Collagen Peptide Mass Fingerprinting Research Articles

ZooMS confirms geometric morphometrics species identification of ancient sheep and goat.

Geometric morphometrics can effectively distinguish isolated third lower molars of present-day sheep and goat, but its applicability to archaeological specimens has yet to be established. Using a modern reference collection of 743 sheep and goats and a two-dimensional landmark-based geometric morphometric (GMM) protocol, this study aimed to morphometrically identify 109 archaeological specimens, used as case studies, dating from the Late Neolithic to the modern period/era. These morphometric identifications were then compared to molecular identifications via collagen peptide mass fingerprinting, known as Zooarcheology by Mass Spectrometry (ZooMS). ZooMS confirmed the morphometric identifications for 104 specimens, with the five misidentified specimens all morphometrically identified as goat. Modern sheep and goats have larger teeth and distinct shapes compared to their archaeological counterparts, suggesting strong differences between archaeological and modern specimens potentially linked with recent breed improvement or geographical origin of the specimens. In addition, for both species, some of the archaeological dental morphologies do not match with any of our modern references. This study validates the applicability of geometric morphometrics for identifying isolated archaeological sheep and goat teeth. It represents a stepping stone for future, non-destructive, bioarchaeological studies of the two species.

Geometric morphometric analysis of Pleuronectiformes vertebrae: A new tool to identify archaeological fish remains?

Flatfish (Pleuronectiformes) vertebrae are difficult to identify to species due to the lack of diagnostic features. This has resulted in a lack of understanding of the species abundances across archaeological sites, hindering interpretations of historical fisheries in the North Sea area. We use a new approach, utilising a combined 2D landmark-based geometric morphometric analysis as an objective and non-destructive method for species identification of flatfish vertebrae from the North Sea area. Modern specimens were used as a reference to describe the morphological variation between taxa using principal component analysis (PCA) and to trial an automated classification using linear discriminant analysis. Although there is limited distinction between taxa using PCAs, the classification shows high accuracies, indicating that flatfish species identifications using geometric morphometrics are possible. Bone samples (n = 105) from two archaeological sites in the United Kingdom and France were analysed using this approach and their identifications were verified using collagen peptide mass fingerprinting. The success rate of species identification was usually less than 50%, indicating that this technique has limited applicability due to preservation/fragmentation of archaeological fish bone. Nonetheless, this could prove a valuable tool for modern and non-fragmented samples. Furthermore, the technique applied in this study can be easily adapted to work on other landmark datasets.

Rapid collagen peptide mass fingerprinting as a tool to authenticate Pleuronectiformes in the food industry

Reliable species identification of specimens in the food chain is crucial for detecting mislabelling fraud. While visual identification, DNA-analyses, and proteomic approaches have been used in the past, this research provides an alternative proteomic technique that has been trialled here to identify tissue samples from species within the order Pleuronectiformes (flatfish). Previously, archaeological research has used peptide mass fingerprinting to identify fish remains to species by extracting collagen Type I preserved in bones. As this type of collagen can also be found in the fins, skin and muscle of fishes (types of tissue that can be found throughout the food chain), collagen peptide mass fingerprinting has been applied here to identify modern fish specimens to species. In this study, MALDI-ToF MS peptide fingerprints are obtained from enzymatic digestion of collagen Type I from fin (or skin if fin was not available) and muscle tissues from six specimens of Pleuronectiformes. Using the already available diagnostic collagen peptide biomarkers for flatfish, all six specimens could be identified to the correct species for each of the tissue types. We recommend using this rapid approach for future screenings of modern flatfish in the food chain to detect mislabelling fraud, and also in more general applications of modern ecological studies of this order. It is likely that the methods can be applied to other Actinopterygian fishes.

A comparison of minimally-invasive sampling techniques for ZooMS analysis of bone artifacts

Bone and antler are important raw materials for tool manufacture in many cultures, past and present. The modification of osseous features which take place during artifact manufacture frequently make it difficult to identify either the bone element or the host animal, which can limit our understanding of the cultural, economic, and/or symbolic factors which influence raw material acquisition and use. While biomolecular approaches can provide taxonomic identifications of bone or antler artifacts, these methods are frequently destructive, raising concerns about invasive sampling of culturally-important artifacts or belongings. Collagen peptide mass fingerprinting (Zooarchaeology by Mass Spectrometry or ZooMS) can provide robust taxonomic identifications of bone and antler artifacts. While the ZooMS method commonly involves destructive subsampling, multiple minimally-invasive sampling techniques also exist. In this paper, we compare three previously proposed minimally-invasive sampling methods (forced bag, eraser, and polishing film) on an assemblage of 15 bone artifacts from the pre-contact site EjTa-4, a large midden complex located on Calvert Island, British Columbia, Canada. We compare the results of the minimally-invasive methods to 10 fragmentary remains sampled using the conventional destructive ZooMS method. We assess the reliability and effectiveness of these methods by comparing MALDI-TOF spectral quality, the number of diagnostic and high molecular weight peaks as well as the taxonomic resolution reached after identification. We find that coarse fiber-optic polishing films are the most effective of the minimally-invasive techniques compared in this study, and that the spectral quality produced by this minimally-invasive method was not significantly different from the conventional destructive method. Our results suggest that this minimally-invasive sampling technique for ZooMS can be successfully applied to culturally significant artifacts, providing comparable taxonomic identifications to the conventional, destructive ZooMS method.

Your horse is a donkey! Identifying domesticated equids from Western Iberia using collagen fingerprinting

Skeletal remains of two equid species, Equus caballus (horse) and Equus asinus (donkey), have been found in archaeological contexts throughout Iberia since the Palaeolithic and Chalcolithic periods, respectively. These two species play different economic and cultural roles, and therefore it is important to be able to distinguish between the two species to better understand their relative importance in the past human societies. The most reliable morphological features for distinguishing between the two domesticated equids are based on cranial measurements and tooth enamel folds, leading to only a small percentage of archaeological remains that can be identified to species. Ancient DNA (aDNA) analysis can be used to reliably distinguish the two equids, but it can be cost prohibitive to apply to large assemblages, and aDNA preservation of non-cranial elements is often low. Collagen peptide mass fingerprinting by matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry, also known as zooarchaeology by mass spectrometry (ZooMS), is a minimally destructive and cost-effective alternative to aDNA analysis for taxonomic determination. However, current ZooMS markers lack resolution below the genus level Equus. In this paper, we report a novel ZooMS peptide marker that reliably distinguishes between horses and donkeys using the enzyme chymotrypsin. We apply this peptide marker to taxonomically identify bones from the Iberian Peninsula ranging from the Iron Age to the Late Modern Period. The peptide biomarker has the potential to facilitate the collection of morphological data for zooarchaeological studies of equids in Iberia and throughout Eurasia and Africa.

A primer for ZooMS applications in archaeology

Collagen peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, also known as zooarchaeology by mass spectrometry (ZooMS), is a rapidly growing analytical technique in the fields of archaeology, ecology, and cultural heritage. Minimally destructive and cost effective, ZooMS enables rapid taxonomic identification of large bone assemblages, cultural heritage objects, and other organic materials of animal origin. As its importance grows as both a research and a conservation tool, it is critical to ensure that its expanding body of users understands its fundamental principles, strengths, and limitations. Here, we outline the basic functionality of ZooMS and provide guidance on interpreting collagen spectra from archaeological bones. We further examine the growing potential of applying ZooMS to nonmammalian assemblages, discuss available options for minimally and nondestructive analyses, and explore the potential for peptide mass fingerprinting to be expanded to noncollagenous proteins. We describe the current limitations of the method regarding accessibility, and we propose solutions for the future. Finally, we review the explosive growth of ZooMS over the past decade and highlight the remarkably diverse applications for which the technique is suited.

Medieval fish remains on the Newport ship identified by ZooMS collagen peptide mass fingerprinting

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.

The earliest Denisovans and their cultural adaptation.

Since the initial identification of the Denisovans a decade ago, only a handful of their physical remains have been discovered. Here, we analyse ~3800 non-diagnostic bone fragments using collagen peptide mass fingerprinting to locate new hominin remains from Denisova Cave (Siberia, Russia). We identify five new hominin bones, four of which contained sufficient DNA for mitochondrial analysis. Three carry mtDNA of the Denisovan type and one is found to carry mtDNA of the Neanderthal type. The former come from the same archaeological layer, near the base of the cave’s sequence, and are the oldest securely dated evidence of Denisovans, at 200 ka (205-192 ka at 68.2% or 217-187 ka at 95% probability). The stratigraphic context in which they were located contains a wealth of archaeological material in the form of lithics and faunal remains, allowing us to determine the material culture associated with these early hominins and explore their behavioural and environmental adaptations. The combination of bone collagen fingerprinting and genetic analyses has so far more-than-doubled the number of hominin bones at Denisova Cave and has expanded our understanding of Denisovan and Neanderthal interactions as well as their archaeological signatures.

High-throughput microCT and ZooMS collagen fingerprinting of Scombrid bone from the Marquesas Islands

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 highly-fragmented 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.

Iron Age hunting and herding in coastal eastern Africa: ZooMS identification of domesticates and wild bovids at Panga ya Saidi, Kenya

The morphological differentiation of African bovids in highly fragmented zooarchaeological assemblages is a major hindrance to reconstructing the nature and spread of pastoralism in sub-Saharan Africa. Here we employ collagen peptide mass fingerprinting, known as Zooarchaeology by Mass Spectrometry (ZooMS), coupled with recently published African ZooMS reference datasets, to identify domesticates and wild bovids in Iron Age assemblages at the cave site of Panga ya Saidi in southeast Kenya. Through ZooMS we have identified all three major African livestock—sheep (Ovis aries), goat (Capra hircus) and cattle (Bos taurus)—at the site for the first time. The results provide critical evidence for the use of domesticates by resident foraging populations during the Iron Age, the period associated with the arrival of food production in coastal Kenya. ZooMS results show that livestock at Panga ya Saidi form a minor component of the assemblage compared to wild bovids, demonstrating the persistence of hunting and the secondary role of acquiring livestock in hunter-gatherer foodways during the introduction of agro-pastoralism. This study sheds new light on the establishment of food production in coastal eastern Africa, particularly the role of interactions between hunter-gatherers and neighbouring agro-pastoral groups in what was a protracted regional transition to farming.

Evidence for early dispersal of domestic sheep into Central Asia.

The development and dispersal of agropastoralism transformed the cultural and ecological landscapes of the Old World, but little is known about when or how this process first impacted Central Asia. Here, we present archaeological and biomolecular evidence from Obishir V in southern Kyrgyzstan, establishing the presence of domesticated sheep by ca. 6,000 BCE. Zooarchaeological and collagen peptide mass fingerprinting show exploitation of Ovis and Capra, while cementum analysis of intact teeth implicates possible pastoral slaughter during the fall season. Most significantly, ancient DNA reveals these directly dated specimens as the domestic O. aries, within the genetic diversity of domesticated sheep lineages. Together, these results provide the earliest evidence for the use of livestock in the mountains of the Ferghana Valley, predating previous evidence by 3,000 years and suggesting that domestic animal economies reached the mountains of interior Central Asia far earlier than previously recognized.

Testing the efficacy and comparability of ZooMS protocols on archaeological bone

Collagen peptide mass fingerprinting, best known as Zooarchaeology by Mass Spectrometry (or ZooMS) when applied to archaeology, has become invaluable for the taxonomic identification of archaeological collagenous materials, in particular fragmentary and modified bone remains. Prior to MALDI-based spectrometric analysis, collagen needs to be extracted from the bone's inorganic matrix, isolated and purified. Several protocols are currently employed for ZooMS analysis, however their efficacy and comparability has not been directly tested. Here, we use four different ZooMS protocols to analyze 400 bone samples from seven archaeological sites, dating to between ~500,000–2000 years ago. One of them, single-pot solid-phase-enhance sample preparation (SP3), is used for the first time as a ZooMS protocol. Our results indicate that the least-destructive ZooMS protocol which uses an ammonium bicarbonate buffer as a means of extracting collagen is most suitable for bones with good collagen preservation, whereas the acid-based methodologies can improve success rates for bones with low-to-medium collagen preservation. Since preservation of biomolecules in archaeological bones is highly variable due to age and environmental conditions, we use the percent nitrogen by weight (%N) value as an independent semi-quantitative proxy for assessing collagen content and for predicting which bones will likely result in a successful ZooMS-based identification. We find that 0.26%N as a threshold for screening material could optimize the number of spectra which produce identifications using ZooMS. Significance statementWe present a direct comparison of three previously published ZooMS protocols for the analyses of archaeological bones, and the first use of an SP3-based approach to ZooMS analysis. Our results show that the acid-based ZooMS protocols increase the success rate for bones with low-medium collagen preservation. We identify 0.26%N as a threshold for optimizing the number of samples with enough collagen for successful peptide mass fingerprinting.

Human and cervid osseous materials used for barbed point manufacture in Mesolithic Doggerland

Barbed bone points originally deposited in Doggerland are regularly collected from the shores of the Netherlands. Their typology and direct 14C dating suggest they are of Mesolithic age. However, the species of which the barbed points were made cannot be identified based on morphological criteria. The bones used to produce the barbed points have been intensively modified during manufacture, use, and post-depositional processes. Here, we taxonomically assess ten barbed points found on the Dutch shore using mass spectrometry and collagen peptide mass fingerprinting alongside newly acquired 14C ages and δ13C and δ15N measurements.Our results demonstrate a sufficient preservation of unmodified collagen for mass spectrometry-based taxonomic identifications of bone and antler artefacts which have been preserved in marine environments since the beginning of the Holocene. We show that Homo sapiens bones as well as Cervus elaphus bones and antlers were transformed into barbed points. The 14C dating of nine barbed points yielded uncalibrated ages between 9.5 and 7.3 ka 14C BP. The δ13C and δ15N values of the seven cervid bone points fall within the range of herbivores, recovered from the North Sea, whereas the two human bone points indicate a freshwater and/or terrestrial fauna diet.The wide-scale application of ZooMS is a critical next step towards revealing the selection of species for osseous-tool manufacture in the context of Mesolithic Doggerland, but also further afield. The selection of Cervus elaphus and human bone for manufacturing barbed points in Mesolithic Doggerland is unlikely to have been opportunistic and instead seems to be strategic in nature. Further, the occurrence of Homo sapiens and Cervus elaphus bones in our random and limited dataset suggests that the selection of these species for barbed point production was non-random and subject to specific criteria. By highlighting the transformation of human bones into barbed points – possibly used as weapons – our study provides additional evidence for the complex manipulation of human remains during the Mesolithic, now also evidenced in Doggerland.

Combined visual and biochemical analyses confirm depositor and diet for Neolithic coprolites from Skara Brae

Coprolites (fossilized faeces) can provide valuable insights into species’ diet and related habits. In archaeozoological contexts, they are a potential source of information on human-animal interactions as well as human and animal subsistence. However, despite a broad discussion on coprolites in archaeology, such finds are rarely subject to detailed examination by researchers, perhaps due to the destructive nature of traditional analytical methods. Here, we have examined coprolitic remains from the Neolithic (third millennium BCE) settlement at Skara Brae, Orkney, using a range of modern methods: X-ray computed tomography, scanning electron microscopy, lipid and protein analysis (shotgun proteomics of the coprolite matrix as well as collagen peptide mass fingerprinting of isolated bone fragments). This combined approach minimised destructiveness of sampling, leaving sufficient material for subsequent study, while providing more information than traditional morphological examination alone. Based on gross visual examination, coprolites were predominantly attributed to domestic dogs (Canis familiaris), with morphologically identified bone inclusions derived from domestic sheep (Ovis aries) and common voles (Microtus arvalis). Partial dissection of a coprolite provided bone samples containing protein markers akin to those of domestic sheep. Considering the predominance of vertebral and distal limb bone fragments, Skara Brae dogs were probably consuming human butchery or meal refuse, either routinely fed to them or scavenged. The presumably opportunistic consumption of rodents may also have played a role in pest control.

What's the catch? Archaeological application of rapid collagen-based species identification for Pacific Salmon

Pacific salmon (Oncorhynchus spp.) are ecological and cultural keystone species along the Northwest Coast of North America and are ubiquitous in archaeological sites of the region. The inability to morphologically identify salmonid post-cranial remains to species, however, can limit our understanding of the ecological and cultural role different taxa played in the seasonal subsistence practices of Indigenous groups in the past. Here, we present a rapid, cost-effective ZooMS method to distinguish salmonid species based on collagen peptide mass-fingerprinting. Using modern reference material and an assemblage of 28 DNA-identified salmonid bones from the pre-contact Yup'ik site of Nunalleq, Western Alaska, we apply high-resolution mass spectrometry (LC-MS/MS) to identify a series of potential collagen peptide markers to distinguish Pacific salmon. We then confirm these peptide markers with a blind ZooMS analysis (MALDI-TOF-MS) of the archaeological remains. We successfully distinguish five species of anadromous salmon with this ZooMS approach, including one specimen that could not be identified through ancient DNA analysis. Our biomolecular identification of chum (43%), sockeye (21%), chinook (18%), coho (11%) and pink (7%), confirm the exploitation of all five available species of salmonid at Nunalleq.

Une baleine grise dans le Périgord magdalénien : identification taxinomique d’une pointe de projectile en os de La Madeleine (Dordogne, France) par empreinte peptidique de masse du collagène

The Cetacean bone artifact described in this paper is the result of excavations carried out in La Madeleine (Tursac, Dordogne) under the direction of D. Peyrony, between 1910-1913. It was discovered in the middle layer (“couche moyenne”) of the stratigraphy described by Capitan and Peyrony and subsequently attributed to the “Magdalenian V” by H. Breuil (i.e the beginning of the Upper Magdalenian). This layer corresponds to layers C25 to C20 of the stratigraphy established later by J.-M. Bouvier. Two radiocarbon dates are currently available (C21: 12750 ± 240 BP, Ly 920; and C25: 13070 ± 190 BP, Ly 921). The artifact was recently identified as Cetacean bone during an integrated study of the bones and reindeer antlers kept at the Musee National de Prehistoire (Peyrony and Bouvier collections). It is a fully worked object, elongated in shape (129 X 13.2 X 12.8 mm; 18.6 g) with straight outline and profile, a circular cross-section and parallel sides. It is interpreted as a mesio-proximal fragment of projectile point. Collagen peptide mass fingerprinting (PMF) was applied to taxonomically identify the bone artefact, as a part of the PaleoCet project. Also known as ZooMS, collagen PMF discriminates taxonomic groups based on differences in the collagen protein sequence observed through Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The bone subsamples from the exterior and interior of the artifact produced similar MALDI-TOF spectra, both of which could be confidently identified as gray whale (Eschrichtius robustus). The artifact from La Madeleine is the first evidence of the presence of this species in Atlantic waters at the end of MIS2, suggesting continuity between the Pleistocene and Holocene populations of this species. Moreover, to our knowledge, this gray whale bone artifact represents the oldest identified gray whale in an archaeological context, thus first witness of the millennia-old interactions between humans and this highly coastal whale species. The manufacturing of the La Madeleine artifact on the Basque coast, followed by its circulation through the western Pyrenees and then towards the north at the east of Landes and Gironde up to Perigord (a travel distance of roughly 350 km), might be the most plausible route taken by this artifact to reach La Madeleine.

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.

Sorting the sheep from the goats in the Pastoral Neolithic: morphological and biomolecular approaches at Luxmanda, Tanzania

Large-scale reconstructions of the spread of food production systems require fine-scale analyses of dietary evidence. One current impediment to understanding early African pastoralism is a lack of high-resolution portraits of herd management, specifically with respect to sheep (Ovis aries) and goat (Capra hircus), osteologically similar but behaviorally distinct caprines. In this study, we argue for the anthropological relevance of distinguishing sheep and goat remains in African pastoralist contexts, commenting upon implications for ecological settings and pastoralists’ strategies for production and risk management. We explain why sheep/goat distinction is rare in African zooarchaeological studies, particularly in comparison to Southwest Asia. We then apply three methods to distinguish caprines in an archaeofaunal sample from the Pastoral Neolithic site of Luxmanda, Tanzania, dated to c. 3000 BP: morphological identifications by two independent analysts, collagen-peptide mass fingerprinting (Zooarchaeology by Mass Spectrometry, ZooMS), and carbon stable isotope analyses. A comparative assessment of the results demonstrates the ability of biomolecular methods to improve resolution of faunal records, particularly where preservation is poor. We call for wider application of these methods to legacy collections, in order to refine existing regional models for the spread of herding in Africa, and to better understand ancient pastoralists’ herd-management decisions.

Radiocarbon dating legacy collections: A Bayesian analysis of high-precision AMS 14C dates from the Par-Tee site, Oregon

Archaeological materials in museum collections provide an excellent opportunity for researchers to investigate social, cultural, and environmental change. However, the precision of the archaeological analysis and interpretation is dependent on a firm understanding of the site chronology. The Par-Tee site (35CLT20), located on the northern Oregon Coast, produced a large archaeological collection including artifacts and faunal remains excavated in the 1960s and 1970s. Radiocarbon dates have been obtained on materials from the Par-Tee collections by several different researchers since the 1970s, but these data have not been adequately assessed for chronometric hygiene. To establish a reliable chronology for the Par-Tee site, we obtained new high-resolution accelerator mass spectrometry (AMS) radiocarbon dates and collagen peptide mass fingerprinting of cervid bones from throughout the site. We evaluate these new radiocarbon dates along with previous radiocarbon dates from the site, using chronometric hygiene assessments and Bayesian statistics to build a refined chronology for the Par-Tee site and museum collection. Previous research suggests site habitation occurred between 350 cal BC to cal AD 1150. Our reassessment of the site chronology suggests the primary site habitation occurred from cal AD ~100–800. We also identified evidence of subsequent site occupation around cal AD ~1490–1635 supporting previous interpretations of site habitation after the primary shell midden forming occupation. The latter occupation may be associated with a change in site use from a semi-sedentary village to a cemetery.

Semi-supervised machine learning for automated species identification by collagen peptide mass fingerprinting

BackgroundBiomolecular methods for species identification are increasingly being utilised in the study of changing environments, both at the microscopic and macroscopic levels. High-throughput peptide mass fingerprinting has been largely applied to bacterial identification, but increasingly used to identify archaeological and palaeontological skeletal material to yield information on past environments and human-animal interaction. However, as applications move away from predominantly domesticate and the more abundant wild fauna to a much wider range of less common taxa that do not yet have genetically-derived sequence information, robust methods of species identification and biomarker selection need to be determined.ResultsHere we developed a supervised machine learning algorithm for classifying the species of ancient remains based on collagen fingerprinting. The aim was to minimise requirements on prior knowledge of known species while yielding satisfactory sensitivity and specificity. The algorithm uses iterations of a modified random forest classifier with a similarity scoring system to expand its identified samples. We tested it on a set of 6805 spectra and found that a high level of accuracy can be achieved with a training set of five identified specimens per taxon.ConclusionsThis method consistently achieves higher accuracy than two-dimensional principal component analysis and similar accuracy with hierarchical clustering using optimised parameters, which greatly reduces requirements for human input. Within the vertebrata, we demonstrate that this method was able to achieve the taxonomic resolution of family or sub-family level whereas the genus- or species-level identification may require manual interpretation or further experiments. In addition, it also identifies additional species biomarkers than those previously published.