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Smallpox, caused by the variola virus (VARV), is prominent in modern histories of the ancient Mediterranean world. The disease, or the diagnosis of it, has shaped estimations of the scale and significance of epidemics and pandemics, notably the 2nd-c. Antonine plague, and the burden of disease in large cities and regions densely populated in antiquity. Here we synthesize recent paleogenetic and evolutionary biological literature that casts significant doubt on the existence of a VARV that caused a disease we would recognize – clinically, ecologically, or epidemiologically – as smallpox in antiquity. On the basis of current data, it is time archaeologists and historians began to eradicate smallpox from their histories of the ancient world.

Plague has an enigmatic history as a zoonotic pathogen. This potentially devastating infectious disease will unexpectedly appear in human populations and disappear just as suddenly. As a result, a long-standing line of inquiry has been to estimate when and where plague appeared in the past. However, there have been significant disparities between phylogenetic studies of the causative bacterium, Yersinia pestis, regarding the timing and geographic origins of its reemergence. Here, we curate and contextualize an updated phylogeny of Y. pestis using 601 genome sequences sampled globally. We perform a detailed Bayesian evaluation of temporal signal in subsets of these data and demonstrate that a Y. pestis-wide molecular clock model is unstable. To resolve this, we devised a new approach in which each Y. pestis population was assessed independently. This enabled us to recover significant temporal signal in five populations, including the ancient pandemic lineages which we now estimate may have emerged decades, or even centuries, before a pandemic was historically documented from European sources. Despite this, we only obtain robust divergence dates from populations sampled over a period of at least 90 years, indicating that genetic evidence alone is insufficient for accurately reconstructing the timing and spread of short-term plague epidemics. Finally, we identify key historical data sets that can be used in future research, which will complement the strengths and mitigate the weaknesses of genomic data.

Escherichia coli - one of the most characterized bacteria and a major public health concern - remains invisible across the temporal landscape. Here, we present the meticulous reconstruction of the first ancient E. coli genome from a 16

The Arctic is currently experiencing dramatic ecosystem changes with immediate effects on biodiversity. Sedimentary ancient DNA is a unique and valuable source of information on ecosystem changes over a long temporal scale. Understanding these past changes may help predict the relative impacts of climate change, herbivory, and anthropogenic effects on present ecosystems. In the BiodivERsA project “Future ArcTic Ecosystems” (FATE), we aim to assess changes in past herbivore abundance over large spatial (circumarctic) and temporal (Last Glacial Maximum until today) scales using three (semi-)quantitative methods on sedimentary ancient DNA of plants, herbivores, and herbivore proxies (i.e. coprophilous fungi and parasites) – metabarcoding, hybridization capture enrichment, and droplet digital PCR (ddPCR).Metabarcoding was applied to DNA of plants and also of coprophilous fungi as proxies of herbivore abundance. This approach is an established and important tool for assessing biodiversity from recent environmental DNA; however, quantification of specific taxa may be complicated due to inherent methodological biases (e.g. amplification efficiency due to primer bias), and our current understanding of the factors affecting potential quantification by metabarcoding is still limited. Moreover, ancient DNA is highly fragmented, which may prevent PCR amplification altogether. As an alternative, target enrichment by hybridization capture is a method that does not depend on target PCR amplification and is typically not affected by DNA fragmentation. Furthermore, hybridization capture can be used to target numerous genetic markers of a vast range of highly diverse taxa. We are using hybridization capture to enrich DNA of a range of herbivore species and numerous proxy organisms. Metabarcoding and hybridization capture can be applied to a vast taxonomic range and may be used quantitatively based on relative sequencing read abundance; however, the respective read abundance may be confounded by random and systematic errors and other biases. We are therefore using an additional quantification method – ddPCR – on several selected taxa, which is taxon-specific but facilitates highly accurate quantification of template DNA molecules in a given sample. The combined taxonomic and quantitative results of these three approaches are used to generate highly resolved datasets on past vegetation and herbivores, which allows us to reconstruct past vegetation changes over large spatial (circumarctic) and temporal (Last Glacial Maximum until today) scales.Detailed inferences on herbivore abundance and reconstructing past ecological conditions may be important for ecosystem management and conservation in the face of accelerating changes in Arctic ecosystems due to global climate change.

Paleofeces are the nonmineralized remains of dung from extant and extinct fauna. They represent a surprisingly large proportion of fossil remains recovered from cave sites across the world. Paleofeces contain the DNA of the defecator as well as the DNA of ingested plant and animal remains. To successfully extract DNA from paleofeces, a balance must be achieved between the minimization of DNA loss during extraction and the removal of coeluates that would otherwise inhibit the Taq DNA polymerase during downstream applications. Here we present a simplified version of a protocol to extract DNA from paleofecal remains.

Antibiotic resistance is often linked to human use of antibiotics. However, antibiotics and antibiotic biosynthetic pathways have been evolving for millions of years suggesting that antibiotic resistance is an ancient phenomenon. As of now, there has been no systematic survey of environmental microbes proven to exist in the absence of human influence and Lechuguilla cave offers such environment. Resistance diversity in strains isolated from this cave was analyzed by a phenotypic screen against a panel of 26 different antibiotics. Resistant strains were further investigated through determination of minimal inhibitory concentration (MIC) and inactivation studies. Of particular interest was strain LC044 (Brachybacterium paraconglomeratum), observed to inactivate macrolide antibiotics by phosphorylation. Genome sequencing and bioinformatics (BLAST analysis) identified a putative macrolide phosphotransferase (MPH) in strain LC044 and biochemical characterization of the purified recombinant protein confirmed its macrolide inactivating properties. To investigate if characterized MPH was unique to cave isolate, available terrestrial Brachybacterium faecium DSM 4810 genome was mined for presence of MPH-like protein. The top hit to the MPH from LC044 (a protein with 282 amino acids and 72% identity) was heterologously expressed and purified. Complete biochemical analysis of this enzyme revealed (i) MPH-activity, despite its annotation as aminoglycoside phosphotransferase (APH), and (ii) no significant differences in substrate specificities or kinetic parameters between these two enzymes suggesting that these two enzymes were equally effective resistance enzymes. This work highlights the prevalence of antibiotic resistance in a pristine, cave ecosystem and provides further support for the theory that antibiotic resistance is everywhere. Furthermore, the mph resistance determinant found in cave isolate and closely related terrestrial isolate show homology to clinical mph genes, suggesting that environmental mph genes could have served as reservoir of clinical determinants. Master of Science (MSc)