While the impacts of black ( Rattus rattus ) and brown ( Rattus norvegicus ) rats on human society are well documented—including the spread of disease, broad-scale environmental destruction, and billions spent annually on animal control—little is known about their ecology and behavior in urban areas due to the challenges of studying animals in city environments. We use isotopic and ZooMS analysis of archaeological (1550s–1900 CE) rat remains from eastern North America to provide a large-scale framework for species arrival, interspecific competition, and dietary ecology. Brown rats arrived earlier than expected and rapidly outcompeted black rats in coastal urban areas. This replacement happened despite evidence that the two species occupy different trophic positions. Findings include the earliest molecularly confirmed brown rat in the Americas and show a deep ecological structure to how rats exploit human-structured areas, with implications for understanding urban zoonosis, rat management, and ecosystem planning as well as broader themes of rat dispersal, phylogeny, evolutionary ecology, and climate impacts.

IntroductionElasmobranchs, such as sharks and rays, are among the world’s most endangered vertebrates, with over 70% loss in abundance over the past 50 years due to human impacts. Zooarchaeological baselines of elasmobranch diversity, distribution, and exploitation hold great promise for contributing essential historical contexts in the assessment of contemporary patterns in their taxonomic diversity and vulnerability to human-caused extinction. Yet, the historical ecology of elasmobranchs receives relatively less archaeological attention compared to that of ray-finned fishes or marine mammals, largely due to issues of taxonomic resolution across zooarchaeological identifications.MethodsWe explore the use of Zooarchaeology by Mass Spectrometry (ZooMS) for species identification in this unstudied group, using an archaeological case study from the marine environments of the Florida Keys, a marine biodiversity hotspot that is home to an array of elasmobranch species and conservation efforts. By comparison with 39 modern reference species, we could distinguish 12 taxa within the zooarchaeological assemblage from the Clupper archaeological site (Upper Matecumbe Key) that included nine sharks, two rays and a sawfish.Results and discussionThe results indicate that, through additional complexity of the collagen peptide mass fingerprint, obtained due to the presence of the cartilaginous type II collagen, ZooMS collagen peptide mass fingerprinting provides exceptionally high taxonomic resolution in this group, yielding species-level identifications in all cases where sufficient reference material was used. This case study also highlights the added value of ZooMS for taxa that are more difficult to distinguish in zooarchaeological analyses, such as vertebrae of the Atlantic sharpnose shark (Rhizoprionodon terraenovae) and the hammerhead sharks (Sphyrna spp.) in the Florida Keys. Therefore, the application of collagen peptide mass fingerprinting to elasmobranchs offers great potential to improve our understanding of their archaeological past and historical ecology.

As the vast majority of excavated archaeofaunal skeletal remains are fragmentary to the extent that they cannot be identified by morphological analysis alone, various molecular methods have been considered to retrieve information from an otherwise underutilised resource. The introduction of collagen fingerprinting, known as Zooarchaeology by Mass Spectrometry (ZooMS), has become one of the most popular approaches to improve taxonomic data yields from fragmentary bone. However, few studies have analysed large numbers of samples. Here we test the incorporation of liquid-handling robots to further develop ZooMS into a more automated technique using samples excavated from Grotte Mandrin, France. By increasing the faunal identifications of the B2 layer (~42-44 Ka) at Grotte Mandrin from 55 to 1037, we identified a wider range of taxa, now including bear and mammoth, as well as further remains of hominin. AutoZooMS has the capacity to investigate larger proportions of archaeofaunal assemblages rapidly and cost effectively whilst requiring little human intervention, aiming to improve our understanding of the human past.

We report the first identifications of species and element used to produce Paleolithic bone needles. Archaeologists have used the tailored, fur-fringed garments of high latitude foragers as modern analogs for the clothes of Paleolithic foragers, arguing that the appearance of bone needles and fur bearer remains in archaeological sites c. 40,000 BP is indirect evidence for the advent of tailored garments at this time. These garments partially enabled modern human dispersal to northern latitudes and eventually enabled colonization of the Americas ca. 14,500 BP. Despite the importance of bone needles to explaining global modern human dispersal, archaeologists have never identified the materials used to produce them, thus limiting understanding of this important cultural innovation. We use Zooarchaeology by Mass Spectrometry (ZooMS) and Micro-CT scanning to establish that bone needles at the ca. 12,900 BP La Prele site (Wyoming, USA) were produced from the bones of canids, felids, and hares. We propose that these bones were used by the Early Paleoindian foragers at La Prele because they were scaled correctly for bone needle production and readily available within the campsite, having remained affixed to pelts sewn into complex garments. Combined with a review of comparable evidence from other North American Paleoindian sites, our results suggest that North American Early Paleoindians had direct access to fur-bearing predators, likely from trapping, and represent some of the most detailed evidence yet discovered for Paleoindian garments.

In northern Europe, the first indications of a Neolithic lifestyle appear around 4000 cal BC from northern Germany up to middle Sweden and south-eastern Norway in an apparently short period of time, largely carried by immigrant populations bringing new species of plants and animals into the region. However, the nature of this domestication “package” is not everywhere the same, whereby both environmental and cultural filters acted on the particular set of species cultivated and bred in different regions. In Neolithic Scandinavia, cattle, pigs, and caprines (sheep and goat) are all present in varying proportions, with cattle more prominent in Denmark and pigs more prominent in more northerly areas. However, little is known about the ratio of sheep to goat remains within this region, largely due to difficulties in morphologically separating the two species. In this paper, we report the results from ZooMS analysis of 45 sheep/goat bone samples from two recently excavated Funnel Beaker settlements in Karleby, Falbygden, Sweden. The ZooMS analyses gave a clear and somewhat surprising result: 33 of the samples were classed as sheep, one as deer, and none of them as goat. In all likelihood, goats have not been present at all on these sites. A survey of the literature shows that while small numbers of goats are likely present in Denmark from the Early Neolithic, their presence in Sweden at this time is ambiguous and the few claims merit reassessment. Furthermore, the low numbers in Scandinavia compared to central and southern Europe suggests an overall geographic trend, with decreasing proportions of caprines as well as goats in the north.

ABSTRACT New stable carbon (δ13C) and nitrogen (δ15N) isotope values of charred plant and bone collagen remains from 6th mill. BCE Halai, central Greece, together with datasets from 6th mill. BCE Kouphovouno, southern Greece, and later 6th/early 5th mill. BCE Makriyalos, northern Greece, demonstrate how early farming communities in mainland Greece adapted mixed farming strategies to distinct local environmental and cultural settings. Intra-site similarities and differences in δ13C and δ15N values of distinct crop species, along with the intra-species variabilities in stable isotopic values, are used to assess the cultivation choices that farmers at the three sites made to fulfil distinct economic goals. At Halai, farmers cultivated multiple crops under variable soil conditions, a strategy likely geared towards minimising overall risk in a relatively arid coastal setting. At better-watered Kouphovouno, by contrast, farmers practiced strategic manuring to maximise the yield of free-threshing wheat, likely grown exclusively for human consumption and rotated with nitrogen-fixing pulses. At Makriyalos, the limited sample size of cereal remains suggests a lack of intensive manuring, in contrast to the two other sites. Assessment of which crops may have been consumed by livestock sheds further light on symbiotic relationships between crop cultivation and animal husbandry.

Fish represent a key economic, social and ecological group of species that humans have exploited for tens of thousands of years. However, as many fish stocks are going into decline and with little known about the anthropogenic impacts on the health of the marine ecosystem pre-Industrial Revolution, understanding historical and archaeological exploitation of fish species is key to accurately modelling these changes. Here, we explore the potential of collagen peptide mass fingerprinting (also known as Zooarchaeology by Mass Spectrometry, or ZooMS) for identifying fish remains from the Medieval (fifteenth century) Newport ship wreck (Wales, UK), and in doing so we establish a set of biomarkers we consider useful in discriminating between European fish taxa through the inclusion of over 50 reference taxa. The archaeological results identified nine distinct taxonomic groups, dominated by ling (> 40%), and a substantial amount of cod (> 20%) and hake (~ 20%). The vast majority of samples (> 70%) were identified to species level, and the inability to identify the remaining taxonomic groups with confidence using ZooMS was due to the fact that the reference collection, despite being relatively large in comparison to those presented in mammalian studies, reflects only a small proportion of fish biodiversity from this region. Although the results clearly demonstrate the potential for ZooMS as a means of fish bone identification, the sheer number of different fish species that potentially make up ichthyoarchaeological assemblages leads to obvious requirements for the analysis on much greater numbers of modern reference specimens, or the acquisition of collagen sequences.

People could have hunted Madagascar’s megafauna to extinction, particularly when introduced taxa and drought exacerbated the effects of predation. However, such explanations are difficult to test due to the scarcity of individual sites with unambiguous traces of humans, introduced taxa, and endemic megaherbivores. We excavated three coastal ponds in arid SW Madagascar and present a unique combination of traces of human activity (modified pygmy hippo bone, processed estuarine shell and fish bone, and charcoal), along with bones of extinct megafauna (giant tortoises, pygmy hippos, and elephant birds), extirpated fauna (e.g., crocodiles), and introduced vertebrates (e.g., zebu cattle). The disappearance of megafauna from the study sites at ~ 1000 years ago followed a relatively arid interval and closely coincides with increasingly frequent traces of human foraging, fire, and pastoralism. Our analyses fail to document drought-associated extirpation or multiple millennia of megafauna hunting and suggest that a late combination of hunting, forest clearance, and pastoralism drove extirpations.

Animal symbolism is a prominent feature of many human societies globally. In some cases, these symbolic attributes manifest in the technological domain, influencing the decision to use the bones of certain animals and not others for tool manufacture. In southern Africa, animals feature prominently in the cosmogenic narratives of both hunter-gatherer and Bantu-speaking farmer groups. Whenever these two culturally distinct groups came into contact with each other there would be an assimilation of cosmogenic concepts of power and the adoption of certain symbolically important animals. In this paper, we report on which animals were selected to make bone tools during the first millennium AD contact period in KwaZulu-Natal Province, South Africa, and explore the extent to which this selection may have been influenced by the symbolic associations of specific animals. Our results show selective targeting of specific animals for tool manufacture at some sites, with a narrowing of the range of selected species during the first millennium AD contact period. Certain antelope tribes, such as Aepycerotini, Cephalophini and Antilopini, appear to have been deliberately avoided, thus arguing against opportunistic selection. Nor does the range of selected animals appear to show any obvious mechanical considerations, as has been noted in similar studies. We highlight the potential of ZooMS for understanding the dynamics of animal symbolism in the past.

Archaeological ceramics are a rich source of ancient biomolecules and their analysis provides valuable information with regard to ancient diet. Traditionally, biomolecular analyses on archaeological ceramics have focused on lipid analysis, with milk, ruminant and non-ruminant adipose fats, plant resins, as well as both marine fish and marine mammals among the resources successfully identified. However, lipidomic analyses can underestimate the presence of lipid-poor resources (e.g. cereals and grains) and are usually less specific at tissue and taxonomic levels. Proteomic analysis can be complementary, allowing for the identification of a greater variety of resources and with increased specificity. Here, we analyse eight ceramic sherds from Tell Khaiber, a Sealand Dynasty (mid-2nd millennium BCE) settlement in modern day southern Iraq, using LC-MS/MS based shotgun proteomics. Our results provide the first proteomic evidence of soybeans from archaeological ceramics and confirmation of the early appearance of soybean products in the Middle East by the mid-second millennium BCE.

Denisova Cave, a Pleistocene site in the Altai Mountains of Russian Siberia, has yielded significant fossil and lithic evidence for the Pleistocene in Northern Asia. Abundant animal and human bones have been discovered at the site, however, these tend to be highly fragmented, necessitating new approaches to identifying important hominin and faunal fossils. Here we report the results for 8253 bone fragments using ZooMS. Through the integration of this new ZooMS-based data with the previously published macroscopically-identified fauna we aim to create a holistic picture of the zooarchaeological record of the site. We identify trends associated with climate variability throughout the Middle and Upper Pleistocene as well as patterns explaining the process of bone fragmentation. Where morphological analysis of bones from the site have identified a high proportion of carnivore bones (30.2%), we find that these account for only 7.6% of the ZooMS assemblage, with large mammals between 3 and 5 more abundant overall. Our analysis suggests a cyclical pattern in fragmentation of bones which sees initial fragmentation by hominins using percussive tools and secondary carnivore action, such as gnawing and digestion, likely furthering the initial human-induced fragmentation.

Collagen peptide mass fingerprinting of archaeofaunal remains, or Zooarchaeology by Mass Spectrometry (ZooMS), has increasingly established itself as a valuable tool for improving our understanding of highlyfragmented faunal assemblages. Although there have been developments in sampling strategies that have attempted to reduce damage to precious archaeological specimens, these tend to yield spectra inferior in quality to those yielded by destructive approaches. Also, in an effort to mitigate the impacts on faunal assemblages, researchers are beginning to turn to microCT for the digital preservation of specimens, as this enhances their value for further studies. Here we combine ZooMS and microCT, in application to over one hundred scombrid remains from the Hanamiai site in the Marquesas, which spans the time period from initial human colonization by Polynesians at ca. AD 1250 until around 1900. Almost all of the hypurals that yielded collagen fingerprints (71 of 73) were confirmed as skipjack. The results suggest striking continuity over time in fishing practices with an inferred emphasis on fishing strategies involving sailing canoes being present from the time of initial settlement. The focus appears to be on daytime fishing for skipjack rather than on night fishing for yellowfin, kawakawa and dogtooth tuna as might otherwise have been expected.

Decay experiments have the potential to provide useful analogues in the interpretation of archaeological remains. Previous studies have focused on how physical properties or processing methods can influence fish bone distributions within archaeological sites. However, the means by which intrinsic chemical properties of fish bone, such as baseline collagen type I ['collagen (I)'] chemistry, may affect both biomolecule and whole bone degradation has not been the focus of any prior study. The variation of facies and resulting impact on taphonomy is not a new concept, but an understanding of the discrete relationship between temperature and the breakdown of collagen (I) in bone material has not been well explored. Here, modern fish bone powder is subjected to differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and water-immersed heat experiments. This is to test whether taxa with less thermally stable collagen (I) configurations, such as cold-water species with reduced proline and hydroxyproline concentrations (Pro+Hyp), will generate collagen breakdown products (TOC and TN) more rapidly than those with more thermally stable arrangements, such as warm water fish with typically increased Pro+Hyp. Our results show that bone collagen (I) in the cold-water fish species in this study (cod and herring with lower Pro+Hyp concentrations) display significantly increased decomposition rates than collagen (I) from the warm-water fishes in this study (amberjack and tilapia with higher Pro+Hyp concentrations), given the same experimental conditions (heating in water at 75 degrees C for up to eight days). Initial reaction rate estimates, based on TOC and TN product concentrations, suggest that cod bone (15.6% Pro+Hyp) reacts similar to 9 times faster than tilapia bone (20.3% Pro+Hyp). We suggest that the primary influencer of collagen (I) stability in bone is the concentration of Pro+Hyp residues and not a function of physical bone structure. Our results suggest that a reduction in collagen (I) stability is likely to lead to a decrease in whole bone stability following deposition, due to the intimate association between organic and inorganic phases of bone. Therefore, species composition based upon bone remains may vary in archaeological and palaeontological sites, as a function of the thermal stability of collagen (I).

Agro-pastoral economies of prehistoric populations of Central Tian Shan highlands (2,000 masl and higher) have been poorly studied to date. Currently, we lack a general understanding of mobility and seasonality patterns of livestock herding and also lack knowledge about management strategies for particular productive goals in these high mountain valleys. In this paper, we report the results of the first systematic zooarchaeological analysis from the Final Bronze Age-Early Iron Age settlement Chap I located in Central Tian Shan highlands and discuss the data in relation to zooarchaeological evidence from contemporaneous sites in Central Asia. Our research has shown that Chap I was dominated by four domesticated herbivores: cattle, horses, sheep and goats. Data from Chap I demonstrate a strong focus on the exploitation of sheep and goats for meat and secondary products. Analysis of collagen peptides (ZooMS) from sheep/goats indicated that sheep were kept in greater numbers than goats. Sheep/goat mortality profiles and material evidence point to wool as an important product of highland pastoralism in the Central Tian Shan.

An accurate understanding of biodiversity of the past is critical for contextualizing biodiversity patterns and trends in the present. Emerging techniques are refining our ability to decipher otherwise cryptic human-mediated species translocations across the Quaternary, yet these techniques are often used in isolation, rather than part of an interdisciplinary hypothesis-testing toolkit, limiting their scope and application. Here we illustrate the use of such an integrative approach and report the occurrence of North America’s largest terrestrial mammalian carnivore, the short-faced bear, Arctodus simus, from Daisy Cave (CA-SMI-261), an important early human occupation site on the California Channel Islands. We identified the specimen by corroborating morphological, protein, and mitogenomic lines of evidence, and evaluated the potential natural and anthropogenic mechanisms of its transport and deposition. While representing just a single specimen, our combination of techniques opened a window into the behavior of an enigmatic species, suggesting that A. simus was a wide-ranging scavenger utilizing terrestrial and marine carcasses. This discovery highlights the utility of bridging archaeological and paleontological datasets to disentangle complex biogeographic scenarios and reveal unexpected biodiversity for island systems worldwide.

Despite the longstanding significance of North America's Great Lakes, little is known about their preindustrial ecology. Here, we report on when and how humans first became a main driver of Lake Ontario's nutrient dynamics. Nitrogen isotope analyses of archaeological fish show that, prior to the 1830s, Lake Ontario's nitrogen cycle and the trophic ecology of its top predators had remained stable for at least 800 yrs, despite Indigenous and historical European agricultural land management across the region. An abrupt shift in the nitrogen isotope composition of Lake Ontario's fish community is evident in the early to mid-19(th)century and reflects the initiation of industrial-scale forest clearance. These data show how the nitrogenous nutrient regimes of even the world's largest freshwater ecosystems can be highly sensitive to short-term watershed forest cover disturbances and indicate a profound shift in the relationship between humans and their environment.

Understanding past human settlement of inhospitable regions is one of the most intriguing puzzles in archaeological research, with implications for more sustainable use of marginal regions today. During the Byzantine period in the 4th century CE, large settlements were established in the arid region of the Negev Desert, Israel, but it remains unclear why it did so, and why the settlements were abandoned three centuries later. Previous theories proposed that the Negev was a "green desert" in the early 1st millennium CE, and that the Byzantine Empire withdrew from this region due to a dramatic climatic downturn. In the absence of a local climate archive correlated to the Byzantine/Early Islamic transition, testing this theory has proven challenging. We use stable isotopic indicators of animal dietary and mobility patterns to assess the extent of the vegetative cover in the desert. By doing so, we aim to detect possible climatic fluctuations that may have led to the abandonment of the Byzantine settlements. The findings show that the Negev Desert was not greener during the time period under investigation than it is today and that the composition of the animals' diets, as well as their grazing mobility patterns, remained unchanged through the Byzantine/Early Islamic transition. Favoring a non-climatic explanation, we propose instead that the abandonment of the Negev Byzantine settlements was motivated by restructuring of the Empire's territorial priorities.

The earliest Native Americans have often been portrayed as either megafaunal specialists or generalist foragers, but this debate cannot be resolved by studying the faunal record alone. Stable isotope analysis directly reveals the foods consumed by individuals. We present multi-tissue isotope analyses of two Ancient Beringian infants from the Upward Sun River site (USR), Alaska (~11,500 years ago). Models of fetal bone turnover combined with seasonally-sensitive taxa show that the carbon and nitrogen isotope composition of USR infant bone collagen reflects maternal diets over the summer. Using comparative faunal isotope data, we demonstrate that although terrestrial sources dominated maternal diets, salmon was also important, supported by carbon isotope analysis of essential amino acids and bone bioapatite. Tooth enamel samples indicate increased salmon use between spring and summer. Our results do not support either strictly megafaunal specialists or generalized foragers but indicate that Ancient Beringian diets were complex and seasonally structured.

Bats form the second most diverse mammalian order (Chiroptera), after rodents, and vary widely in their physiology and ecology. Those species that live in temperate climates are generally insectivorous and nocturnal or crepuscular, sheltering in tree hollows, caves, or buildings during the day. They are potentially valuable ecological indicators, due to their dependence on suitable roosting sites and arthropod food, both of which are commonly affected by human activities. Identification of bats from ancient assemblages that are found in caves could therefore provide useful data for palaeoenvironmental reconstructions and show the effect of habitat loss. Here, we apply the recently developed approach of collagen fingerprinting by soft ionisation mass spectrometry to the identification of ancient bat remains in an archaeological assemblage from Pin Hole Cave (Derbyshire, England). Our results show that a simple set of markers can distinguish all seven genera of bats known to be present in either modern or ancient Britain ( Myotis , Nyctalus , Pipistrellus , Barbastella , Plecotus , Eptesicus , and Rhinolophus ). Further analysis indicates that species‐level determination is possible in some of these taxa, but it would more readily be achieved using the more advanced methods of collagen sequence analysis by liquid chromatography coupled with tandem mass spectrometry. Within our assemblage yielding ~6,800 ancient bone collagen fingerprints, we identified only ~1% that derived from chiropterans, and these were predominantly derived from Myotis (two apparent Brandt's bat fingerprints but the majority indistinguishable between the whiskered, Daubenton's and Natterer's bats), Barbastella (the western barbastelle being the only member of this genus known within Europe), and Rhinolophus (identified as the lesser horsehoe bat R. hipposideros rather than the rare greater horseshoe bat R. ferrumequinum ). We infer that the site was likely used by roosting bats throughout the year, and the accumulation of these remains was probably not the result of predator activity. More importantly, the peptide biomarkers provided here could proove valuable in the more systematic analysis of microfaunal remains across many European archaeological and palaeontological sites, preferably those that are collected with well curated stratigraphical information and chronological frameworks.

Attempts to extend methods for dating archaeological bones beyond that of radiocarbon dating, such as amino acid racemization, have met with limited success owing to the dependence on multiple environmental factors and controls. Despite facing similar challenges, deamidation of glutamine has recently been investigated as a potential indicator of ‘thermal age' in archaeological bones, as well as a measure of their preservation quality. In this study, we undertook a series of simulated diagenetic experiments to understand the various factors affecting deamidation. Further, we analysed bones from different Middle Palaeolithic layers from Grotte Mandrin (France), with the results suggesting potential use of deamidation for relative dating, but only in case of extremely well‐preserved layers. The results also suggested the possible use of attenuated total reflectance‐Fourier transform infrared (ATR‐FTIR) spectroscopy as a screening test for soluble collagen before proteomic analysis.