Metagenomics enables the study of complex microbial communities from myriad sources, including the remains of oral and gut microbiota preserved in archaeological dental calculus and paleofeces, respectively. While accurate taxonomic assignment is essential to this process, DNA damage characteristic of ancient samples (e.g., reduction in fragment size and cytosine deamination) may reduce the accuracy of read taxonomic assignment. Using a set of in silico-generated metagenomic data sets, we investigated how the addition of ancient DNA (aDNA) damage patterns influences microbial taxonomic assignment by five widely used profilers: QIIME/UCLUST, MetaPhlAn2, MIDAS, CLARK-S, and MALT. In silico-generated data sets were designed to mimic dental plaque, consisting of 40, 100, and 200 microbial species/strains, both with and without simulated aDNA damage patterns. Following taxonomic assignment, the profiles were evaluated for species presence/absence, relative abundance, alpha diversity, beta diversity, and specific taxonomic assignment biases. Unifrac metrics indicated that both MIDAS and MetaPhlAn2 reconstructed the most accurate community structure. QIIME/UCLUST, CLARK-S, and MALT had the highest number of inaccurate taxonomic assignments; false-positive rates were highest by CLARK-S and QIIME/UCLUST. Filtering out species present at <0.1% abundance greatly increased the accuracy of CLARK-S and MALT. All programs except CLARK-S failed to detect some species from the input file that were in their databases. The addition of ancient DNA damage resulted in minimal differences in species detection and relative abundance between simulated ancient and modern data sets for most programs. Overall, taxonomic profiling biases are program specific rather than damage dependent, and the choice of taxonomic classification program should be tailored to specific research questions. IMPORTANCE Ancient biomolecules from oral and gut microbiome samples have been shown to be preserved in the archaeological record. Studying ancient microbiome communities using metagenomic techniques offers a unique opportunity to reconstruct the evolutionary trajectories of microbial communities through time. DNA accumulates specific damage over time, which could potentially affect taxonomic classification and our ability to accurately reconstruct community assemblages. It is therefore necessary to assess whether ancient DNA (aDNA) damage patterns affect metagenomic taxonomic profiling. Here, we assessed biases in community structure, diversity, species detection, and relative abundance estimates by five popular metagenomic taxonomic classification programs using in silico-generated data sets with and without aDNA damage. Damage patterns had minimal impact on the taxonomic profiles produced by each program, while false-positive rates and biases were intrinsic to each program. Therefore, the most appropriate classification program is one that minimizes the biases related to the questions being addressed.

For 10,000 years pigs and humans have shared a close and complex relationship. From domestication to modern breeding practices, humans have shaped the genomes of domestic pigs. Here we present the assembly and analysis of the genome sequence of a female domestic Duroc pig (Sus scrofa) and a comparison with the genomes of wild and domestic pigs from Europe and Asia. Wild pigs emerged in South East Asia and subsequently spread across Eurasia. Our results reveal a deep phylogenetic split between European and Asian wild boars ∼1 million years ago, and a selective sweep analysis indicates selection on genes involved in RNA processing and regulation. Genes associated with immune response and olfaction exhibit fast evolution. Pigs have the largest repertoire of functional olfactory receptor genes, reflecting the importance of smell in this scavenging animal. The pig genome sequence provides an important resource for further improvements of this important livestock species, and our identification of many putative disease-causing variants extends the potential of the pig as a biomedical model.

Elucidating the process of speciation requires an in-depth understanding of the evolutionary history of the species in question. Studies that rely upon a limited number of genetic loci do not always reveal actual evolutionary history, and often confuse inferences related to phylogeny and speciation. Whole-genome data, however, can overcome this issue by providing a nearly unbiased window into the patterns and processes of speciation. In order to reveal the complexity of the speciation process, we sequenced and analyzed the genomes of 10 wild pigs, representing morphologically or geographically well-defined species and subspecies of the genus Sus from insular and mainland Southeast Asia, and one African common warthog.

Understanding the demographic history of a population is critical to conservation and to our broader understanding of evolutionary processes. For many tropical large mammals, however, this aim is confounded by the absence of fossil material and by the misleading signal obtained from genetic data of recently fragmented and isolated populations. This is particularly true for the lion which as a consequence of millennia of human persecution, has large gaps in its natural distribution and several recently extinct populations.

The independent domestication of local wild boar populations in Asia and Europe about 10,000 years ago led to distinct European and Asian pig breeds, each with very different phenotypic characteristics. During the Industrial Revolution, Chinese breeds were imported to Europe to improve commercial traits in European breeds. Here we demonstrate the presence of introgressed Asian haplotypes in European domestic pigs and selection signatures on some loci in these regions, using whole genome sequence data. The introgression signatures are widespread and the Asian haplotypes are rarely fixed. The Asian introgressed haplotypes are associated with regions harbouring genes involved in meat quality, development and fertility. We identify Asian-derived non-synonymous mutations in the AHR gene that associate with increased litter size in multiple European commercial lines. These findings demonstrate that increased fertility was an important breeding goal for early nineteenth century pig farmers, and that Asian variants of genes related to this trait were preferentially selected during the development of modern European pig breeds.

Bos primigenius, the aurochs, is the wild ancestor of modern cattle breeds and was formerly widespread across Eurasia and northern Africa. After a progressive decline, the species became extinct in 1627. The origin of modern taurine breeds in Europe is debated. Archaeological and early genetic evidence point to a single Near Eastern origin and a subsequent spread during the diffusion of herding and farming. More recent genetic data are instead compatible with local domestication events or at least some level of local introgression from the aurochs. Here we present the analysis of the complete mitochondrial genome of a pre-Neolithic Italian aurochs.

Mesolithic populations throughout Europe used diverse resource exploitation strategies that focused heavily on collecting and hunting wild prey. Between 5500 and 4200 cal BC, agriculturalists migrated into northwestern Europe bringing a suite of Neolithic technologies including domesticated animals. Here we investigate to what extent Mesolithic Ertebølle communities in northern Germany had access to domestic pigs, possibly through contact with neighbouring Neolithic agricultural groups. We employ a multidisciplinary approach, applying sequencing of ancient mitochondrial and nuclear DNA (coat colour-coding gene MC1R) as well as traditional and geometric morphometric (molar size and shape) analyses in Sus specimens from 17 Neolithic and Ertebølle sites. Our data from 63 ancient pig specimens show that Ertebølle hunter-gatherers acquired domestic pigs of varying size and coat colour that had both Near Eastern and European mitochondrial DNA ancestry. Our results also reveal that domestic pigs were present in the region ~500 years earlier than previously demonstrated.

The aurochs (Bos primigenius) was a large bovine that ranged over almost the entirety of the Eurasian continent and North Africa. It is the wild ancestor of the modern cattle (Bos taurus), and went extinct in 1627 probably as a consequence of human hunting and the progressive reduction of its habitat. To investigate in detail the genetic history of this species and to compare the population dynamics in different European areas, we analysed Bos primigenius remains from various sites across Italy.

Yellow skin is an abundant phenotype among domestic chickens and is caused by a recessive allele (W*Y) that allows deposition of yellow carotenoids in the skin. Here we show that yellow skin is caused by one or more cis-acting and tissue-specific regulatory mutation(s) that inhibit expression of BCDO2 (beta-carotene dioxygenase 2) in skin. Our data imply that carotenoids are taken up from the circulation in both genotypes but are degraded by BCDO2 in skin from animals carrying the white skin allele (W*W). Surprisingly, our results demonstrate that yellow skin does not originate from the red junglefowl (Gallus gallus), the presumed sole wild ancestor of the domestic chicken, but most likely from the closely related grey junglefowl (Gallus sonneratii). This is the first conclusive evidence for a hybrid origin of the domestic chicken, and it has important implications for our views of the domestication process.

Lions are one of the world's most iconic megafauna, yet little is known about their temporal and spatial demographic history and population differentiation. We analyzed a genomic dataset of 20 specimens: two ca. 30,000-y-old cave lions (Panthera leo spelaea), 12 historic lions (Panthera leo leo/Panthera leo melanochaita) that lived between the 15th and 20th centuries outside the current geographic distribution of lions, and 6 present-day lions from Africa and India. We found that cave and modern lions shared an ancestor ca. 500,000 y ago and that the 2 lineages likely did not hybridize following their divergence. Within modern lions, we found 2 main lineages that diverged ca. 70,000 y ago, with clear evidence of subsequent gene flow. Our data also reveal a nearly complete absence of genetic diversity within Indian lions, probably due to well-documented extremely low effective population sizes in the recent past. Our results contribute toward the understanding of the evolutionary history of lions and complement conservation efforts to protect the diversity of this vulnerable species.

Grey wolves (Canis lupus) are one of the few large terrestrial carnivores that have maintained a wide geographical distribution across the Northern Hemisphere throughout the Pleistocene and Holocene. Recent genetic studies have suggested that, despite this continuous presence, major demographic changes occurred in wolf populations between the Late Pleistocene and early Holocene, and that extant wolves trace their ancestry to a single Late Pleistocene population. Both the geographical origin of this ancestral population and how it became widespread remain unknown. Here, we used a spatially and temporally explicit modelling framework to analyse a data set of 90 modern and 45 ancient mitochondrial wolf genomes from across the Northern Hemisphere, spanning the last 50,000 years. Our results suggest that contemporary wolf populations trace their ancestry to an expansion from Beringia at the end of the Last Glacial Maximum, and that this process was most likely driven by Late Pleistocene ecological fluctuations that occurred across the Northern Hemisphere. This study provides direct ancient genetic evidence that long-range migration has played an important role in the population history of a large carnivore, and provides insight into how wolves survived the wave of megafaunal extinctions at the end of the last glaciation. Moreover, because Late Pleistocene grey wolves were the likely source from which all modern dogs trace their origins, the demographic history described in this study has fundamental implications for understanding the geographical origin of the dog.

The distribution of the black rat (Rattus rattus) has been heavily influenced by its association with humans. The dispersal history of this non-native commensal rodent across Europe, however, remains poorly understood, and different introductions may have occurred during the Roman and medieval periods. Here, in order to reconstruct the population history of European black rats, we first generate a de novo genome assembly of the black rat. We then sequence 67 ancient and three modern black rat mitogenomes, and 36 ancient and three modern nuclear genomes from archaeological sites spanning the 1st-17th centuries CE in Europe and North Africa. Analyses of our newly reported sequences, together with published mitochondrial DNA sequences, confirm that black rats were introduced into the Mediterranean and Europe from Southwest Asia. Genomic analyses of the ancient rats reveal a population turnover in temperate Europe between the 6th and 10th centuries CE, coincident with an archaeologically attested decline in the black rat population. The near disappearance and re-emergence of black rats in Europe may have been the result of the breakdown of the Roman Empire, the First Plague Pandemic, and/or post-Roman climatic cooling.

Across Europe, the genetics of the Chalcolithic/Bronze Age transition is increasingly characterized in terms of an influx of Steppe-related ancestry. The effect of this major shift on the genetic structure of populations in the Italian Peninsula remains underexplored. Here, genome-wide shotgun data for 22 individuals from commingled cave and single burials in Northeastern and Central Italy dated between 3200 and 1500 BCE provide the first genomic characterization of Bronze Age individuals (n = 8; 0.001-1.2× coverage) from the central Italian Peninsula, filling a gap in the literature between 1950 and 1500 BCE. Our study confirms a diversity of ancestry components during the Chalcolithic and the arrival of Steppe-related ancestry in the central Italian Peninsula as early as 1600 BCE, with this ancestry component increasing through time. We detect close patrilineal kinship in the burial patterns of Chalcolithic commingled cave burials and a shift away from this in the Bronze Age (2200-900 BCE) along with lowered runs of homozygosity, which may reflect larger changes in population structure. Finally, we find no evidence that the arrival of Steppe-related ancestry in Central Italy directly led to changes in frequency of 115 phenotypes present in the dataset, rather that the post-Roman Imperial period had a stronger influence, particularly on the frequency of variants associated with protection against Hansen's disease (leprosy). Our study provides a closer look at local dynamics of demography and phenotypic shifts as they occurred as part of a broader phenomenon of widespread admixture during the Chalcolithic/Bronze Age transition.

The European wildcat population in Scotland is considered critically endangered as a result of hybridization with introduced domestic cats,1,2 though the time frame over which this gene flow has taken place is unknown. Here, using genome data from modern, museum, and ancient samples, we reconstructed the trajectory and dated the decline of the local wildcat population from viable to severely hybridized. We demonstrate that although domestic cats have been present in Britain for over 2,000 years,3 the onset of hybridization was only within the last 70 years. Our analyses reveal that the domestic ancestry present in modern wildcats is markedly over-represented in many parts of the genome, including the major histocompatibility complex (MHC). We hypothesize that introgression provides wildcats with protection against diseases harbored and introduced by domestic cats, and that this selection contributes to maladaptive genetic swamping through linkage drag. Using the case of the Scottish wildcat, we demonstrate the importance of local ancestry estimates to both understand the impacts of hybridization in wild populations and support conservation efforts to mitigate the consequences of anthropogenic and environmental change.

Zooarcheological evidence suggests that pigs were domesticated in Southwest Asia ~8,500 BC. They then spread across the Middle and Near East and westward into Europe alongside early agriculturalists. European pigs were either domesticated independently or more likely appeared so as a result of admixture between introduced pigs and European wild boar. As a result, European wild boar mtDNA lineages replaced Near Eastern/Anatolian mtDNA signatures in Europe and subsequently replaced indigenous domestic pig lineages in Anatolia. The specific details of these processes, however, remain unknown. To address questions related to early pig domestication, dispersal, and turnover in the Near East, we analyzed ancient mitochondrial DNA and dental geometric morphometric variation in 393 ancient pig specimens representing 48 archeological sites (from the Pre-Pottery Neolithic to the Medieval period) from Armenia, Cyprus, Georgia, Iran, Syria, and Turkey. Our results reveal the first genetic signatures of early domestic pigs in the Near Eastern Neolithic core zone. We also demonstrate that these early pigs differed genetically from those in western Anatolia that were introduced to Europe during the Neolithic expansion. In addition, we present a significantly more refined chronology for the introduction of European domestic pigs into Asia Minor that took place during the Bronze Age, at least 900 years earlier than previously detected. By the 5th century AD, European signatures completely replaced the endemic lineages possibly coinciding with the widespread demographic and societal changes that occurred during the Anatolian Bronze and Iron Ages.

Near Eastern wild boars possess a characteristic DNA signature. Unexpectedly, wild boars from Israel have the DNA sequences of European wild boars and domestic pigs. To understand how this anomaly evolved, we sequenced DNA from ancient and modern pigs from Israel. Pigs from Late Bronze Age (until ca. 1150 BCE) in Israel shared haplotypes of modern and ancient Near Eastern pigs. European haplotypes became dominant only during the Iron Age (ca. 900 BCE). This raises the possibility that European pigs were brought to the region by the Sea Peoples who migrated to the Levant at that time. Then, a complete genetic turnover took place, most likely because of repeated admixture between local and introduced European domestic pigs that went feral. Severe population bottlenecks likely accelerated this process. Introductions by humans have strongly affected the phylogeography of wild animals, and interpretations of phylogeography based on modern DNA alone should be taken with caution.

Pigs (Sus scrofa) have played an important cultural role in Hawaii since Polynesians first introduced them in approximately AD 1200. Additional varieties of pigs were introduced following Captain Cook's arrival in Hawaii in 1778 and it has been suggested that the current pig population may descend primarily, or even exclusively, from European pigs. Although populations of feral pigs today are an important source of recreational hunting on all of the major islands, they also negatively impact native plants and animals. As a result, understanding the origins of these feral pig populations has significant ramifications for discussions concerning conservation management, identity and cultural continuity on the islands. Here, we analysed a neutral mitochondrial marker and a functional nuclear coat colour marker in 57 feral Hawaiian pigs. Through the identification of a new mutation in the MC1R gene that results in black coloration, we demonstrate that Hawaiian feral pigs are mostly the descendants of those originally introduced during Polynesian settlement, though there is evidence for some admixture. As such, extant Hawaiian pigs represent a unique historical lineage that is not exclusively descended from feral pigs of European origin.

Domestic dogs have been central to life in the North American Arctic for millennia. The ancestors of the Inuit were the first to introduce the widespread usage of dog sledge transportation technology to the Americas, but whether the Inuit adopted local Palaeo-Inuit dogs or introduced a new dog population to the region remains unknown. To test these hypotheses, we generated mitochondrial DNA and geometric morphometric data of skull and dental elements from a total of 922 North American Arctic dogs and wolves spanning over 4500 years. Our analyses revealed that dogs from Inuit sites dating from 2000 BP possess morphological and genetic signatures that distinguish them from earlier Palaeo-Inuit dogs, and identified a novel mitochondrial clade in eastern Siberia and Alaska. The genetic legacy of these Inuit dogs survives today in modern Arctic sledge dogs despite phenotypic differences between archaeological and modern Arctic dogs. Together, our data reveal that Inuit dogs derive from a secondary pre-contact migration of dogs distinct from Palaeo-Inuit dogs, and probably aided the Inuit expansion across the North American Arctic beginning around 1000 BP.

Helminth infections are among the World Health Organization's top neglected diseases with significant impact in many Less Economically Developed Countries. Despite no longer being endemic in Europe, the widespread presence of helminth eggs in archaeological deposits indicates that helminths represented a considerable burden in past European populations. Prevalence of infection is a key epidemiological feature that would influence the elimination of endemic intestinal helminths, for example, low prevalence rates may have made it easier to eliminate these infections in Europe without the use of modern anthelminthic drugs. To determine historical prevalence rates we analysed 589 grave samples from 7 European sites dated between 680 and 1700 CE, identifying two soil transmitted nematodes (Ascaris spp. and Trichuris trichiura) at all locations, and two food derived cestodes (Diphyllobothrium latum and Taenia spp.) at 4 sites. The rates of nematode infection in the medieval populations (1.5 to 25.6% for T. trichiura; 9.3-42.9% for Ascaris spp.) were comparable to those reported within modern endemically infected populations. There was some evidence of higher levels of nematode infection in younger individuals but not at all sites. The genetic diversity of T. trichiura ITS-1 in single graves was variable but much lower than with communal medieval latrine deposits. The prevalence of food derived cestodes was much lower (1.0-9.9%) than the prevalence of nematodes. Interestingly, sites that contained Taenia spp. eggs also contained D. latum which may reflect local culinary practices. These data demonstrate the importance of helminth infections in Medieval Europe and provide a baseline for studies on the epidemiology of infection in historical and modern contexts. Since the prevalence of medieval STH infections mirror those in modern endemic countries the factors affecting STH decline in Europe may also inform modern intervention campaigns.

Extant Canis lupus genetic diversity can be grouped into three phylogenetically distinct clades: Eurasian and American wolves and domestic dogs.1 Genetic studies have suggested these groups trace their origins to a wolf population that expanded during the last glacial maximum (LGM)1-3 and replaced local wolf populations.4 Moreover, ancient genomes from the Yana basin and the Taimyr peninsula provided evidence of at least one extinct wolf lineage that dwelled in Siberia during the Pleistocene.35 Previous studies have suggested that Pleistocene Siberian canids can be classified into two groups based on cranial morphology. Wolves in the first group are most similar to present-day populations, although those in the second group possess intermediate features between dogs and wolves.67 However, whether this morphological classification represents distinct genetic groups remains unknown. To investigate this question and the relationships between Pleistocene canids, present-day wolves, and dogs, we resequenced the genomes of four Pleistocene canids from Northeast Siberia dated between >50 and 14 ka old, including samples from the two morphological categories. We found these specimens cluster with the two previously sequenced Pleistocene wolves, which are genetically more similar to Eurasian wolves. Our results show that, though the four specimens represent extinct wolf lineages, they do not form a monophyletic group. Instead, each Pleistocene Siberian canid branched off the lineage that gave rise to present-day wolves and dogs. Finally, our results suggest the two previously described morphological groups could represent independent lineages similarly related to present-day wolves and dogs.