Jacob Enk

Yersinia pestis and the Plague of Justinian 541–543 AD: a genomic analysis

BACKGROUND Yersinia pestis has caused at least three human plague pandemics. The second (Black Death, 14-17th centuries) and third (19-20th centuries) have been genetically characterised, but there is only a limited understanding of the first pandemic, the Plague of Justinian (6-8th centuries). To address this gap, we sequenced and analysed draft genomes of Y pestis obtained from two individuals who died in the first pandemic. METHODS Teeth were removed from two individuals (known as A120 and A76) from the early medieval Aschheim-Bajuwarenring cemetery (Aschheim, Bavaria, Germany). We isolated DNA from the teeth using a modified phenol-chloroform method. We screened DNA extracts for the presence of the Y pestis-specific pla gene on the pPCP1 plasmid using primers and standards from an established assay, enriched the DNA, and then sequenced it. We reconstructed draft genomes of the infectious Y pestis strains, compared them with a database of genomes from 131 Y pestis strains from the second and third pandemics, and constructed a maximum likelihood phylogenetic tree. FINDINGS Radiocarbon dating of both individuals (A120 to 533 AD [plus or minus 98 years]; A76 to 504 AD [plus or minus 61 years]) places them in the timeframe of the first pandemic. Our phylogeny contains a novel branch (100% bootstrap at all relevant nodes) leading to the two Justinian samples. This branch has no known contemporary representatives, and thus is either extinct or unsampled in wild rodent reservoirs. The Justinian branch is interleaved between two extant groups, 0.ANT1 and 0.ANT2, and is distant from strains associated with the second and third pandemics. INTERPRETATION We conclude that the Y pestis lineages that caused the Plague of Justinian and the Black Death 800 years later were independent emergences from rodents into human beings. These results show that rodent species worldwide represent important reservoirs for the repeated emergence of diverse lineages of Y pestis into human populations. FUNDING McMaster University, Northern Arizona University, Social Sciences and Humanities Research Council of Canada, Canada Research Chairs Program, US Department of Homeland Security, US National Institutes of Health, Australian National Health and Medical Research Council.

Second-pandemic strain of Vibrio cholerae from the Philadelphia cholera outbreak of 1849.

In the 19th century, there were several major cholera pandemics in the Indian subcontinent, Europe, and North America. The causes of these outbreaks and the genomic strain identities remain a mystery. We used targeted high-throughput sequencing to reconstruct the Vibrio cholerae genome from the preserved intestine of a victim of the 1849 cholera outbreak in Philadelphia, part of the second cholera pandemic. This O1 biotype strain has 95 to 97% similarity with the classical O395 genome, differing by 203 single-nucleotide polymorphisms (SNPs), lacking three genomic islands, and probably having one or more tandem cholera toxin prophage (CTX) arrays, which potentially affected its virulence. This result highlights archived medical remains as a potential resource for investigations into the genomic origins of past pandemics.

Ancient whole genome enrichment using baits built from modern DNA.

We report metrics from complete genome capture of nuclear DNA from extinct mammoths using biotinylated RNAs transcribed from an Asian elephant DNA extract. Enrichment of the nuclear genome ranged from 1.06- to 18.65-fold, to an apparent maximum threshold of ∼80% on-target. This projects an order of magnitude less costly complete genome sequencing from long-dead organisms, even when a reference genome is unavailable for bait design.

Shotgun Mitogenomics Provides a Reference Phylogenetic Framework and Timescale for Living Xenarthrans

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 into two distinct families Dasypodidae (dasypodines) and Chlamyphoridae (euphractines, chlamyphorines, and tolypeutines) to better reflect their ancient divergence, estimated around 42 Ma. 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 My. Biogeographic reconstructions identified the tropical rainforest biome of Amazonia and the Guiana Shield as the cradle of xenarthran evolutionary history with subsequent dispersions into more open and dry habitats.

Complete Columbian mammoth mitogenome suggests interbreeding with woolly mammoths

BackgroundLate Pleistocene North America hosted at least two divergent and ecologically distinct species of mammoth: the periglacial woolly mammoth (Mammuthus primigenius) and the subglacial Columbian mammoth (Mammuthus columbi). To date, mammoth genetic research has been entirely restricted to woolly mammoths, rendering their genetic evolution difficult to contextualize within broader Pleistocene paleoecology and biogeography. Here, we take an interspecific approach to clarifying mammoth phylogeny by targeting Columbian mammoth remains for mitogenomic sequencing.ResultsWe sequenced the first complete mitochondrial genome of a classic Columbian mammoth, as well as the first complete mitochondrial genome of a North American woolly mammoth. Somewhat contrary to conventional paleontological models, which posit that the two species were highly divergent, the M. columbi mitogenome we obtained falls securely within a subclade of endemic North American M. primigenius.ConclusionsThough limited, our data suggest that the two species interbred at some point in their evolutionary histories. One potential explanation is that woolly mammoth haplotypes entered Columbian mammoth populations via introgression at subglacial ecotones, a scenario with compelling parallels in extant elephants and consistent with certain regional paleontological observations. This highlights the need for multi-genomic data to sufficiently characterize mammoth evolutionary history. Our results demonstrate that the use of next-generation sequencing technologies holds promise in obtaining such data, even from non-cave, non-permafrost Pleistocene depositional contexts.

Ancient pathogen DNA in archaeological samples detected with a Microbial Detection Array.

Ancient human remains of paleopathological interest typically contain highly degraded DNA in which pathogenic taxa are often minority components, making sequence-based metagenomic characterization costly. Microarrays may hold a potential solution to these challenges, offering a rapid, affordable, and highly informative snapshot of microbial diversity in complex samples without the lengthy analysis and/or high cost associated with high-throughput sequencing. Their versatility is well established for modern clinical specimens, but they have yet to be applied to ancient remains. Here we report bacterial profiles of archaeological and historical human remains using the Lawrence Livermore Microbial Detection Array (LLMDA). The array successfully identified previously-verified bacterial human pathogens, including Vibrio cholerae (cholera) in a 19th century intestinal specimen and Yersinia pestis (“Black Death” plague) in a medieval tooth, which represented only minute fractions (0.03% and 0.08% alignable high-throughput shotgun sequencing reads) of their respective DNA content. This demonstrates that the LLMDA can identify primary and/or co-infecting bacterial pathogens in ancient samples, thereby serving as a rapid and inexpensive paleopathological screening tool to study health across both space and time.

Quantitative PCR as a predictor of aligned ancient DNA read counts following targeted enrichment.

Targeted DNA enrichment through hybridization capture (EHC) is rapidly replacing PCR as the method of choice for enrichment prior to genomic resequencing. This is especially true in the case of ancient DNA (aDNA) from long-dead organisms, where targets tend to be highly fragmented and outnumbered by contaminant DNA. However, the behavior of EHC using aDNA has been quite variable, making success difficult to predict and preventing efficient sample equilibration during multiplexed sequencing runs. Here, we evaluate whether quantitative PCR (qPCR) measurements of aDNA samples correlate with on-target read counts before and after EHC. Our data indicate that not only do simple target qPCRs correlate strongly with high-throughput sequencing (HTS) data but that certain sample characteristics, such as overall target abundance as well as experimental parameters (e.g., bait concentration and secondary structure propensity), consistently influenced enrichment of our diverse set of aDNA samples. Taken together, our results should help guide experimental design, screening strategies, and multiplexed sample equilibration, increasing yield and reducing the expected and actual cost of aDNA EHC high-throughput sequencing projects in the future.

Mammuthus Population Dynamics in Late Pleistocene North America: Divergence, Phylogeography, and Introgression

After evolving in Africa at the close of the Miocene, mammoths (Mammuthus sp.) spread through much of the northern hemisphere, diversifying morphologically as they entered various habitats. Paleontologically, these morphs are conventionally recognized as species. In Pleistocene North America alone, several mammoth species have been recognized, inhabiting environments as different as cold tundra-steppe in the north and the arid grasslands or temperate savanna-parklands of the south. Yet mammoth phylogeographic studies have overwhelmingly focused on permafrost-preserved remains of only one of these species, Mammuthus primigenius (woolly mammoth). Here we challenge this bias by performing a geographically and taxonomically wide survey of mammoth genetic diversity across North America. Using a targeted enrichment technique, we sequenced 67 complete mitochondrial genomes from non-primigenius specimens representing M. columbi (Columbian mammoth), M. jeffersonii (Jeffersonian mammoth), and M. exilis (pygmy mammoth), including specimens from contexts not generally associated with good DNA preservation. While we uncovered clear phylogeographic structure in mammoth matrilines, their phylogeny as recovered from mitochondrial DNA is not compatible with existing systematic interpretations of their paleontological record. Instead, our results strongly suggest that various nominal mammoth species interbred, perhaps extensively. We hypothesize that at least two distinct stages of interbreeding between conventional paleontological species are likely responsible for this pattern – one between Siberian woolly mammoths and resident American populations that introduced woolly mammoth phenotypes to the continent, and another between ecomorphologically distinct populations of woolly and Columbian mammoths in North America south of the ice.

Ancient DNA and the tropics: a rodent's tale

Most genetic studies of Holocene fauna have been performed with ancient samples from dry and cold regions, in which preservation of fossils is facilitated and molecular damage is reduced. Ancient DNA work from tropical regions has been precluded owing to factors that limit DNA preservation (e.g. temperature, hydrolytic damage). We analysed ancient DNA from rodent jawbones identified as Ototylomys phyllotis, found in Holocene and Late Pleistocene stratigraphic layers from Loltún, a humid tropical cave located in the Yucatan peninsula. We extracted DNA and amplified six short overlapping fragments of the cytochrome b gene, totalling 666 bp, which represents an unprecedented success considering tropical ancient DNA samples. We performed genetic, phylogenetic and divergence time analyses, combining sequences from ancient and modern O. phyllotis, in order to assess the ancestry of the Loltún samples. Results show that all ancient samples fall into a unique clade that diverged prior to the divergence of the modern O. phyllotis, supporting it as a distinct Pleistocene form of the Ototylomys genus. Hence, this rodents tale suggests that the sister group to modern O. phyllotis arose during the Miocene–Pliocene, diversified during the Pleistocene and went extinct in the Holocene.

A molecular portrait of maternal sepsis from Byzantine Troy

Pregnancy complications are poorly represented in the archeological record, despite their importance in contemporary and ancient societies. While excavating a Byzantine cemetery in Troy, we discovered calcified abscesses among a woman’s remains. Scanning electron microscopy of the tissue revealed ‘ghost cells’, resulting from dystrophic calcification, which preserved ancient maternal, fetal and bacterial DNA of a severe infection, likely chorioamnionitis. Gardnerella vaginalis and Staphylococcus saprophyticus dominated the abscesses. Phylogenomic analyses of ancient, historical, and contemporary data showed that G. vaginalis Troy fell within contemporary genetic diversity, whereas S. saprophyticus Troy belongs to a lineage that does not appear to be commonly associated with human disease today. We speculate that the ecology of S. saprophyticus infection may have differed in the ancient world as a result of close contacts between humans and domesticated animals. These results highlight the complex and dynamic interactions with our microbial milieu that underlie severe maternal infections. DOI: http://dx.doi.org/10.7554/eLife.20983.001