Pleuronectidae (flatfish) are not always identifiable to the species level on the basis of isolated bones found in archaeological assemblages, and one of the most targeted species in northwestern Europe, flounder (Platichthys flesus), has an extremely large plasticity in habitat use. These issues limit the interpretation of the presence of flatfish bones with regard to fishing ground exploitation and fish trade in the past. This study aims to infer the fishing grounds of flatfish consumed at the site of Abbeville Chauss & eacute;e du Bois, in northwestern france, dated to the 16th century, based on the analysis of examples of two unpaired bones, the first anal-fin pterygi-ophore or "os anale" and the urohyal. The city of Abbeville was an important port during Middle Ages and the Early Modern period. We used an integrated approach combining osteology, osteometrics (n=84), and stable isotope analysis of bone collagen (n=31). We were able to confirm the presence of both flounder and plaice (Pleuronectes platessa). We estimated their size and age at capture thanks to metric models that we developed for modern plaice (n=34) and flounder (n=55) from in- and offshore environments in the Somme estuary. The presence of only adult fish remains at the site and the ethology of the two species exclude exploitation of only the immediate vicinity of the city, i.e., the River Somme itself or the very non-saline part of the estuary close to the river outlet. Although the delta 13C and delta 15N values of the flounder and plaice from the site partially overlap, the two groups present distinctive delta 13C and delta 15N values and bi-isotope variability. Their isotope signatures were com-pared with those of archaeological marine (n=14) and freshwater (n=26) fish remains used as references. The flatfish identified from the site had spent time living in habitats ranging from marine to brackish or even fresh-water. Our combined approach suggests the contemporaneous exploitation of several fishing grounds or alterna-tively marine waters at spawning time to provide the menu served at the site. Our approach appears a promising means to explore the trade in flatfish inland during historical times.

Age profiling of archaeological bone assemblages can inform on past animal management practices, but is limited by the fragmentary nature of the fossil record and the lack of universal skeletal markers for age. DNA methylation clocks offer new, albeit challenging, alternatives for estimating the age-at-death of ancient individuals. Here, we take advantage of the availability of a DNA methylation clock based on 31,836 CpG sites and dental age markers in horses to assess age predictions in 84 ancient remains. We evaluate our approach using whole-genome sequencing data and develop a capture assay providing reliable estimates for only a fraction of the cost. We also leverage DNA methylation patterns to assess castration practice in the past. Our work opens for a deeper characterization of past husbandry and ritual practices and holds the potential to reveal age mortality profiles in ancient societies, once extended to human remains.

Although as a species sheep are commonly found in archaeological deposits and occupy a major position in the history of animal husbandry, it remains difficult to discriminate the sexual type (male, female or castrate) of individuals from observation of the remains. However, understanding sheep husbandry through sex ratio analysis, contributes to a better understanding of the history of animal husbandry. Current criteria for determining are limited to visual characters on coxal and skull. This study explores the variability of sheep skeletons according to their sex to submit hypotheses for methods capable of individual sexing. Using 58 complete skeletons and 428 isolated Soay sheep bones from the collection of the Natural History Museum (UK), we initially tested the sexual distinction using the so-called classical methods: the estimates of shoulder heights and the slenderness index. We then evaluated the influence of sex on the shape and size of selected bones with geometric morphometric, using a protocol that we had previously developed and validated. Finally, we classified these data using the K-Nearest Neighbors (KNN) method, quantifying the reliability of sex attribution. We were able to test its application on an archaeological sample, after selecting bones with the best rate of sexual assignment. Our results demonstrate the difficulty of using traditional methods to sex sheep whilst proposing solutions for sex assignment (88% reliable) using geometric morphometric and KNN. In addition, we were able to highlight the influence of sex according to bone type, though this is strongly qualified by various criteria (such as the age of castration). By characterising and quantifying the sex ratio within archaeological assemblages, we may better understand herd management, procurement strategies and feeding habits, and therefore improve our global knowledge of animal husbandry history.

Ancient DNA preserved in the dental pulp offers the opportunity to characterize the genome of some of the deadliest pathogens in human history. However, while DNA capture technologies help, focus sequencing efforts, and therefore, reduce experimental costs, the recovery of ancient pathogen DNA remains challenging. Here, we tracked the kinetics of ancient Yersinia pestis DNA release in solution during a pre-digestion of the dental pulp. We found that most of the ancient Y. pestis DNA is released within 60 min at 37°C in our experimental conditions. We recommend a simple pre-digestion as an economical procedure to obtain extracts enriched in ancient pathogen DNA, as longer digestion times release other types of templates, including host DNA. Combining this procedure with DNA capture, we characterized the genome sequences of 12 ancient Y. pestis bacteria from France dating to the second pandemic outbreaks of the 17th and 18th centuries Common Era.

Ancient DNA preservation in subfossil specimens provides a unique opportunity to retrieve genetic information from the past. As ancient DNA extracts are generally dominated by molecules originating from environmental microbes, capture techniques are often used to economically retrieve orthologous sequence data at the population scale. Post-mortem DNA damage, especially the deamination of cytosine residues into uracils, also considerably inflates sequence error rates unless ancient DNA extracts are treated with the USER enzymatic mix prior to library construction. While both approaches have recently gained popularity in ancient DNA research, the impact of USER-treatment on capture efficacy still remains untested. In this study, we applied hyRAD capture to eight ancient equine subfossil specimens from France (1st-17th century CE), including horses, donkeys and their first-generation mule hybrids. We found that USER-treatment could reduce capture efficacy and introduce significant experimental bias. It differentially affected the size distribution of on-target templates following capture with two distinct hyRAD probe sets in a manner that was not driven by differences in probe sizes and DNA methylation levels. Finally, we recovered unbalanced proportions of donkey-specific and horse-specific alleles in mule capture sequence data, due to the combined effects of USER-treatment, probe sets and reference bias. Our work demonstrates that while USER-treatment can improve the quality of ancient DNA sequence data, it can also significantly affect hyRAD capture outcomes, introducing bias in the sequence data that is difficult to predict based on simple molecular probe features. Such technical batch effects may prove easier to model and correct for using capture with synthetic probes of controlled sizes and diversity content.

Sex identification from fragmentary archeozoological assemblages is particularly challenging in the Equid family, including for horses, donkeys and their hybrids. This limitation has precluded in-depth investigations of sex-ratio variation in various temporal, geographic and social contexts. Recently, shallow DNA sequencing has offered an economical solution to equine sex determination, even in environments where DNA preservation conditions is not optimal. In this study, we applied state-of-the-art methods in ancient DNA-based equine sex determination to 897 osseous remains in order to assess whether equal proportions of males and females could be found in a range of archeological contexts in France. We found Magdalenian horse hunt not focused on isolated bachelors, and Upper Paleolithic habitats and natural traps equally balancing sex ratios. In contrast, Iron Age sacrificial rituals appeared to have been preferentially oriented to male horses and this practice extended into the Roman Period. During Antiquity, the Middle Ages and the Modern Period, cities emerged as environments largely dominated by horse males. This strong sex-bias was considerably reduced, and sometimes even absent, in various rural contexts. Combined with previous archaeozoological work and textual evidence, our results portray an urban economy fueled by adult, often old, males, and rural environments where females and subadults of both sexes were maintained to sustain production demands.

Boves Castle, located in northern France, was occupied between the 9th and 16th centuries AD. Under excavation since 1996, this high-status site has yielded more than six tons of faunal remains. Until now, archaeozoological studies have focused on the early occupation of the site (10th and 11th centuries AD), revealing specific supply and consumption strategies, widely based around birds. Aside from the very large amount of domestic fowl, Boves Castle is distinguished by the number and diversity of wild birds from various environments, including wetland, coastal, forested and anthropized areas. However, the special feature of this site is the importance of freshwater birds, especially large waders, which remained a status symbol until modern times in this region. On the basis of this outstanding example, and other high-status sites from the same region, we aim to discuss the importance and status of various domestic and game bird categories in northern France during the High Middle Ages. We will also address the subject of controlled hunting areas around castles and other high-status consumer sites, for a better understanding of the link between power and consumption during the medieval period.

Donkeys transformed human history as essential beasts of burden for long-distance movement, especially across semi-arid and upland environments. They remain insufficiently studied despite globally expanding and providing key support to low- to middle-income communities. To elucidate their domestication history, we constructed a comprehensive genome panel of 207 modern and 31 ancient donkeys, as well as 15 wild equids. We found a strong phylogeographic structure in modern donkeys that supports a single domestication in Africa ~5000 BCE, followed by further expansions in this continent and Eurasia and ultimately returning to Africa. We uncover a previously unknown genetic lineage in the Levant ~200 BCE, which contributed increasing ancestry toward Asia. Donkey management involved inbreeding and the production of giant bloodlines at a time when mules were essential to the Roman economy and military.

Horses and donkeys have had a far-reaching impact on human history, providing mechanical power for agriculture and transportation. Their F1-hybrids, especially mules, have also been of considerable importance due to their exceptional strength, endurance and resistance. The reconstruction of the respective role that horses, donkeys and mules played in past societies requires prior identification of their osseous elements in archaeological assemblages. This, however, remains difficult on the basis of morphological data alone and in the absence of complete skeletal elements. While DNA sequencing provides almost certain identification success, this approach requires dedicated infrastructure and sufficient ancient DNA (aDNA) preservation. Here, we assessed the performance of a cost-effective alternative approach based on geometric morphometric (GMM) analysis of the bony labyrinth, a structure carried within the petrosal bone. This extremely compact osseous structure provides good aDNA preservation and is frequently found in archaeological assemblages. To assess the GMM performance, we first used High-throughput DNA sequencing to identify 41 horses, 24 donkeys, 36 mules and one hinny from 11 archaeological sites from France and Turkey spanning different time periods. This provided a panel of 102 ancient equine remains for micro-computed tomography (microCT) and GMM assessment of the variation of the bony labyrinth shape, including the cochlea and the semicircular canals. Our new method shows good-to-excellent prediction rates (85.7%–95.2%) for the identification of species and hybrids when considering the cochlea and semicircular canals together. It provides a cheap, non-destructive alternative to aDNA for the taxonomic identification of past equine assemblages.

The field of ancient DNA is dominated by studies focusing on terrestrial vertebrates. This taxonomic bias limits our understanding of endogenous DNA preservation for species with different bone physiology, such as teleost fish. Teleost bone is typically brittle, porous, lightweight, and is characterized by a lack of bone remodeling during growth. All of these factors potentially affect DNA preservation. Using high-throughput shotgun sequencing, we here investigate the preservation of DNA in a range of different bone elements from over 200 archaeological Atlantic cod (Gadus morhua) specimens from 38 sites in northern Europe, dating up to 8000 years before present. We observe that the majority of archaeological sites (79%) yield endogenous DNA, with 40% of sites providing samples containing high levels (>20%). Library preparation success and levels of endogenous DNA depend mainly on excavation site and pre-extraction laboratory treatment. The use of pre-extraction treatments lowers the rate of libraries that can be sequenced, although ? if successful ? the fraction of endogenous DNA can be improved by several orders of magnitude. This trade-off between library preparation success and levels of endogenous DNA allows for alternative extraction strategies depending on the requirements of down-stream analyses and research questions. Finally, we do not find particular bone elements to yield higher levels of endogenous DNA, as is the case for denser bones in mammals. Our results highlight the potential of archaeological fish bone as a source for ancient DNA and suggest a possible role of bone remodeling in the preservation of endogenous DNA.

L’analyse archéozoologique se concentre sur trois structures contenant des restes d’équidés équarris, toutes trois découvertes dans une enceinte conventuelle et datées de l’époque moderne. Deux des assemblages osseux proviennent du couvent des Cordeliers de Beauvais (xvie et xviie siècles) et le troisième du couvent des Capucins de Reims (xviie-xviiie siècle). L’analyse du matériel faunique conduit d’abord à quelques réflexions sur les liens plus ou moins étroits unissant les équidés équarris et les communautés religieuses, notamment en fonction des modalités d’abandon des carcasses et des espèces concernées. Le rôle des occupants des couvents dans la constitution de ces dépôts d’équarrissage, et en particulier les circonstances amenant ces derniers à les autoriser, sera ensuite discuté.