Inferring diet, disease and antibiotic resistance from ancient human oral microbiomes.

The interaction between a host and its microbiome is an area of intense study. For the human host, it is known that the various body-site-associated microbiomes impact heavily on health and disease states. For instance, the oral microbiome is a source of various pathogens and potential antibiotic resistance gene pools. The effect of historical changes to the human host and environment to the associated microbiome, however, has been less well explored. In this review, we characterize several historical and prehistoric events which are considered to have impacted the oral environment and therefore the bacterial communities residing within it. The link between evolutionary changes to the oral microbiota and the significant societal and behavioural changes occurring during the pre-Neolithic, Agricultural Revolution, Industrial Revolution and Antibiotic Era is outlined. While previous studies suggest the functional profile of these communities may have shifted over the centuries, there is currently a gap in knowledge that needs to be filled. Biomolecular archaeological evidence of innate antimicrobial resistance within the oral microbiome shows an increase in the abundance of antimicrobial resistance genes since the advent and widespread use of antibiotics in the modern era. Nevertheless, a lack of research into the prevalence and evolution of antimicrobial resistance within the oral microbiome throughout history hinders our ability to combat antimicrobial resistance in the modern era.

A revisited history of cacao domestication in pre-Columbian times revealed by archaeogenomic approaches.

Humans have a long history of transporting and trading plants, contributing to the evolution of domesticated plants. Theobroma cacao originated in the Neotropics from South America. However, little is known about its domestication and use in these regions. In this study, ceramic residues from a large sample of pre-Columbian cultures from South and Central America were analyzed using archaeogenomic and biochemical approaches. Here we show, for the first time, the widespread use of cacao in South America out of its native Amazonian area of origin, extending back 5000 years, likely supported by cultural interactions between the Amazon and the Pacific coast. We observed that strong genetic mixing between geographically distant cacao populations occurred as early as the middle Holocene, in South America, driven by humans, favoring the adaptation of T. cacao to new environments. This complex history of cacao domestication is the basis of today's cacao tree populations and its knowledge can help us better manage their genetic resources.

Dental calculus in the industrial age: Human dental calculus in the Post-Medieval period, a case study from industrial Manchester.

The analysis of dental calculus (mineralised dental plaque) has become an increasingly important facet of bioarchaeological research. Although microscopic analysis of microdebris entrapped within dental calculus has revealed important insights into the diet, health, and environment of multiple prehistoric populations, relatively few studies have examined the contributions of this approach to more recent historical periods. In this study, we analyze dental calculus from an English Post-Medieval, middle-class urban skeletal assemblage from Manchester, England using light microscopy. We characterize all types of microremains, observing heavily damaged starch and plant material, high quantities of fungal and yeast spores, the presence of wood particles, plant (cotton) and animal (wool) fibres, as well as limited quantities of microcharcoal and burnt debris. We observe the presence of non-native, imported plant products, including New World maize and potentially tapioca starch. We compare our results to similar studies from earlier time periods to reveal the impacts of the significant economic, social and environmental changes occurring during the Industrial period in England, including changes in food processing, food access, food storage, and air quality. We conclude by outlining important methodological considerations for the future study of Post-Medieval dental calculus and propose potential areas of future research.

The prelude to industrial whaling: identifying the targets of ancient European whaling using zooarchaeology and collagen mass-peptide fingerprinting.

Taxonomic identification of whale bones found during archaeological excavations is problematic due to their typically fragmented state. This difficulty limits understanding of both the past spatio-temporal distributions of whale populations and of possible early whaling activities. To overcome this challenge, we performed zooarchaeology by mass spectrometry on an unprecedented 719 archaeological and palaeontological specimens of probable whale bone from Atlantic European contexts, predominantly dating from ca 3500 BCE to the eighteenth century CE. The results show high numbers of Balaenidae (many probably North Atlantic right whale (Eubalaena glacialis)) and grey whale (Eschrichtius robustus) specimens, two taxa no longer present in the eastern North Atlantic. This discovery matches expectations regarding the past utilization of North Atlantic right whales, but was unanticipated for grey whales, which have hitherto rarely been identified in the European zooarchaeological record. Many of these specimens derive from contexts associated with mediaeval cultures frequently linked to whaling: the Basques, northern Spaniards, Normans, Flemish, Frisians, Anglo-Saxons and Scandinavians. This association raises the likelihood that early whaling impacted these taxa, contributing to their extirpation and extinction. Much lower numbers of other large cetacean taxa were identified, suggesting that what are now the most depleted whales were once those most frequently used.

Archaeology demonstrates sustainable ancestral Coast Salish salmon stewardship over thousands of years.

Salmon are an essential component of the ecosystem in Tsleil-Waututh Nation's traditional, ancestral, and contemporary unceded territory, centred on present-day Burrard Inlet, BC, Canada, where Tsleil-Waututh people have been harvesting salmon, along with a wide variety of other fishes, for millennia. Tsleil-Waututh Nation is a Coast Salish community that has called the Inlet home since time immemorial. This research assesses the continuity and sustainability of the salmon fishery at təmtəmíxÊ·tən, an ancestral Tsleil-Waututh settlement in the Inlet, over thousands of years before European contact (1792 CE). We apply Zooarchaeology by Mass Spectrometry (ZooMS) analysis to 245 archaeological salmon vertebrae to identify the species that were harvested by the ancestral Tsleil-Waututh community that lived at təmtəmíxÊ·tən. The results demonstrate that Tsleil-Waututh communities consistently and preferentially fished for chum salmon (Oncorhynchus keta) over the period of almost 3,000 years. The consistent abundance indicates a sustainable chum salmon fishery over that time, and a strong salmon-to-people relationship through perhaps 100 generations. This research supports Tsleil-Waututh Nation's stewardship obligations under their ancestral legal principles to maintain conditions that uphold the Nation's way of life.

North and South: Exploring isotopic analysis of bone carbonates and collagen to understand post-medieval diets in London and northern England.

OBJECTIVES: We evaluate the potential of paired isotopic analysis of bone carbonate and collagen to examine the diet of post-medieval human and animal populations from England (17th-19th c.), including, for the first time, manufacturing towns in northern England. The potential for identifying C4 crop consumption is explored alongside regional and local patterning in diet by sex and socioeconomic status. MATERIALS AND METHODS: Humans (n = 216) and animals (n = 168) were analyzed from sites in London and northern England for both carbon and nitrogen isotopes of bone collagen (𝛿13 Ccoll , 𝛿15 Ncoll ). Isotopic analysis of bone carbonates (𝛿13 Ccarb , 𝛿18 Ocarb ) was carried out on all humans and 27 animals, using Fourier transform infrared spectroscopy-attenuated total reflectance to assess diagenesis. RESULTS: Variations in diet were observed between and within different populations by geographical location and socioeconomic status. Three pigs and one cow consumed C4 resources, indicating the availability of C4 -fed animal protein. Londoners consumed more animal and marine protein and C4 resources. Middle- and upper-class populations from both London and northern populations also had greater access to these foods compared to those of lower status in the same regions. DISCUSSION: This substantial multi-isotope dataset deriving from bone carbonate and collagen combined from diverse post-medieval urban communities enabled, for the first time, the biomolecular identification of the dynamics of C4 consumption (cane sugar/maize) in England, providing insight into the dynamics of food globalization during this period. We also add substantially to the animal dataset for post-medieval England, providing further insight into animal management during a key moment of agricultural change.

Ancient DNA reveals phenological diversity of Coast Salish herring harvests over multiple centuries.

Phenological diversity in food resources prolongs foraging opportunities for consumers and buffers them against environmental disturbances. Such diversity is particularly important in forage fish such as Pacific herring (Clupea pallasii), which are foundational to coastal food webs and fisheries. While the importance of phenological diversity is well-known from contemporary studies, the extent to which different populations contribute to fisheries over long time scales is mostly unknown. In this study, we investigated the relative contributions of genetically and phenologically distinct herring populations to Indigenous Peoples' food systems over multiple centuries, using ancient DNA extracted from archaeological herring bones. These bones were excavated from two Coast Salish archaeological sites (Burton Acres Shell Midden and Bay Street Shell Midden) in the Puget Sound region, USA. Using genetic stock identification from seven nuclear DNA markers, we showed that catches at the two sites in central Puget Sound were dominated by January-February and March-April spawners, which are the contemporary spawning groups in the vicinity of the sites. However, May spawners were detected in the older Burton Acres assemblage (dated to 910-685 cal BP), and a mixed stock analysis indicated that catches at this site consisted of multiple populations. These results suggest that Coast Salish ancestors used a portfolio of herring populations and benefited from the ecological resource wave created by different spawning groups of herring. This study of ancient DNA allowed us to glimpse into Indigenous traditional food and management systems, and it enabled us to investigate long-term patterns of biodiversity in an ecologically important forage fish species.

Palaeogenomic analysis of black rat (Rattus rattus) reveals multiple European introductions associated with human economic history.

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.

Isotope analysis of human dental calculus δ13 CO3 2- : Investigating a potential new proxy for sugar consumption.

RATIONALE: Dental calculus (mineralised dental plaque) is composed primarily of hydroxyapatite. We hypothesise that the carbonate component of dental calculus will reflect the isotopic composition of ingested simple carbohydrates. Therefore, dental calculus carbonates may be an indicator for sugar consumption, and an alternative to bone carbonate in isotopic palaeodiet studies. METHODS: We utilised Fourier transform infrared attenuated total reflectance analysis to characterise the composition and crystallisation of bone and dental calculus before isotope analysis of carbonate. Using a Sercon 20-22 mass spectrometer coupled with a Sercon GSL sample preparation system and an IsoPrime 100 dual inlet mass spectrometer plus Multiprep device to measure carbon, we tested the potential of dental calculus carbonate to identify C4 resources in diet through analysis of δ13 C values in paired bone, calculus and teeth mineral samples. RESULTS: The modern population shows higher δ13 C values in all three tissue carbonates compared to both archaeological populations. Clear differences in dental calculus δ13 C values are observed between the modern and archaeological individuals suggesting potential for utilising dental calculus in isotope palaeodiet studies. The offset between dental calculus and either bone or enamel carbonate δ13 C values is large and consistent in direction, with no consistent offset between the δ13 C values for the three tissues per individual. CONCLUSIONS: Our results support dental calculus carbonate as a new biomaterial to identify C4 sugar through isotope analysis. Greater carbon fractionation in the mouth is likely due to the complex formation of dental calculus as a mineralized biofilm, which results in consistently high δ13 C values compared to bone and enamel.

Indigenous sex-selective salmon harvesting demonstrates pre-contact marine resource management in Burrard Inlet, British Columbia, Canada.

To gain insight into pre-contact Coast Salish fishing practices, we used new palaeogenetic analytical techniques to assign sex identifications to salmonid bones from four archaeological sites in Burrard Inlet (Tsleil-Waut), British Columbia, Canada, dating between about 2300-1000 BP (ca. 400 BCE-CE 1200). Our results indicate that male chum salmon (Oncorhynchus keta) were preferentially targeted at two of the four sampled archaeological sites. Because a single male salmon can mate with several females, selectively harvesting male salmon can increase a fishery's maximum sustainable harvest. We suggest such selective harvesting of visually distinctive male spawning chum salmon was a common practice, most effectively undertaken at wooden weirs spanning small salmon rivers and streams. We argue that this selective harvesting of males is indicative of an ancient and probably geographically widespread practice for ensuring sustainable salmon populations. The archaeological data presented here confirms earlier ethnographic accounts describing the selective harvest of male salmon.

Palaeoproteomic analyses of dog palaeofaeces reveal a preserved dietary and host digestive proteome.

The domestic dog has inhabited the anthropogenic niche for at least 15 000 years, but despite their impact on human strategies, the lives of dogs and their interactions with humans have only recently become a subject of interest to archaeologists. In the Arctic, dogs rely exclusively on humans for food during the winter, and while stable isotope analyses have revealed dietary similarities at some sites, deciphering the details of provisioning strategies have been challenging. In this study, we apply zooarchaeology by mass spectrometry (ZooMS) and liquid chromatography tandem mass spectrometry to dog palaeofaeces to investigate protein preservation in this highly degradable material and obtain information about the diet of domestic dogs at the Nunalleq site, Alaska. We identify a suite of digestive and metabolic proteins from the host species, demonstrating the utility of this material as a novel and viable substrate for the recovery of gastrointestinal proteomes. The recovered proteins revealed that the Nunalleq dogs consumed a range of Pacific salmon species (coho, chum, chinook and sockeye) and that the consumed tissues derived from muscle and bone tissues as well as roe and guts. Overall, the study demonstrated the viability of permafrost-preserved palaeofaeces as a unique source of host and dietary proteomes.

Assessing the degradation of ancient milk proteins through site-specific deamidation patterns.

The origins, prevalence and nature of dairying have been long debated by archaeologists. Within the last decade, new advances in high-resolution mass spectrometry have allowed for the direct detection of milk proteins from archaeological remains, including ceramic residues, dental calculus, and preserved dairy products. Proteins recovered from archaeological remains are susceptible to post-excavation and laboratory contamination, a particular concern for ancient dairying studies as milk proteins such as beta-lactoglobulin (BLG) and caseins are potential laboratory contaminants. Here, we examine how site-specific rates of deamidation (i.e., deamidation occurring in specific positions in the protein chain) can be used to elucidate patterns of peptide degradation, and authenticate ancient milk proteins. First, we characterize site-specific deamidation patterns in modern milk products and experimental samples, confirming that deamidation occurs primarily at low half-time sites. We then compare this to previously published palaeoproteomic data from six studies reporting ancient milk peptides. We confirm that site-specific deamidation rates, on average, are more advanced in BLG  recovered from ancient dental calculus and pottery residues. Nevertheless, deamidation rates displayed a high degree of variability, making it challenging to authenticate samples with relatively few milk peptides. We demonstrate that site-specific deamidation is a useful tool for identifying modern contamination but highlight the need for multiple lines of evidence to authenticate ancient protein data.

Scottish soldiers from the Battle of Dunbar 1650: A prosopographical approach to a skeletal assemblage.

After the Battle Dunbar between English and Scottish forces in 1650, captured Scottish soldiers were imprisoned in Durham and many hundreds died there within a few weeks. The partial skeletal remains of 28 of these men were discovered in 2013. Building on previous osteological work, here we report wide-ranging scientific studies of the remains to address the following questions: Did they have comparable diet, health and disease throughout their lives? Did they have common histories of movement (or lack of movement) during their childhoods? Can we create a collective biography of these men? Strontium and oxygen isotope analysis of tooth enamel investigated childhood movement. Carbon and nitrogen isotope analysis of incrementally sampled dentine addressed childhood diet and nutrition. Metaproteomic analysis of dental calculus investigated oral microbiomes and food residues; this was complemented by microscopic analysis of debris in calculus from ingested materials. Selected individuals were examined for dental microwear. The extent of hydroxylation of proline in collagen was examined as a potential biomarker for scurvy. An osteobiography for each man was created using the full range of data generated about him, and these were synthesised using an approach based on the historical method for a collective biography or prosopography. The childhood residences of the men were primarily within the Midland Valley of Scotland, though some spent parts of their childhood outside the British Isles. This is concordant with the known recruitment areas of the Scottish army in 1650. Their diets included oats, brassicas and milk but little seafood, as expected for lowland rather than highland diets of the period. Childhood periods of starvation or illness were almost ubiquitous, but not simultaneous, suggesting regionally variable food shortages in the 1620s and 1630s. It is likely there was widespread low-level scurvy, ameliorating in later years of life, which suggests historically unrecorded shortages of fruit and vegetables in the early 1640s. Almost all men were exposed to burnt plant matter, probably as inhaled soot, and this may relate to the high proportion of them with of sinusitis. Interpersonal violence causing skeletal trauma was rare. Based on commonalities in their osteobiographies, we argue that these men were drawn from the same stratum of society. This study is perhaps the most extensive to date of individuals from 17th century Scotland. Combined with a precise historical context it allows the lives of these men to be investigated and compared to the historical record with unprecedented precision. It illustrates the power of archaeological science methods to confirm, challenge and complement historical evidence.

Retroviral analysis reveals the ancient origin of Kihnu native sheep in Estonia: implications for breed conservation.

Native animal breeds constitute an invaluable pool of genetic resources in a changing environment. Discovering native breeds and safeguarding their genetic diversity through specific conservation programs is therefore of high importance. Endogenous retroviruses have proved to be a reliable genetic marker for studying the demographic history of sheep (Ovis aries). Previous research has revealed two migratory episodes of domesticated sheep from the Middle East to Europe. The first episode included predominantly 'primitive populations', while the second and most recent is hypothesised to have included sheep with markedly improved wool production. To examine whether the recently discovered Kihnu native sheep in Estonia have historically been part of the first migratory episode and to what extent they have preserved primitive genetic characters, we analysed retroviral insertions in 80 modern Kihnu sheep and 83 ancient sheep from the Bronze Age to Modern Period (850 BCE-1950 CE). We identified that the Kihnu sheep have preserved 'primitive', 'Nordic', and other 'ancient' retrotypes that were present both in archaeological and modern samples, confirming their shared ancestry and suggesting that contemporary Kihnu native sheep originate from the first migratory episode. However, over the course of history, there has been a gradual decrease in the frequency of primitive retrotypes. Furthermore, Kihnu sheep possessed several 'novel' retrotypes that were absent in archaeological individuals, but were shared with improvement breeds, suggesting recent crossing within the last two centuries. To preserve these ancient lineages, our results are being applied in the conservation program of the Kihnu Native Sheep Society.

Living off the land: Terrestrial-based diet and dairying in the farming communities of the Neolithic Balkans.

The application of biomolecular techniques to archaeological materials from the Balkans is providing valuable new information on the prehistory of the region. This is especially relevant for the study of the neolithisation process in SE Europe, which gradually affected the rest of the continent. Here, to answer questions regarding diet and subsistence practices in early farming societies in the central Balkans, we combine organic residue analyses of archaeological pottery, taxonomic and isotopic study of domestic animal remains and biomolecular analyses of human dental calculus. The results from the analyses of the lipid residues from pottery suggest that milk was processed in ceramic vessels. Dairy products were shown to be part of the subsistence strategies of the earliest Neolithic communities in the region but were of varying importance in different areas of the Balkan. Conversely, milk proteins were not detected within the dental calculus. The molecular and isotopic identification of meat, dairy, plants and beeswax in the pottery lipids also provided insights into the diversity of diet in these early Neolithic communities, mainly based on terrestrial resources. We also present the first compound-specific radiocarbon dates for the region, obtained directly from absorbed organic residues extracted from pottery, identified as dairy lipids.

Predicting habitat use by the Argentine hake Merluccius hubbsi in a warmer world: inferences from the Middle Holocene.

Fish skeletal remains recovered from two archaeological sites dated in the Middle Holocene of Tierra del Fuego (Argentina) were analysed to describe habitat use patterns by hake in the past and predict changes in a warmer world. Mitochondrial DNA was successfully extracted and amplified from 42 out of 45 first vertebra from ancient hake and phylogenetic analysis assigned all haplotypes to Argentine hake (Merluccius hubbsi). According to osteometry, the Argentine hake recovered from the archaeological site were likely adults ranging 37.2-58.1 cm in standard length. C and N stable isotope analysis showed that currently Argentine hake use foraging grounds deeper than those of Patagonian blenny and pink cusk-eel. Argentine hake, however, had a much broader isotopic niche during the Middle Holocene, when a large part of the population foraged much shallower than contemporary pink cusk-eel. The overall evidence suggests the presence of large numbers of Argentine hake onshore Tierra del Fuego during the Middle Holocene, which allowed exploitation by hunter-gatherer-fisher groups devoid of fishing technology. Interestingly, average SST off Tierra del Fuego during the Middle Holocene was higher than currently (11 °C vs 7 °C) and matched SST in the current southernmost onshore spawning aggregations, at latitude 47 °S. This indicates that increasing SST resulting from global warming will likely result into an increased abundance of adult Argentine hake onshore Tierra del Fuego, as during the Middle Holocene. Furthermore, stable isotope ratios from mollusc shells confirmed a much higher marine primary productivity during the Middle Holocene off Tierra del Fuego.

Multidisciplinary investigations of the diets of two post-medieval populations from London using stable isotopes and microdebris analysis.

This paper presents the first multi-tissue study of diet in post-medieval London using both the stable light isotope analysis of carbon and nitrogen and analysis of microdebris in dental calculus. Dietary intake was explored over short and long timescales. Bulk bone collagen was analysed from humans from the Queen's Chapel of the Savoy (QCS) (n = 66) and the St Barnabas/St Mary Abbots (SB) (n = 25). Incremental dentine analysis was performed on the second molar of individual QCS1123 to explore childhood dietary intake. Bulk hair samples (n = 4) were sampled from adults from QCS, and dental calculus was analysed from four other individuals using microscopy. In addition, bone collagen from a total of 46 animals from QCS (n = 11) and the additional site of Prescot Street (n = 35) was analysed, providing the first animal dietary baseline for post-medieval London. Overall, isotopic results suggest a largely C3-based terrestrial diet for both populations, with the exception of QCS1123 who exhibited values consistent with the consumption of C4 food sources throughout childhood and adulthood. The differences exhibited in δ15Ncoll across both populations likely reflect variations in diet due to social class and occupation, with individuals from SB likely representing wealthier individuals consuming larger quantities of animal and marine fish protein. Microdebris analysis results were limited but indicate the consumption of domestic cereals. This paper demonstrates the utility of a multidisciplinary approach to investigate diet across long and short timescales to further our understanding of variations in social status and mobility.

Identifying Archaeological Bone via Non-Destructive ZooMS and the Materiality of Symbolic Expression: Examples from Iroquoian Bone Points.

Today, practical, functional and symbolic choices inform the selection of raw materials for worked objects. In cases where we can discern the origin of worked bone, tooth, ivory and antler objects in the past, we assume that similar choices are being made. However, morphological species identification of worked objects is often impossible due to the loss of identifying characteristics during manufacture. Here, we describe a novel non-destructive ZooMS (Zooarchaeology by Mass Spectrometry) method which was applied to bone points from Pre-Contact St. Lawrence Iroquoian village sites in southern Quebec, Canada. The traditional ZooMS technique requires destructive analysis of a sample, which can be problematic when dealing with artefacts. Here we instead extracted proteins from the plastic bags in which the points had been stored. ZooMS analysis revealed hitherto unexpected species, notably black bear (Ursus americanus) and human (Homo sapiens sapiens), used in point manufacture. These surprising results (confirmed through genomic sequencing) highlight the importance of advancing biomolecular research in artefact studies. Furthermore, they unexpectedly and exceptionally allow us to identify and explore the tangible, material traces of the symbolic relationship between bears and humans, central to past and present Iroquoian cosmology and mythology.

Recent dating of extinct Atlantic gray whale fossils, (Eschrichtius robustus), Georgia Bight and Florida, western Atlantic Ocean.

The Atlantic gray whale (Eschrichtius robustus) presents an interesting case study of climate related dispersal and extinction. While (limited) fossil records confirm its presence in the Atlantic up until the 18th Century, its abundance and distribution within the Eastern and Western basins are still not well understood. The discovery of presumed gray whale fossil remains from the Georgia Bight and the Atlantic coast of Florida, from the mid-1980s to late-2000s, provides a new opportunity to recover additional data regarding their chronology within the Western basin. Here, we apply accelerator mass spectrometry radiocarbon techniques to six fossil whale finds, identifying dates within marine isotope stage 3 (59-24 ka) and the late Holocene, ∼2,000 yr BP. We additionally confirm the taxonomic identification of two fossil bone samples as E. robustus using collagen peptide mass fingerprinting (ZooMS). The obtained dates, when combined with a larger corpus of previously published Atlantic gray whale fossil dates, support the hypothesis for the decline of the Atlantic gray whale in the late Pleistocene and the late Holocene. These new data augment the findings of the Eastern Atlantic Basin and better incorporate the Western Atlantic Basin into a pan-ocean understanding for the species.