showed evidence of interbreeding, likely following a latitudinal cline across North America. While hybridization events have shaped elephantid history in profound ways, isolation also appears to have played an important role. Our data reveal nearly complete isolation between the ancestors of the African forest and savanna elephants for ∼500,000 y, providing compelling justification for the conservation of forest and savanna elephants as separate species.

Yersinia pestis , the causative agent of plague, evolved from the closely related pathogen Yersinia pseudotuberculosis . During its emergence, Y. pestis is believed to have acquired its unique pathogenic characteristics through numerous gene gains/losses, genomic rearrangements, and single nucleotide polymorphism (SNP) changes. One such SNP creates a single amino acid variation in the DNA binding domain of PhoP, the response regulator in the PhoP/PhoQ two-component system. Y. pseudotuberculosis and the basal human-avirulent strains of Y. pestis harbor glycines at position 215 of PhoP, whereas the modern human-virulent strains (e.g., KIM and CO92) harbor serines at this residue. Since PhoP plays multiple roles in the adaptation of Y. pestis to stressful host conditions, we tested whether this amino acid substitution affects PhoP activity or the ability of Y. pestis to survive in host environments. Compared to the parental KIM6+ strain carrying the modern allele of phoP ( phoP-S215 ), a derivative carrying the basal allele ( phoP-G215 ) exhibited slightly defective growth under a low-Mg 2+ condition and decreased transcription of a PhoP target gene, ugd , as well as an ∼8-fold increase in the susceptibility to the antimicrobial peptide polymyxin B. The phoP-G215 strain showed no apparent defect in flea colonization, although a phoP -null mutant showed decreased flea infectivity in competition experiments. Our results suggest that the amino acid variation at position 215 of PhoP causes subtle changes in the PhoP activity and raise the possibility that the change in this residue have contributed to the evolution of increased virulence in Y. pestis . IMPORTANCE Y. pestis acquired a single nucleotide polymorphism (SNP) in phoP when the highly human-virulent strains diverged from less virulent basal strains, resulting in an amino acid substitution in the DNA binding domain of the PhoP response regulator. We show that Y. pestis carrying the modern phoP allele has an increased ability to induce the PhoP-regulated ugd gene and resist antimicrobial peptides compared to an isogenic strain carrying the basal allele. Given the important roles PhoP plays in host adaptation, the results raise an intriguing possibility that this amino acid substitution contributed to the evolution of increased virulence in Y. pestis . Additionally, we present the first evidence that phoP confers a survival fitness advantage to Y. pestis inside the flea midgut.

Ancient bacterial and viral genomes provide a window into the tempo, chronology, and rate of evolutionary processes of pathogens that have accompanied humans throughout history, from catastrophic pandemics (e.g., Yersinia pestis and the Black Death) to diseases associated with “everyday” morbidity and mortality (e.g., tuberculosis, leprosy, hepatitis B virus). Excitingly, the scope of pathogens that can be explored using ancient DNA methods is expanding, largely due to advances in the recovery of these typically minute molecular fractions. Increasingly, ancient DNA is applied to the study of rapidly or slowly evolving pathogens across significant time transects (hundreds to thousands of years ago) enabling us to investigate ancient genomic diversity through a comparative lens that can potentially inform our understanding of how a pathogen has changed over time. In this chapter, we highlight the impact of changing molecular strategies in recovering and analyzing ancient pathogen genomes alongside the wealth of information within the historical record that both informs and challenges the framework used to explore pathogens and human disease in the past. The power of ancient DNA to detect the signatures of ancient pathogens is also tempered by recognized limitations in characterizing the relative “outcome” of complex human-pathogen interactions in diverse archaeological contexts.

DNA damage in the form of abasic sites, chemically altered nucleotides, and strand fragmentation is the foremost limitation in obtaining genetic information from many ancient samples. Upon cell death, DNA continues to endure various chemical attacks such as hydrolysis and oxidation, but repair pathways found in vivo no longer operate. By incubating degraded DNA with specific enzyme combinations adopted from these pathways, it is possible to reverse some of the post-mortem nucleic acid damage prior to downstream analyses such as library preparation, targeted enrichment, and high-throughput sequencing. Here, we evaluate the performance of two available repair protocols on previously characterized DNA extracts from four mammoths. Both methods use endonucleases and glycosylases along with a DNA polymerase-ligase combination. PreCR Repair Mix increases the number of molecules converted to sequencing libraries, leading to an increase in endogenous content and a decrease in cytosine-to-thymine transitions due to cytosine deamination. However, the effects of Nelson Repair Mix on repair of DNA damage remain inconclusive.

Summary The processes leading up to species extinctions are typically characterized by prolonged declines in population size and geographic distribution, followed by a phase in which populations are very small and may be subject to intrinsic threats, including loss of genetic diversity and inbreeding [1]. However, whether such genetic factors have had an impact on species prior to their extinction is unclear [2, 3]; examining this would require a detailed reconstruction of a species' demographic history as well as changes in genome-wide diversity leading up to its extinction. Here, we present high-quality complete genome sequences from two woolly mammoths ( Mammuthus primigenius ). The first mammoth was sequenced at 17.1-fold coverage and dates to ∼4,300 years before present, representing one of the last surviving individuals on Wrangel Island. The second mammoth, sequenced at 11.2-fold coverage, was obtained from an ∼44,800-year-old specimen from the Late Pleistocene population in northeastern Siberia. The demographic trajectories inferred from the two genomes are qualitatively similar and reveal a population bottleneck during the Middle or Early Pleistocene, and a more recent severe decline in the ancestors of the Wrangel mammoth at the end of the last glaciation. A comparison of the two genomes shows that the Wrangel mammoth has a 20% reduction in heterozygosity as well as a 28-fold increase in the fraction of the genome that comprises runs of homozygosity. We conclude that the population on Wrangel Island, which was the last surviving woolly mammoth population, was subject to reduced genetic diversity shortly before it became extinct.

High-throughput sequencing (HTS) has radically altered approaches to human evolutionary research. Recent contributions highlight that HTS is able to reach depths of the human lineage previously thought to be impossible. In this paper, we outline the methodological advances afforded by recent developments in DNA recovery, data output, scalability, speed, and resolution of the current sequencing technology. We review and critically evaluate the 'DNA pipeline' for ancient samples: from DNA extraction, to constructing immortalized sequence libraries, to enrichment strategies (e.g., polymerase chain reaction [PCR] and hybridization capture), and finally, to bioinformatic analyses of sequence data. We argue that continued evaluations and improvements to this process are essential to ensure sequence data validity. Also, we highlight the role of contamination and authentication in ancient DNA-HTS, which is particularly relevant to ancient human genomics, since sequencing the genomes of hominins such as Homo erectus and Homo heidelbergensis may soon be within the realm of possibility.

Although smallpox has been known for centuries, the oldest available variola virus strains were isolated in the early 1940s. At that time, large regions of the world were already smallpox-free. Therefore, genetic information of these strains can represent only the very last fraction of a long evolutionary process. Based on the genomes of 48 strains, two clades are differentiated: Clade 1 includes variants of variola major, and clade 2 includes West African and variola minor (Alastrim) strains. Recently, the genome of an almost 400-year-old Lithuanian mummy was determined, which fell basal to all currently sequenced strains of variola virus on phylogenetic trees. Here, we determined two complete variola virus genomes from human tissues kept in a museum in Prague dating back 60 and 160 years, respectively. Moreover, mass spectrometry-based proteomic, chemical, and microscopic examinations were performed. The 60-year-old specimen was most likely an importation from India, a country with endemic smallpox at that time. The genome of the 160-year-old specimen is related to clade 2 West African and variola minor strains. This sequence likely represents a new endemic European variant of variola virus circulating in the midst of the 19th century in Europe.