Keywords: Ancient DNA, Archaeozoology, Horse, Donkey, Mule, Iron Age, Roman Period, Middle Ages, Hunting, Husbandry, Breeding, City

First, I am thankful to my supervisor Ludovic Orlando for giving me the opportunity to be in his group and the constant support and patience he showed me during my time here. I am immensely grateful for his problem-solving ability and dedication to any difficulties

Résume Français

La domestication des chevaux a commencé il y a environ 5,500 ans dans les steppes d'Asie Centrale. Ce processus a eu un impact profond sur l'histoire de l'humanité, car les chevaux nous fournissent un transport rapide et facilitent la propagation de nos biens, de nos maladies et de nos cultures. Une fois la domestication lancée, les humains n'ont eu de cesse que de façonner l'histoire évolutive des chevaux, pour aboutir, au moyen de la sélection artificielle et de mélanges savants, à la formation des centaines de races domestiques vivant aujourd'hui sur terre, mais aussi à l'extinction de toutes les populations sauvages. Depuis quelques siècles, le rôle des mâles et des femelles dans la reproduction équine s'est considérablement déséquilibré, puisque très souvent, et en particulier chez les races à haute valeur économique ajoutée, les étalons sont soumis à une sélection particulièrement intensive, donnant à quelques poignées d'individus un immense pouvoir reproducteur. Il n'est cependant pas certain que des stratégies de gestion similaires furent déjà mises en place dès les premiers stades de la domestication du cheval. Cette thèse de doctorat vise à éclairer la question de la contribution des mâles et des femelles dans le processus d'élevage des chevaux, depuis leur domestication initiale à nos jours. Pour se faire, des analyses paléogénétiques se sont attachées, dans un premier temps, à suivre les patrons de variation de l'ADN mitochondrial et du chromosome Y dans l'espace et le temps. L'analyse en parallèle de 266 génomes mitochondriaux complets et de 76 chromosomes Y partiels de chevaux anciens par la méthode de modélisation bayésienne Skyline, directement calibrée par datation radiocarbone, nous a permis de reconstituer les trajectoires démographiques des deux sexes au cours des dix derniers milliers d'années.

Nos résultats suggèrent que les lignées maternelles et paternelles qui ont conduit à l'essor du cheval domestique moderne ont connu une expansion démographique massive et parallèle à partir de la fin du 3ème millénaire avant notre ère. Les profils démographiques des lignées maternelles et paternelles s'épousent l'un l'autre, ce qui semble indiquer que des logiques de production balancées entre mâles et femelles à l'époque. Au-delà des chevaux, cette thèse s'est ensuite attachée à évaluer les stratégies de gestion d'autres ressources équines, comme les ânes et leurs hybrides de première génération, les mules, qui sont connus pour leur force de travail et pour avoir joué des rôles économiques et symboliques de premier plan en France. Ce sont ici des données de séquençage d'ADN ancien peu profondes qui nous ont permis, par analyse bioinformatique via le programme Zonkey, de révéler que si les chevaux sont restés l'espèce équine la plus populaire depuis l'Âge du Fer, les ânes et les mules, tantôt d'un sexe, tantôt de l'autre, ont pu occuper les devants de la scène selon les contextes sociaux, géographiques et temporels.

INTRODUCTION

Animal and plant domestication during the Neolithic drastically changed the daily life of humankind and led to building the modern world as we experience it today. Animal domestication facilitates human life, providing a protein-rich diet as well as a range of primary resources directly derived from these animals, such as bone, leather, and wool.

Horses represent a special case amongst domestic animals. While they were sporadically used as a food source in western countries (and remain an important part of the diet in some non-western countries), they were mainly used as a means of transportation that allowed people to travel long distances considerably faster than on their own legs. As a result, the whole dynamics of human migration, trade and circulations of goods and cultures accelerated and the world felt smaller, and probably globalized, for the first time. In addition to transportation, horses represent key assets in the battlefield and have revolutionized warfare from their early domestication stages and until the total mechanization of military weaponry following World War II.

Over the last decade, our understanding of the horse domestication process has made important progress thanks to the multiplication of studies that have mapped spatial and temporal patterns of genomic variation in ancient horses ( [START_REF] Librado | Genomics and the Evolutionary History of Equids[END_REF] recent reviews). The attention has been such that the number and the scope of ancient DNA studies on horses is only second to humans (see [START_REF] Orlando | Ancient DNA analysis[END_REF] for a review).

A primer to horse domestication

The history of the relationship between horses and humans started millennia before the initiation of horse husbandry, in the form of hunters and preys. The first solid archaeological evidence of horse hunting has been found in Schöningen, Germany, and dates back roughly 300,000 years [START_REF] Lang | The Pleistocene of Schöningen, Germany: A complex tunnel valley fill revealed from 3D subsurface modelling and shear wave seismics[END_REF]. In no more than 5 years, between 1994 and 1998,

there were not less than around 10,000 bones that could be excavated on site, together with wooden spears and other human artifacts. Many of the horse bones showed characteristic cut marks: particular parts of the hunted body were prepared so as to collect meat [START_REF] Lang | The Pleistocene of Schöningen, Germany: A complex tunnel valley fill revealed from 3D subsurface modelling and shear wave seismics[END_REF]. The human-horse relationship was, however, not just that of a predator and its prey. It was also a strongly symbolic relationship as horses represent the most common animal represented in the parietal art of western Europe and we can still admire powerful horse pictures in many Iberian sites, including at Altamira (Spain, 36,000-22,000 years ago), and many

French caves, such as Chauvet (37,000 -28,000 years ago), Pech Merle (27,000 years ago), Lascaux (17,000 years ago) and Niaux (17,000-11,000 years ago).

The symbolic nature of our relationship with horses is not limited to the Upper Paleolithic period but extends to the Holocene, especially as early as the sixth millennium BCE when horses associated with funerary rituals appear in the Pontic-Caspian steppes [START_REF] Anthony | theWheel, andLanguage: HowBronze-AgeRiders fromthe Eurasian Steppes Shaped the Modern World[END_REF]. The Khvalynsk cemetery, for example, dates back to 4700-3800 BCE and is located on the west bank of the Volga river in present-day Samara, Russia. There, the remains of 11 horses were discovered alongside the remains of other domestic livestock and 158 buried individuals. At S'yezzhe, a contemporaneous site near Samara, this is not only horse skulls and lower limb bones, but also carved bone figurines of horses, that were discovered in what looks like a sacrificial deposit atop human tombs. The same type of horse images could be found at Varfolomievka, another Neolithic site located east of the Volga River.

Combined, these findings attest to horses' ritual role in Samara culture, which suggested that horses could have started to be domesticated at the time. [START_REF] Anthony | theWheel, andLanguage: HowBronze-AgeRiders fromthe Eurasian Steppes Shaped the Modern World[END_REF] Other types of evidence have suggested the same, for example, mace heads that have been excavated at the Cucuteni-Trypillia sites in present-day Moldova, Romania, and Ukraine, dating back to the Late Neolithic, 4200-3800 BCE, and are interpreted as horseshaped, another sign of increasingly intimate relationships building up with the animal at the animal [START_REF] Anthony | theWheel, andLanguage: HowBronze-AgeRiders fromthe Eurasian Steppes Shaped the Modern World[END_REF] [5] Even pleading for the highly symbolic value of horses in the Neolithic of the Pontic-Caspian steppes and the Eastern European steppes, pictures and maces do not tell whether the animals depicted were wild or farmed [START_REF] Anthony | The Secondary Products Revolution, Horse-Riding, and Mounted Warfare[END_REF]. Archaeozoologists have, thus, scrutinized the area and beyond to obtain more direct evidence, such as at the Eneolithic site of Dereivka, in present-day Ukraine. Out of the 4,000 animal bones that were discovered on this habitation site, more than 60% belonged to horses, mostly 4 to 10-yearold males [START_REF] Levine | Dereivka and the problem of horse domestication[END_REF] Horses were undoubtedly a driving force of the subsistence economy at the time, and the predominance of rather young males could suggest that culling was practiced on site, hence, that horses were domesticated. The horse mortality patterns, however, have remained hotly debated, and could equally fit models of stalked and hunted bachelor groups [START_REF] Levine | Dereivka and the problem of horse domestication[END_REF], instead of evidence for controlled slaughter of domestic animals for ritual purposes [5] [7] Radiocarbon dating finally closed the debates by showing that what was taken as the body of a seven to eight-year-old male found in a Neolithic sacrifice deposit lived in fact some 3,000 years later, between 700 and 200 BCE [START_REF] Mair | Reconfiguring the Silk Road : new research on east-west exchange in antiquity[END_REF]. Dereivka was, thus, not the smoking gun for horse domestication that archeozoologists were looking for.

The oldest archaeozoological evidence for horse domestication is in fact multifarious and comes from the Eneolithic Botai settlements from Kazakhstan, dated 5,500 years ago [START_REF] Outram | The earliest horse harnessing and milking[END_REF].

There are indeed not less than 300,000 skeletal osseous elements that have been uncovered

in Botai bone assemblages, over 99% of which are horses, suggesting horses as instrumental subsistence resources at the time. Additionally, biometrical analysis revealed equine metacarpal bones at Botai more similar to Bronze Age domestic horses than to pre-Botai horses. Isotopic signatures of Botai ceramic vessels were suggestive of not being used to process horse fats [START_REF] Copley | Direct chemical evidence for widespread dairying in prehistoric Britain[END_REF], but horse milk [START_REF] Outram | The earliest horse harnessing and milking[END_REF] Dental pathologies interpreted as caused by the use of bridle [START_REF] Outram | The earliest horse harnessing and milking[END_REF], [START_REF] Olsen | NORWEGIAN JOURNAL OF GEOLOGY AMS radiocarbon dating of glacigenic sediments with low organic carbon content-an important tool for reconstructing the history of glacial variations in Norway radiocarbon dating of glacigenic sediments with low orga nic carbon content-an important tool for reconstructing the history of glacial variations in Norway[END_REF] added harnessing to the milking evidence to support horse domestication by the Botai people, and soon enough corralling as archaeological traces of enclosures with soils enriched in horse dung were uncovered [START_REF] Olsen | NORWEGIAN JOURNAL OF GEOLOGY AMS radiocarbon dating of glacigenic sediments with low organic carbon content-an important tool for reconstructing the history of glacial variations in Norway radiocarbon dating of glacigenic sediments with low orga nic carbon content-an important tool for reconstructing the history of glacial variations in Norway[END_REF], [START_REF] Gaunitz | Ancient genomes revisit the ancestry of domestic and Przewalski's horses[END_REF].

While this remains debated [START_REF] Taylor | Rethinking the evidence for early horse domestication at Botai[END_REF] most archaeologists consider that the economy of the settled Botai people combined horse hunting and husbandry. It has been suggested that plummeting horse populations at the time [START_REF] Gaunitz | Ancient genomes revisit the ancestry of domestic and Przewalski's horses[END_REF] may have provided the incentive for engaging along the prey-pathway of horse domestication [START_REF] Zeder | Pathways to Animal Domestication[END_REF]. That horses were domesticated at Botai does not, however, rules out that other domestication processes were initiated in other regions of the world. In particular, two other regions have received special attention and have been proposed as alternative domestication centers. The first is the Iberian peninsula, combining various types of evidence: a higher genetic diversity amongst present-day Iberian breeds [START_REF] Warmuth | European Domestic Horses Originated in Two Holocene Refugia[END_REF] potentially reflecting this region as a source and neighboring regions as sinks, and; the long-lasting presence of horses from across the Upper-Paleolithic into the Holocene and the Neolithic in the region, contrasting with their almost disappearance in most other regions of Europe following the Last Glacial Maximum [START_REF] Leonardi | Late Quaternary horses in Eurasia in the face of climate and vegetation change[END_REF] Another region often considered as a possible candidate for horse domestication is Anatolia.

Overall, despite decades of archaeological research, both the number, the location and timing of horse domestication remain debated, mostly for the lack of unambiguous morphological indicators signifying the domestication status of ancient animals in horses, in contrast to cattle and other ruminants. In the absence of such indicators, researchers turned to ancient DNA, as another source of data that can reveal the genealogical history of ancient and present-day horses and, thus, could perhaps help finally map the true centre(s) of horse domestication. The following section provides an overview of the methods, concepts and applications of ancient DNA research.

A primer on ancient DNA research

Ancient DNA (aDNA) can be defined as a sample of DNA isolated from ancient specimens, with the word 'ancient' equally fitting for any pre-modern period, including historical but also prehistorical, proto-historical and paleontological. DNA can be extracted from samples such as sub-fossilized bones, teeth, hair or other mummified soft tissues, but also from herbarium leaves [START_REF] Green | A Draft Sequence of the Neandertal Genome[END_REF] seeds [START_REF] Jaenicke-Després | Early allelic selection in maize as revealed by ancient DNA[END_REF] and wood [START_REF] Wagner | High-Throughput DNA sequencing of ancient wood[END_REF] and perhaps even more surprisingly, directly from lake [START_REF] Willerslev | Fifty thousand years of Arctic vegetation and megafaunal diet[END_REF] or cave [START_REF] Slon | Neandertal and Denisovan DNA from Pleistocene sediments[END_REF] sediments.

The history of ancient DNA research started in 1984 when Russell Higuchi and his colleagues reported the successful molecular cloning and sequencing of a short fragment of mitochondrial DNA from the dried muscle of the quagga, an extinct member of the horse family whose remains were preserved in a natural history museum [START_REF] Higuchi | DNA sequences from the quagga, an extinct member of the horse family[END_REF] This showed the world that DNA can be obtained 140 years after the animal died, and opened up the possibility to sequence DNA to time travel the past and catch evolution red-handed at the molecular level. Since then, a series of technological and methodological milestones have marked the history of ancient DNA research, to now allow the sequencing of complete genomes from ancient and even extinct organisms, beyond the one-million year time scale [START_REF] Van Der Valk | Million-year-old DNA sheds light on the genomic history of mammoths[END_REF].

The first such milestone was reached with the development of the Polymerase Chain Reaction (PCR) [START_REF] Mullis | Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction[END_REF] which quickly replaced the then traditional molecular cloning procedures that showed success on the quagga specimen. PCR became widely used in ancient DNA research because it allowed the direct amplification of targeted DNA molecules that were preserved only in minute amounts and showed extensive chemical degradation and physical fragmentation [START_REF] Paabo | Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification[END_REF], [START_REF] Poinar | Amino Acid Racemization and the Preservation of Ancient DNA[END_REF]. It was, however, not devoid of potential issues, including fresh contaminants outcompeting ancient DNA templates during amplification the co-extraction of molecules acting as polymerase inhibitors), in vitro recombination of various DNA templates and introduction of artefactual mutations during amplification [START_REF] Paabo | Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification[END_REF], [START_REF] Hansen | Statistical evidence for miscoding lesions in ancient DNA templates[END_REF], [START_REF] Hofreiter | DNA sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA[END_REF] and limited throughput capacities as whole extracts could be exhausted before regions of a few hundred base pairs could be characterized.

The second milestone in ancient DNA research was reached when researchers reported the successful sequencing of ancient DNA material retrieved from archaeological bones [START_REF] Hagelberg | Ancient bone DNA amplified[END_REF] as this indicated that a considerably larger paleontological record could be amenable to DNA analyses than just mummified tissues and/or museum specimens. DNA studies produced altogether over two decades [START_REF] Poinar | Metagenomics to paleogenomics: large-scale sequencing of mammoth DNA[END_REF]). It is, thus, no exaggeration to state that this third milestone was truly revolutionary.

None of the above technologies would have had the success we know without advances in understanding the chemical and physical characteristics of ancient DNA templates. This is so because a whole range of techniques could then be optimized to overcome the inherent limitations of ancient DNA molecules, as they are strikingly different from fresh DNA templates. Ancient DNA molecules have experienced extensive DNA fragmentation after death, mostly through a depurination process causing the loss of adenine and/or guanine residues and then driving through beta-elimination the formation of an abasic site. While complete human chromosomes can extend over fifty to a couple of hundred millions of nucleotides, the size of typical ancient DNA molecules rarely exceeds fifty base pairs [START_REF] Green | The Neandertal genome and ancient DNA authenticity[END_REF] A whole range of other degradation reactions take place after death, cleaving further individual DNA strands (nicks) and even transforming nucleotide bases into others. The most prevalent such reaction consists of the hydrolytic loss of the amino group present in cytosine residues, which introduces uracil residues in DNA sequences, mostly within single-stranded overhanging ends [START_REF] Briggs | Patterns of damage in genomic DNA sequences from a Neandertal[END_REF] Even though post-mortem degradation is what limits access and manipulation of ancient DNA molecules, this leaves specific signatures that ancient DNA researchers can leverage to authenticate their data as such signatures are barely present in fresh DNA contaminants. The over-representation of purines at genomic sites preceding read starts as well as the concentration of C-to-T misincorporations towards read termini (and their complementary G-to-A misincorporations, depending on the molecular techniques used while preparing DNA libraries, [START_REF] Seguin-Orlando | Pros and cons of methylation-based enrichment methods for ancient DNA[END_REF] offer such authentication signatures.

In addition to authentication procedures, ancient DNA researchers have developed their lab infrastructures and procedures to minimize contamination (see [START_REF] Orlando | Ancient DNA analysis[END_REF] for a review). These include the use of ultra-clean facilities, with positive air-pressure, UV and bleach-based decontamination procedures, disposable personal protective equipment and more. At the molecular level, new techniques are continuously developed to enhance our capacity to remove contamination, extract DNA from bones (e.g. [START_REF] Korlević | Reducing microbial and human contamination in DNA extractions from ancient bones and teeth[END_REF]- [START_REF] Boessenkool | Combining bleach and mild predigestion improves ancient DNA recovery from bones[END_REF]) and convert them into DNA libraries [START_REF] Meyer | A High-Coverage Genome Sequence from an Archaic Denisovan Individual A High-Coverage Genome Sequence from an Archaic Denisovan Individual A High-Coverage Genome Sequence from an Archaic Denisovan Individual[END_REF] incorporate into DNA libraries only those molecules most damaged [START_REF] Gansauge | Selective enrichment of damaged DNA molecules for ancient genome sequencing[END_REF] or focus sequencing efforts on pre-defined genomic regions of interest [START_REF] Mathieson | Genome-wide patterns of selection in 230 ancient Eurasians[END_REF] At the computational level, new statistical models are developed to facilitate data processing, such as the PALEOMIX [START_REF] Schubert | Characterization of ancient and modern genomes by SNP detection and phylogenomic and metagenomic analysis using PALEOMIX[END_REF] and Eager [START_REF] Peltzer | EAGER: efficient ancient genome reconstruction[END_REF] pipelines which produce high-quality alignments from raw DNA sequencing data, but also to authenticate data on the basis of post-mortem DNA damage signatures (e.g. mapDamage, [START_REF] Jónsson | mapDamage2.0: fast approximate Bayesian estimates of ancient DNA damage parameters[END_REF], quantify contamination levels (e.g. [START_REF] Renaud | Schmutzi: Estimation of contamination and endogenous mitochondrial consensus calling for ancient DNA[END_REF]), filter contamination (e.g. PMDtools, [START_REF] Krzewińska | Mitochondrial DNA variation in the Viking age population of Norway[END_REF] and account for often highlevel of data missingness and limited coverage (e.g. Admixtools, [START_REF] Patterson | Ancient admixture in human history[END_REF], or ANGSD, [START_REF] Korneliussen | ANGSD: Analysis of Next Generation Sequencing Data[END_REF]).

Combined, these developments have paved the explosion of high-profile ancient DNA studies that we have seen over the last 10 years.

It was no later than 2010 when the first complete ancient human genome and when the first Neanderthal genome [START_REF] Reich | Genetic history of an archaic hominin group from Denisova cave in Siberia[END_REF] were sequenced. It was also the same year that Denisovans, a new group of archaic hominins was discovered on the basis of carrying a genome sequence too divergent to fall within the gene pool of anatomically modern humans or Neanderthals [START_REF] Reich | Genetic history of an archaic hominin group from Denisova cave in Siberia[END_REF] The first Middle Pleistocene genome could be sequenced no later than in 2013 from a piece of bone that was preserved in the Yukon permafrost for 560,000 -780,000 years, and now in 2021, the world record for the oldest DNA was broken when genome-data could be sequenced from 1.6 million year-old mammoth remains belonging to the previous geological sub-era, the early Middle Pleistocene [START_REF] Van Der Valk | Million-year-old DNA sheds light on the genomic history of mammoths[END_REF]. Thanks to the technical developments underlying such breakthroughs, and the discovery that specific types of remains (such as the petrosal bone [START_REF] Gamba | Genome flux and stasis in a five millennium transect of European prehistory[END_REF] it has become not uncommon that hundreds of ancient individuals can be analyzed within a single ancient DNA studies Furthermore, not only specimens that were once considered unsuitable for DNA analyses as either discovered in environments accelerating degradation or highly contaminated [START_REF] Fu | An early modern human from Romania with a recent Neanderthal ancestor[END_REF] but also sediments [START_REF] Slon | Neandertal and Denisovan DNA from Pleistocene sediments[END_REF], [START_REF] Vernot | Unearthing Neanderthal population history using nuclear and mitochondrial DNA from cave sediments[END_REF]) are increasingly attracting interest and success.

The time had, thus, come to harness the full power of this technology to revisit crucial question about the human past, including the history of animal domestication, especially that of the horse.

Ancient DNA contribution to understanding horse domestication

In the last few years, the application of ancient DNA techniques to the zooarchaeological record has proved excellent for studying the process of animal domestication in dogs, sheep, goats, pigs and horses (see [START_REF] Frantz | Animal domestication in the era of ancient genomics[END_REF] for a review). Focusing on horses, ancient DNA evidence has resulted in not less than a full rethinking of the standard paradigm, starting from the legacy of Botai horses to the modern world.

The sequencing of 20 complete genomes from Botai indeed did not confirm those horses as the direct ancestors of all, or any in fact, modern domestic horses but as the direct ancestors of Przewalski's horses [START_REF] Gaunitz | Ancient genomes revisit the ancestry of domestic and Przewalski's horses[END_REF] Therefore, the horse domestication process at play within the Botai culture apparently remained a local phenomenon. This was another horse type that was domesticated somewhere else that was successfully spread across the world until today. Genomic ancestry profiles and patterns of mitochondrial DNA variation suggested an origins during the third millennium BCE, but as this PhD work started, the true centre of modern horse domestication remained unknown. Neither the DNA data sequenced at Botai, nor in the Iberian peninsula [START_REF] Fages | Tracking Five Millennia of Horse Management with Extensive Ancient Genome Time Series[END_REF] nor again in Anatolia [START_REF] Guimaraes | Ancient DNA shows domestic horses were introduced in the southern Caucasus and Anatolia during the Bronze Age[END_REF] supported models of local domestication. Iberian horses dating from the 3rd and early 2nd millennium BCE showed ancestry profiles drastically different than that present in modern domestic horses and contributed no more than around 5% of the genetic diversity present in modern breeds [START_REF] Fages | Tracking Five Millennia of Horse Management with Extensive Ancient Genome Time Series[END_REF]. The mitochondrial and Y-chromosomal diversity present in pre-Bronze Age horses from Anatolia was also found dramatically different than during and after the Bronze Age, suggesting the replacement of wild lineages by domestic lineages during the Early Bronze Age [START_REF] Guimaraes | Ancient DNA shows domestic horses were introduced in the southern Caucasus and Anatolia during the Bronze Age[END_REF]. At the same time as candidate regions for horse domestication were dismissed, new ancient and now-extinct lineages of horses were uncovered, for example, in Iberia [START_REF] Fages | Tracking Five Millennia of Horse Management with Extensive Ancient Genome Time Series[END_REF] but also in North East Siberia where a lineage morphologically identified as Equus lenensis (the Lena horse) was identified based on patterns of genome variation to have diverged from all modern horse lineages some 120,000 years ago [START_REF] Schubert | Characterization of ancient and modern genomes by SNP detection and phylogenomic and metagenomic analysis using PALEOMIX[END_REF]. The exact extinction times of this lineage is unknown but the sequencing of a specimen from Batagai (Republic of Sakha) that was radiocarbon dated to ~5,000 years ago, revealed that they may have survived when the domestication process was initiated.

Ancient DNA studies have not simply focused on early domestication stages. They have also helped better understand how past herders and breeders further transformed the horse following their domestication. For example, a whole genome study of Scythian Pazyryk horses at Be'rel (~2,300 years ago, Kazakhstan) identified selection signatures for genes involved in the forelimb development, suggesting ongoing breeding at the time for more robust anatomy [START_REF] Librado | Ancient genetic changes associated with domestication of the horse[END_REF] The DNA characterization of over 250 ancient horse remains spread across Eurasia and mostly spanning the last 4,000 years has provided the largest collection of ancient genomes for a non-human animal species [START_REF] Fages | Tracking Five Millennia of Horse Management with Extensive Ancient Genome Time Series[END_REF]. This work revealed important changes in the genetic structure of domestic horse population by the 7th-9th centuries CE (Common Era), possibly in relation to the Muslim expansion. It also identified a sharp decline in genetic diversity during the last few centuries, concomitantly with the rise of close stud breeding and the formation of modern breeds.

While whole genome studies have received much attention, other studies more focused on single markers, such as uniparental loci or markers causative for coat coloration phenotypes speed [START_REF] Bower | The genetic origin and history of speed in the Thoroughbred racehorse[END_REF] or ambling [START_REF] Andersson | Mutations in DMRT3 affect locomotion in horses and spinal circuit function in mice[END_REF] have provided important insights on changing breeding practices through space and time. Considering uniparental markers, most ancient DNA studies thus far have consistently failed to identify strong phylogeographic patterns in mitochondrial DNA variation, including when whole mitochondrial genomes were sequenced [START_REF] Fages | Tracking Five Millennia of Horse Management with Extensive Ancient Genome Time Series[END_REF] as opposed to shorter partial control regions [START_REF] Guimaraes | Ancient DNA shows domestic horses were introduced in the southern Caucasus and Anatolia during the Bronze Age[END_REF], [START_REF] Cieslak | Origin and History of Mitochondrial DNA Lineages in Domestic Horses[END_REF]. This has generally be taken as support for the narrative that, as domestic horses expanded outside of their original homeland, mares extensively admixed with local wild stallions, thereby introgression divergent lineages and blurring away phylogeographic that may have existed earlier (e.g. [START_REF] Vilà | Widespread origins of domestic horse lineages[END_REF]) The lack of strong phylogeographic patterns in mitochondrial variation may, alternatively support models of incomplete lineage sorting in which the ancestral horse population was large at the time most lineages diverged, hence, the chances of multiple haplotypes co-segregating in many independent lineages were increased [START_REF] Librado | The evolutionary origin and genetic makeup of domestic horses[END_REF].

Strikingly, the amount of genetic variation found on the Y-chromosome of modern horses is extremely limited, which contrasts with the extensive amount of genetic variation found on the mitochondrial genome. Until ancient DNA data were generated on ancient domestic horses, this was taken to support heavy male-biased reproductive success from early domestication stages, resulting in only a handful of Y-chromosomal haplotypes being passed to descending generations [START_REF] Lindgren | Limited number of patrilines in horse domestication[END_REF], [START_REF] Wallner | Identification of Genetic Variation on the Horse Y Chromosome and the Tracing of Male Founder Lineages in Modern Breeds[END_REF]. However, our understanding changed dramatically as the first ancient Y-chromosome haplotypes were sequenced, showing extensive levels of diversity during the Iron Age [START_REF] Librado | Ancient genetic changes associated with domestication of the horse[END_REF], [START_REF] Lippold | Whole mitochondrial genome sequencing of domestic horses reveals incorporation of extensive wild horse diversity during domestication[END_REF]. More complete time transects further indicated that Y-chromosomal diversity mostly dropped within the last 2,000 years, especially in the last 1,000 years following the expansion of Oriental bloodlines [START_REF] Fages | Tracking Five Millennia of Horse Management with Extensive Ancient Genome Time Series[END_REF].

Considering SNP variants underlying important phenotypic traits, the vast majority of the studies have focused on predicting the color of the coat of ancient horses, revealing astounding shifts in the frequency of causative variants from the early stages of horse domestication (sensu lato, i.e. the 3rd millennium BCE) [START_REF] Ludwig | Coat color variation at the beginning of horse domestication[END_REF]). Such shifts took place on standing variation as a diversity of such alleles could be identified in Upper Paleolithic remains, such as the TRPM1 insertion driving leopard spotted phenotypes [START_REF] Bellone | Evidence for a Retroviral Insertion in TRPM1 as the Cause of Congenital Stationary Night Blindness and Leopard Complex Spotting in the Horse[END_REF] or deletions driving non-dun coat colorations [START_REF] Imsland | Regulatory mutations in TBX3 disrupt asymmetric hair pigmentation that underlies Dun camouflage color in horses[END_REF]. It was also discovered, as more samples kept being analyzed, that allelic trajectories were not unidirectional either but could in fact fluctuate in space and time, as past breeders fancied different types of horses in various parts and cultures of the world, such as leopard spotting post-Iron Age [START_REF] Wutke | The origin of ambling horses[END_REF] or chestnut coloration in Europe during the Medieval times [START_REF] Wutke | Decline of genetic diversity in ancient domestic stallions in Europe[END_REF] Besides coat coloration phenotypes, a handful of studies have started to uncover past allelic variation for other phenotypes, including locomotory, such as speed [START_REF] Bower | The genetic origin and history of speed in the Thoroughbred racehorse[END_REF] and ambling [START_REF] Sandoval-Castellanos | Coat colour adaptation of post-glacial horses to increasing forest vegetation[END_REF]. Although limited, the data currently available suggested that most such phenotypes, even though they seem iconic of the essence of the horse, only rose in frequency during the last 1,000 years. Combined, these types of studies have provided great examples that migration dynamics as well as strategies for horse herding and breeding have been extremely dynamic from the early days of domestication onwards and until the modern times. Incidentally, this demonstrated the whole power of ancient DNA data, which also fully justifies why approaches, such as the one developed in this PhD work, are important to enhance our understanding of past animal domestication processes.

The research questions

Our understanding of horse domestication has considerably advanced over the last few years. However, there remain many gaps in our knowledge, not least of which the center for their domestication remained unknown at the time this PhD work started. Together with the other laboratory members, I endeavored over the last four years to find an answer to this question. Our approach has consisted in mapping the horse population structure during the 2nd and 3rd millennium BCE at the scale of Eurasia using complete genome sequencing. My specific role to this project was to carry the sequence analysis of uniparental markers represented by the mitochondrial genome and the Y-chromosome so as to (1) reconstruct past demographic trajectories of both maternal and paternal lineages and (2) assess whether males and females played a symmetric role during early stages of horse domestication. Our rationale was to look for sudden significant increases in the horse effective size that could be taken as the signature of the domestication onset. Any shift in the timing and/or magnitude of the demographic trajectory reconstructed for maternal and paternal lineages may be indicative of sex-biased roles for males and females.

Even though horses have represented the most popular equine species following their domestication, other species in the Equid family, such as donkeys and mules, have also provided important force and transportation power to ancient societies. These equids have, however, received much less archaeological attention, perhaps due to the difficulty to identify their remains based on a generally fragmentary record and the lack of proper taxonomically diagnostic morphological criteria. In this PhD, we thus endeavored to use shallow ancient DNA sequencing to unambiguously assign 896 ancient equine specimens from France. This has allowed us to chart through space, time and social contexts, patterns of horse, donkey and mule management in the country during the last 2,500 years. Our work reveals that rather than being managed as single, isolated resources, horses, donkeys and mules of both sexes were assigned to specific purposes reflecting rationalized utilization of their working capacity. Even though most of the recent attention in ancient DNA research was focused on the analysis of genome-wide patterns of SNP variation, the two uni parentally inherited markers, mitochondrial DNA (mtDNA) and the Y chromosome, have not been completely superseded as they can provide important insights about past population structures and demographic trajectories, both in humans and domestic animals. Their unique pattern of inheritance, evolutionary rates and non-recombining characteristics have also provided technical assets to gain resolution from more limited sequencing efforts.

Mitochondrial DNA

The horse reference mitochondrial genome of the horse is ~16,660 bp long (Accession Number = NC_001640) and contains 13 coding proteins in 5 protein complexes, 22

transfer RNAs, and 2 ribosomal RNAs of all 13 mitochondrial genes [START_REF] Xu | The complete mitochondrial DNA sequence of the horse, Equus caballus: extensive heteroplasmy of the control region[END_REF] . A highly variable regulatory region in the horse reference mitochondrial genome includes multiple repeats of the GTGCACCT motif, and frequently exhibits heteroplasmy. It remains poorly characterized through re-sequencing experiments with short DNA reads. There are multiple aspects in the biology of the mitochondrial genome that makes this genome extremely useful for population genetics but also underpin some important limitations.

First, the lack of recombination resulting from its strict non-Mendelian maternal inheritance provides access to a single genealogy amongst all those underlying the population history. This translates in limited statistical power, potentially limiting the resolution and precision of some types of inference on the one hand, but this also provides direct access to the phylogenetic history of matrilineages. Second, the mitochondrial genome is haploid and has a high mutation rate, particularly in the control region (also called D-loop). As a result, even for recently diverged species or populations, mtDNA provides a high resolution for revealing evolutionary dynamics. Third, because mitochondrial DNA is present in each mitochondrion with hundreds or thousands of copies per cell, it can be accessed relatively more easily than single copy genes [START_REF]Defining the equine genome: the nuclear genome and the mitochondrial genome[END_REF] Altogether, these features have proved decisive in the early days of ancient DNA research when for the lack of alternative technologies, researchers had to PCR target specific loci that were particularly informative to maximize chances of successful outcomes. This has also had important positive consequences following the advent of next-generation sequencing since nearly complete mitochondrial genomes can be routinely be obtained from shallow sequencing efforts [START_REF] Gilbert | EDITORIAL[END_REF] and the complex mitochondrial content of ancient DNA libraries also often increases the chance of mitogenome characterization through target-enrichment [START_REF] Cruz-Dávalos | Experimental conditions improving in-solution target enrichment for ancient DNA[END_REF]. Finally, the limited size of this genome often makes it possible to characterize genetic variation at the whole population level, and not restrict inference from a few individuals only.

Modern horses show a high level of mitochondrial DNA diversity and are characterized by a lack of phylogeographic mitochondrial structure, resulting in little correspondence between mtDNA haplotypes, breeds, and geography [START_REF] Vilà | Widespread origins of domestic horse lineages[END_REF], [START_REF] Librado | The evolutionary origin and genetic makeup of domestic horses[END_REF], [START_REF] Achilli | Mitochondrial genomes from modern horses reveal the major haplogroups that underwent domestication[END_REF]. Early mitochondrial studies in horses aimed at characterizing the control region variation present across a worldwide panel of breeds to infer global maternal diversity and at timing their most recent common ancestor (MRCA) [START_REF] Vilà | Widespread origins of domestic horse lineages[END_REF], [START_REF] Jansen | Mitochondrial DNA and the origins of the domestic horse[END_REF]. These were further complemented with complete mitochondrial genome sequences [START_REF] Lippold | Whole mitochondrial genome sequencing of domestic horses reveals incorporation of extensive wild horse diversity during domestication[END_REF], [START_REF] Achilli | Mitochondrial genomes from modern horses reveal the major haplogroups that underwent domestication[END_REF]. Other studies were focused on statistical inference of past demographic variation leveraging the then nascent and developing methodologies as part of the BEAST inference framework [START_REF] Drummond | Relaxed phylogenetics and dating with confidence[END_REF] , specifically methods of the skyline family [START_REF] Ho | Skyline-plot methods for estimating demographic history from nucleotide sequences[END_REF], following early success with bisons [START_REF] Shapiro | Rise and fall of the Beringian steppe bison[END_REF]. Different studies differed regarding the inclusion of ancient DNA data or not, which opened for tip-calibration or limited inference to external calibration, such as the horse-donkey divergence, which was almost doubled to 4.0-4.5 million years ago following the sequencing of the first Middle

Pleistocene genome [START_REF] Orlando | Recalibrating equus evolution using the genome sequence of an early Middle Pleistocene horse[END_REF]. While the different demographic trajectories differed prior to 10,000 years ago, they converged to support a massive demographic within the last 4,000 to 8,000 years, which largely overlap with horse domestication starting during that time.

The magnitude of the expansion, its exact timing and the time to when all mitochondrial horse haplotypes coalesced differed depending on the amount of data considered and the calibration method.

More recent work from our laboratory relied on a larger panel of pseudo-complete ancient mitochondrial genomes attached to radiocarbon dating information for tip-calibration [START_REF] Gaunitz | Ancient genomes revisit the ancestry of domestic and Przewalski's horses[END_REF],

[53], [START_REF] Librado | Ancient genetic changes associated with domestication of the horse[END_REF]. These studies identified an expansion-collapse cycle during the Late Pleistocene, peaking prior to the Last Glacial Maximum, some 30-35 thousand years ago [START_REF] Orlando | Recalibrating equus evolution using the genome sequence of an early Middle Pleistocene horse[END_REF] and refined the timing of the most recent demographic expansion to the 3rd millennium BCE. More data were, thus, necessary to gain precision, which provided some of the motivation behind my work. The almost lack of similar studies for another uniparentally-inherited marker, the Y chromosome, also precluded direct comparisons with the demographic dynamics of paternal lineages, and their role during the horse domestication process.

Y chromosome

Mammalian sex determination is based on Y chromosomes, but their repeat-rich nature has impeded sequencing and related evolutionary studies for long time [START_REF]Origins and functional evolution of Y chromosomes across mammals[END_REF]. As many inversion rearrangements made it loose the ability to recombine with the X chromosome for most of its part , only a pseudo-autosomal region of extremely limited size [START_REF] Raudsepp | Ten years of the horse reference genome: insights into equine biology, domestication and population dynamics in the post-genome era[END_REF] preserved sequence similarity for pairing and recombining with the homologous X chromosome region. It follows that the majority of the Y chromosome is male specific (MSY), haploid, and does not cross over. With such characteristics, the Y chromosome shrank in size and gene content but has also accumulated many male-beneficial genes and large ampliconic areas [START_REF] Lahn | Functional Coherence of the Human Y Chromosome[END_REF]- [START_REF] Hughes | Chimpanzee and human Y chromosomes are remarkably divergent in structure and gene content[END_REF].

Apart from the intrinsic challenging nature of the Y chromosome, the horse reference genome was generated from an inbred female individual, and therefore is devoid of a Y chromosomal assembly [START_REF] Wade | Genome sequence, comparative analysis, and population genetics of the domestic horse[END_REF]. However, the application of a range of next-generation DNA sequencing techniques, including to Y-chromosome specific BAC [START_REF] Raudsepp | Ten years of the horse reference genome: insights into equine biology, domestication and population dynamics in the post-genome era[END_REF], but also DNA extracts and libraries enriched in Y-chromosomal content [START_REF] Felkel | The horse Y chromosome as an informative marker for tracing sire lines[END_REF] contributed to the first comprehensive annotated assemblies for the horse Y chromosome. As a result, the EquCab3.0 release of the horse nuclear reference genome [START_REF] Kalbfleisch | Improved reference genome for the domestic horse increases assembly contiguity and composition[END_REF] of the male-specific region of the Y chrosomosome (MSY), originally published by Janečka and colleagues [START_REF] Janečka | Horse Y chromosome assembly displays unique evolutionary features and putative stallion fertility genes[END_REF] The most recent assembly now covers 6.46 Mbp across 764 contigs spanning the MSY [START_REF] Felkel | The horse Y chromosome as an informative marker for tracing sire lines[END_REF].

Early studies addressing the sequence variation present in the horse Y chromosome have focused on relatively small portions of the chromosome, but yet reported a striking pattern that was further confirmed using longer sequences and larger panels of breeds: only limited, if any, sequence variation, implying that most stallions living in the world today shared identical or close haplotypes, except for Przewalski's horses which carry their own divergent Y chromosomal haplogroup [START_REF] Lindgren | Limited number of patrilines in horse domestication[END_REF]. The authors interpreted this limited variety, which contrasts with the high mtDNA diversity, as severe sexual bias in early domestication procedures, with only a few stallion patrilines incorporated in the domestic breed. Investigated chromosomal sections of increasing sizes, Wallner and colleagues ended up discovering evidence for some haplotypes segregating at rather low frequencies in modern breeds of western origins, and additional variation could be obtained in nonwestern breeds, especially in Asia [START_REF] Wallner | Identification of Genetic Variation on the Horse Y Chromosome and the Tracing of Male Founder Lineages in Modern Breeds[END_REF], [START_REF] Felkel | The horse Y chromosome as an informative marker for tracing sire lines[END_REF], [START_REF] Wallner | Y Chromosome Uncovers the Recent Oriental Origin of Modern Stallions[END_REF], [START_REF] Der Sarkissian | Evolutionary genomics and conservation of the endangered Przewalski's horse[END_REF]. Haplotype network based inference revealed the extent of the Oriental bloodlines during the last 700-1,300 years and indicated the co-existence and incorporation of several paternal lineages during early domestication, in line with ancient DNA evidence also discovering additional haplotypic diversity in pre-Roman ancient horses [START_REF] Fages | Tracking Five Millennia of Horse Management with Extensive Ancient Genome Time Series[END_REF], [START_REF] Librado | Ancient genetic changes associated with domestication of the horse[END_REF], [START_REF] Lippold | Whole mitochondrial genome sequencing of domestic horses reveals incorporation of extensive wild horse diversity during domestication[END_REF]. Combined, these studies challenged earlier theories positing extreme bias in the stallion reproductive success during early domestication stages. They also revealed the considerable drop of genetic diversity that accompanied the most recent stages of horse evolution, thus, the limited potential of the locus for inferring past demographic trajectories of paternal lineages pre-dating this collapse. This motivated my work to time travel before that time and leverage the Ychromosomal sequence data generated in an extensive panel of ancient horses generated in the laboratory to gain insights on the diversity of paternal lineages effectively mobilized during early stages of domestication.

Bayesian Skyline Plots for Inferring demographic changes

In this study, I tried to construct maternal and paternal demographic changes in horses by using bayesian skyline method. Underlying data is already collected and created by other team members. I prepare already generated mtDNA and Y chromosome sequences to implement in to BEAST and I run the skyline anyleses. The ideal data set should consist of a sufficient number of randomly selected individuals from the population of interest to capture the population's diversity [START_REF] Drummond | Bayesian Coalescent Inference of Past Population Dynamics from Molecular Sequences[END_REF]. The samples are selected from populations that are in the group of DOM2 (domesticated lineage) and across Eurasia. Radiocarbon dated samples are used for that study in order to be precise about timing. In addition to data quantity, data quality is also an important aspect for skyline plots. Another important factor in the skyline plot method is the data quality. Heled and Drummond (2008) [START_REF] Heled | Bayesian inference of population size history from multiple loci[END_REF] discovered that even low levels of sequence error (0.01 percent) can result in a doubling of estimation error. Also, Axelsson et al. (2009) [START_REF] Axelsson | The Effect of Ancient DNA Damage on Inferences of Demographic Histories[END_REF] discovered that because of postmortem damage in ancient sequence data, spurious demographic trends could be reconstructed. For example, due to demage singleton accumulation is increasing and that could cause a misinterpretation of demographic expansion. Thanks to high-throughput sequencing methods, manifold sequencing coverage is achieved and so the risk and presence of errors are reduced. Moreover, we applied strict quality pipelines for both mtDNA and Y chromosome sequences in order to reduce misinterpretation.

A population's demographic history leaves signatures on the DNA of its modern descendants. There are statistical and computational difficulties in getting the necessary historical signals such as population size changes, population splits, admixture, and migration from the population of interest. There are several methods to identify population demographic changes by using nucleotide sequence data [START_REF] Emerson | The prevalence of challenging behaviors: a total population study[END_REF] The . majority of them presume that a basic parametric model, such as exponential or logistic growth, appropriately describes the population history. The demographic history can then be deduced by assessing support for the candidate models or calculating the parameters in a flexible population model [START_REF] Harpending | Genetic traces of ancient demography[END_REF]. One commonly used method for parametric inference in population is based on the sample frequency spectrum. It is computationally efficient but has fundamental limits on its accuracy as a function of the sample size and ignores linkage information [START_REF] Kamm | Efficient computation of the joint sample frequency spectra for multiple populations[END_REF] However, parametric models generally fail to explain complex population histories [START_REF] Ho | Skyline-plot methods for estimating demographic history from nucleotide sequences[END_REF].

In actuality, population histories are frequently more complicated than simple parametric models can represent. This problem has prompted the development of nonparametric and semiparametric approaches for inferring demographic history from sequencing data or estimated genealogy. From genomic data, coalescent-based approaches can be used to infer the demographic change in the sense of population size change [START_REF] Drummond | Bayesian Coalescent Inference of Past Population Dynamics from Molecular Sequences[END_REF]. Coalescent approaches for inferring demographic histories involve the usage of a "demographic model," which describes not only the change in effective population size through time but also deme fragmentation and more. All skyline-plot approaches are basically based on coalescent theory, which quantifies the link between sequence genealogy and population demographic history [START_REF] Kingman | The coalescent[END_REF]. Coalescent framework trace backwards the lineages from sample of sequences,with pairings of lineages randomly merging until a single common ancestor is reached. Any specific locus' genealogy reflects a single realisation of this stochastic process, the circumstances of which are dictated by population history, natural selection, and other variables [START_REF] Donnelly | Coalescents and genealogical structure under neutrality[END_REF]. Estimating the genealogy and inferring the effective population size at various stages along the genealogical timeline are required for reconstructing this demographic history.

There are 2 steps for reconstructing demosgraphic history from a sequence alignment. First one is estimating the genealogy from the sequence data and the second one is estimating the population history based on the genealogy. The genealogy provides the relationships among the individuals (tree topology) as well as their times of divergence (node times).

Skyline-plot approaches use a very straightforward relationship between population size and the estimated duration of the coalescent interval to reconstruct demographic history. In each coalescent interval, they give estimate of the population size.

The Bayesian skyline plot model incorporates standard Markov chain Monte Carlo (MCMC) sampling processes to estimate a posterior distribution of effective population size through time directly from a sample of sequences [START_REF] Drummond | Bayesian Coalescent Inference of Past Population Dynamics from Molecular Sequences[END_REF]. This way they produce a piecewise reconstruction of the demographic history [START_REF] Pybus | An Integrated Framework for the Inference of Viral Population History From Reconstructed Genealogies[END_REF] Previous skyline methods are omitting the phylogenetic error in inferred genealogy which is impracticable. [START_REF] Drummond | Bayesian Coalescent Inference of Past Population Dynamics from Molecular Sequences[END_REF] proposed the Bayesian skyline plot to solve this issue, which is implemented in a framework in which the genealogy, demographic history, and substitution-model parameters are all coestimated in a single study. The plot of population history that results, which is averaged across the posterior sample of population sizes across time (as in the Bayesian multiple-change-point technique), contains credibility intervals that represent the combined phylogenetic and coalescent uncertainty. Because the extended Bayesian skyline method should be used if sequences from multiple independent loci are available [START_REF] Heled | Bayesian inference of population size history from multiple loci[END_REF] we used Bayesian Skyline method which implemented in BEAST used for this study. Also as suggesed a relaxed molecular clock can be employed to accommodate rate variation among lineages [START_REF] Drummond | Relaxed phylogenetics and dating with confidence[END_REF] is used in our analysis.

Reconstructing maternal demeograpghy

We reconstructed the mitochondrial phylogeny of domestic horses in two stages.First, 457

mtDNA sequences from all ancient horse specimens were used regardless of whether they belong to the DOM2 group or not for the phylogenetic tree construction, using RAxML [START_REF] Stamatakis | RAxML version 8: A tool for phylogenetic analysis and postanalysis of large phylogenies[END_REF]. In the second part, 266 radiocarbon-dated ancient specimens were used to reconstruct demographic trajectories, using Bayesian skyline plotting in BEAST2.5 [START_REF] Bouckaert | BEAST 2: A Software Platform for Bayesian Evolutionary Analysis[END_REF].

Workflow for phylogenetic tree construction is as follows:

-Individual sequences were called with BCFtools pipeline with majority rule and as long as at least five individual high-quality reads are present.

-PCR duplicates and sequence alignments with mapping qualities inferior to 25 were filtered and bases showing individual quality scores lower than 30 were also disregarded.

-All sequences are aligned together by using mafft aligner.

Next, the aligned sequences were further divided into six sequence partitions, following previous work [START_REF] Gaunitz | Ancient genomes revisit the ancestry of domestic and Przewalski's horses[END_REF] and consisting of the Control Region, all tRNAs, both rRNAs, and 1st, 2nd, and 3rd codon positions.

There are sequence overlaps between different partitions of mtDNA. In order to prevent same positions repeating in the data set overlaps between partitions and repeat regions were removed with custom R-scripts.

The resulting partitioned alignment is used as input for phylogenetic inference with RAxML[ with one hundred bootstrap pseudo-replicates to assess node support.

The best substitution model for each partition was identified using ModelGenerator v0.8

and the input files for BEAST (v2.5.1 67 ) were prepared in BEAUTi (version 2.5.2 64)

with the following parameters:

(1)specifying individual sequence radio carbon dates in years before present to provide temporal calibrating information,

(2) reporting each individual partition substitution model according to the ModelGenerator results,

(3) selecting the Bayesian Skyline demographic model, (4) assuming uncorrelated log-Normal relaxed molecular clocks -BEAST was run for a total of 1,000,000,000 generations. Convergence was checked using Tracer (version 1.7.1 68 ). Posterior distributions were obtained using 30% as burnin.

Y-chromosomal phylogeny reconstruction

The datasets used for the Y chromosomal reconstruction followed those used above for the mtDNA/. First, all the samples were used for Bayesian skylines (n=x). In order to focus on domestic linage, only the samples from the Dom2 lineage were used for Bayesian skyline construction. Because of the reasons mentioned before, dealing with Y-chromosome alignments requires careful attention. For this reason, different filtering parameters were applied to find an optimal pipeline to obtain high-quality variants along the Y-chromosome assembly to use as input for phylogenetic tree and Bayesian Skyline construction -First, all the individual sequences re-mapped using the new Y-chromosome reference (which consists of 764 contigs) with Bowtie2. [START_REF] Felkel | The horse Y chromosome as an informative marker for tracing sire lines[END_REF], [START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF]. Next, 17 modern male horse genomes were used to identify potentially problematic regions which could be confidently called heterozygous (using ANGSD [START_REF] Korneliussen | ANGSD: Analysis of Next Generation Sequencing Data[END_REF] -Positions that are not shared among half of the individuals are disregarded.

-Transitions were excluded from the final set of polymorphic sites. In addition to transitions, r C<->A and G<->A mutations were also excluded due to the observation of high error rates at these sites..

-142 specimens are used for phylogenetic tree construction with Raxml and 72 radiocarbon-dated individuals which belong to Dom2 groups are used for bayesian skyline construction -Finally, we used 51 ancient and 13 modern horses with 200 polymorphic sites.

-Input files for BEAST (version 2.5.2; REF) were prepared in BEAUti (version 2.5.2;

Bouckaert et al. 2014 [START_REF] Bouckaert | BEAST 2: A Software Platform for Bayesian Evolutionary Analysis[END_REF]);

(1) specifying individual sequence dates in years before present

(2)selecting the Jukes-Cantor mutational model (as transitions were disregarded)

(3)selecting the Bayesian Skyline demographic model [START_REF] Drummond | Bayesian Coalescent Inference of Past Population Dynamics from Molecular Sequences[END_REF] (4)assuming uncorrelated log-Normal relaxed molecular clocks [START_REF] Drummond | Relaxed phylogenetics and dating with confidence[END_REF] (5)BEAST was run for a total of 1,000,000,000 generations. Convergence was checked using Tracer (version 1.7.1; [START_REF] Rambaut | Posterior Summarization in Bayesian Phylogenetics Using Tracer 1.7[END_REF]. Posterior distributions were obtained using 30% as burn-in Figure 2 is showing respective mtDNA and Chromosomal Bayesian skylines.

Figure 2 Y chromosomal and mtDNA Bayesian Skylines

However, at the final stage a different and more efficient pipeline is applied for Y chromosome.

The absence of a full-length assembly for the Ychromosome implies that some nonoptimal read alignments could pass our mapping criteria. For example, paralogs that are not yet present as multiple copies in the available set of Y-chromosomal contigs would result in reads aligning against an apparently unique position. To limit the impact of such situations, we excluded (1) Y-contigs shorter than 5 kb, as usually representing highlyrepetitive unassembled regions, or encompassing (2) more than 1% of sites identified as multicopy, or (3) more than one percent of the sites potentially present in non-MSY chromosomal regions. Criteria 2 and 3 were following the estimates reported by Felkel and colleagues [START_REF] Felkel | The horse Y chromosome as an informative marker for tracing sire lines[END_REF]. This provided a total of 967,543 high-quality sites allegedly present on the MSY region of the horse Y chromosome. We then applied the strict filtering strategy described below for autosomal data (see section 'Pseudo-haploidizing ancient genomes'), provided that the MSY-linked site was present in at least 20% of the horses, while Posterior distributions were obtained using 30% as burn-in. The resulting Bayesian skylines and phylogenetic trees are shown in Figure 3.

A history of equine management in France over last 2,500 years

Besides horses, donkeys (the other domesticated species from the equid family) have had a significant impact on human history, serving as mechanical power for agriculture and transportation [START_REF] Blench | The origins and development of African livestock : archaeology, genetics, linguistics and ethnography[END_REF]- [START_REF] Kelekna | The horse in human history[END_REF]. Their first-generation hybrids have also played an important role due to their great strength, endurance, and resilience. Donkeys, unlike horses, are not very fast animals; yet, have outstanding resilience and carrying capacities and are well adapted to arid and semi-arid deserts [START_REF] Maloiy | Thermoregulation and metabolism in a small desert carnivore: the Fennec fox (Fennecus zerda) (Mammalia)[END_REF]. As a result, they have been extensively utilized as pack and draught animals along significant historical economic routes such as the trans-Sahelian and trans-Saharan roadways [START_REF] Blench | The origins and development of African livestock : archaeology, genetics, linguistics and ethnography[END_REF] and they continue to be vital for the economies of many developing countries today where they are used principally as draught or pack animals. Although the exact location and timing of donkey domestication are still unclear, there is a broad consensus that the domestication of donkeys began in the tropics or subtropics of Africa in Egypt and Nubia [START_REF] Beja-Pereira | African origins of the domestic donkey[END_REF], [START_REF] Rossel | Domestication of the donkey: Timing, processes, and indicators[END_REF].

Mules represent first-generation hybrids between a female horse (mare) and a male donkey (jack). They were highly prized for their strength, resilience, stamina, and sure-footedness on narrow mountain paths, as well as their even temperament. Although mule breeding requires considerable financial investment due to its infertile nature, they have been invaluable animals throughout history. The earliest time for mule breeding stated until now is 2,200 years ago[ [START_REF] Fages | Tracking Five Millennia of Horse Management with Extensive Ancient Genome Time Series[END_REF] With the light of this study, the date of this sample was revised to 1st century BCE and the early 1st century CE . Among the donkeys and horses, identifying mules from fossil records using morphology alone is difficult due to morphological similarity between the bones of a mule and those of their parental [START_REF] Clavel | Assessing the predictive taxonomic power of the bony labyrinth 3D shape in horses, donkeys and their F1-hybrids[END_REF]. There are alternative approaches to morphological identification. One of them is based on geometric morphometric analysis of the bony labyrinth of petrosal bone for identification of horses, donkeys and their hybrids [START_REF] Clavel | Assessing the predictive taxonomic power of the bony labyrinth 3D shape in horses, donkeys and their F1-hybrids[END_REF]. However, this method is not sufficient enough to differentiate between female and male, and also different hybrid species from each other [START_REF] Clavel | Assessing the predictive taxonomic power of the bony labyrinth 3D shape in horses, donkeys and their F1-hybrids[END_REF].

Although archeological data shows textual and iconographic evidence of past equine management, DNA-based methods are required for precise conclusions about presence of specific equine species in the area and the time zone. A notable tool for this purpose is the Zonkey pipeline, which can properly identify all living equid members and their hybrids from small amounts of high-throughput DNA sequencing data. It is specialized for equine genomes because of the reference panel, which includes Przewalski's horse, Franches-Montagnes horse, domestic donkeys, African wild ass, Grant's zebra, Grevy's zebra, Hartmann's mountain zebra, onager, and Tibetan kiang [START_REF] Librado | Ancient genetic changes associated with domestication of the horse[END_REF]. The usage of the pipeline requires at least one sample alignment file (BAM) against the mitochondrial and nuclear genome, but it is possible to carry partial analysis even if only one alignment is available.

Principal component analysis (PCA) using EIGENSOFT SmartPCA Admixture [START_REF] Price | Principal components analysis corrects for stratification in genome-wide association studies[END_REF]- [START_REF] Patterson | Population structure and eigenanalysis[END_REF] for estimation of ancestry components, Treemix [START_REF] Pickrell | Inference of Population Splits and Mixtures from Genome-Wide Allele Frequency Data[END_REF] for the nuclear phylogeny and mitochondrial phylogenetic analysis [START_REF] Stamatakis | RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models[END_REF] are conducted inside of the pipeline in order to determine the relation of the sample of interest with the ones in the reference pane.

Moreover, the pipeline outputs coverage statistics of autosomes and X-chromosomes to determine the sex of the sample. Males and females are expected to show X-to autosomal coverage ratios of ∼0.5 and ∼1.0, respectively. All the analyses are carried on with 2 sets of SNP panels with transitions and without transitions to reduce the impact of post-mortem DNA damage, which are pervasive in ancient DNA studies. The most important feature that makes the Zonkey pipeline important is that even 1,000 sequences mapped to the horse reference genome is enough to have clear results for identification. That is why in that study we leveraged our already generated minimal amount of high-throughput DNA sequencing data with the Zonkey pipeline.. Together with archeological information collected for the samples, this study has begun to shed light on the chronological, cultural, and sociological elements that have shaped horse (Equus caballus), donkey (Equus asinus), and mule management over the previous 2,500 years. Later, the data set was complemented with 27 additional ancient horse remains from 6 Upper Paleolithic sites by using the same Zonkey pipeline.

In this study, I used already generated data by the other members of the team and tabulated the Zonkey outputs in order to answer the questions about their relative importance and role in rural and urban contexts. to horses, mules are not mentioned before the mid-13 th century CE (Bautier, 1981).

Unveiling Equine resource management in historical

Overall, available sources depict a global medieval Northern France centered on horses and a global medieval Southern France where donkeys and mules were more common.

This regional polarity may even have strengthened during the 14 th -15 th centuries CE as mule trading intensified along the Pyrenean range and the Massif Central [START_REF] Pinto | Le commerce des chevaux et des mules entre la France et les pays catalans (xive-xve siècle)[END_REF] and mules were depicted as central to the rural economies of the Alps [START_REF] Mouthon | L'inventaire du bétail dans une communauté alpine à la fin du XIVe siècle[END_REF]. Mules may also have become more widespread at the time given their increasing textual occurrence in Paris or Normandy (for examples, see Contamine, 2008).

Archaeological data hold the potential to fill the gaps in knowledge on past equine management resulting from textual and iconographic evidence. For example, skeletal remains can inform on micro-regional patterns of horse, donkey and mule production

Material and methods

Archaeological Samples

A total of 874 archaeological remains were sampled for ancient DNA analysis. 1.

Results

Shifting temporal preferences in equine management

The collection of samples available for this study was largely dominated by horses (718/874 ~ 82.1%) but also led to the identification of 100 (~ 11.4%) donkeys, 55 (~ 6.3%) mules and a single hinny, dated to the 16 th -17 th centuries CE (Fontdouce Abbey (Saint-Bris-des-Bois)). Interestingly, the different taxa identified are not distributed evenly through space and time, but showed striking differences between proto-historic, Roman and Medieval periods (Fig. 3). More specifically, no hybrids were identified during the proto-historic period, which was only represented by horses. Hybrids were almost exclusively restricted to the Roman Period

Donkey management

The earliest donkey identified in our dataset was found in the urban context of Chartres (Boulevard de la Courtille), which is dated to 100-110 CE. Donkeys were no longer detected in the following two centuries until they reappeared again at Boinville-en-Woëvre. This late Antiquity site (3 rd -6 th centuries CE) was characterized by the presence of 22 (almost) complete skeletons in over a dozen of burials, both single and multiple, located in the Pars Rustica of a villa (Fig. 4A). Thirteen such animals were tested positive for DNA and included two horses and eleven donkeys (eight males, three females). Three additional donkeys showing poor DNA preservation were also suggested (data not shown). Such a predominance of donkeys is unprecedented and indicates management strategies largely focused on this species. Furthermore, those donkey specimens identified genetically at Boinville-en-Woëvre showed exceptional osteological characteristics that are suggestive of particularly large sizes, ranging 141-155 centimeters at the withers (Fig. 4B). The temporal changes in the relative prominence of donkeys in our dataset were not uniform across the different socio-economical contexts (Fig. 5B). They reflected contrasted patterns of resource management in which the animal was most common in rural elite contexts during the 7 th and 10 th century CE (chi-squared test, p-value = 0.044), but in urban elite contexts between the 11 th and 13 th centuries CE (chi-squared test, p-value = 0.010).

The relative importance of donkeys found in rural civil contexts remained secondary across the whole Medieval Period.

Mule Management

The earliest mule identified in our dataset (G'VA-219) was excavated at the site Saint-Justen-Chaussée (Plainval). It was found within a ditch connected to the borders of an Iron Age Celtic sanctuary and was revised to the Augustean Period, between the late 1 st century BCE and the early 1 st century CE, since its original report by Fages and colleagues (2019) in the terminal La Tène period. Another remarkable mule belonging to the the Northern France. However, our dataset suggests that mule management in Northern France remained only significant during Antiquity and was particularly important between the 1 st and 3 rd centuries CE in both rural and urban contexts.

Horse Management

Horses are the only species of equids identified in Iron Age France. They have remained the most frequently used equine species across all time periods and socio-economic contexts investigated in this study (718/874 ~ 82.1%; Fig. 2). This indicates a general preference for horses in France during almost the last 2,500 years.

A number of situations, however, reflect ritual rather than husbandry-related management practices. This is the case for the two sites of Gondole Les Piots and Orcet La Roche Blanche, dated between the 1 st century BCE and the 1 st century CE. These famous archaeological sites are located at the foothills of the Gergovia plateau and play host to a number of multiple horse burials, sometimes accompanied by humans. These are generally interpreted in connection with the battle of Gergovia or as sacrificial rituals specific to the Arvernes political center (Foucras et al., 2019). Regardless, our work confirms that only horses were inhumated on site, even though previous morphological analyses had suggested mules (Foucras et al., 2019). The same holds true for the sanctuaries of Vertault (1 st century BCE) and Saint-Just-en-Chaussée (La Tène D1-D2 -2 nd -1 st century BCE), that only comprised horses. The Roman site at Longueil Sainte-Marie (L'Orméon) is also worth mentioning, as it is interpreted as a rural sanctuary dated to the 2 nd -3 rd centuries CE (Gaudefroy & Lepetz, 2000). While mules were most prevalent at the time in our global dataset, none of the 26 samples analyzed consisted of mules, which supports common contentious of intentional horse sacrifice at the site.

It is important to emphasize that our analysis only revealed the presence of horses at the Iron Age site of Pech-Maho, dated to the early 2 nd century BCE, while donkeys had been identified morphologically there (Boulbes & Gardeisen 2014, Boulbes & Gardeisen 2018).

Our sampling strategy was strictly designed to exclude the latter specimens from our analyses, as our original aim was focused on the genetic characterization of horses exclusively. The apparent absence of donkeys resulting from our analysis hence only reflects this sampling bias, and shall not be taken against previous morphological reports. In fact, our analyses confirm previous morphological identification for all 25 horse specimens genetically tested. Importantly, this site is the only one of those analyzed in this study where sampling was implemented to avoid other equine species than the horse. While no other situations can be expected to reflect similar bias, the proportion of non-horse taxa may be considered as conservative.

Discussion

This study provides the first genetic attempt to characterize the geographic, temporal and socio-economic patterns of equine resource management in France. It confirms the prevalence of horses across all contexts and time periods, which is also reflected in textual evidence and iconography. Interestingly, this study reveals important temporal shifts that were not immediately apparent from other sources, including previous zooarchaeological The large proportion of mules identified during the Roman Period contrasts with the almost absence of donkeys and opens questions about the underlying production models.

Indeed, no mules can be bred without any donkey jacks. Taken at face value, the absence of donkeys may support models assuming exogenous production, with mules representing imported commodities. Interestingly, the existence of true breeding centers specialized in mule production was reported by Varro [START_REF] Orlando | Ancient DNA analysis[END_REF][START_REF] Mair | Reconfiguring the Silk Road : new research on east-west exchange in antiquity[END_REF] in Italy and recent isotopic work suggested non-local mule breeding in Bavaria [START_REF] Berger | Life history of a mule (c. 160 AD) from the Roman fort Biriciana Weißenburg (Upper Bavaria) as revealed by serial stable isotope analysis of dental tissues : Life History of a Mule[END_REF]. However, other regions must also have developed such a production across the whole Empire since these Italian production areas are unlikely to have produced animals in sufficient numbers to supply all the northern territories of the Empire. Therefore, the near absence of donkeys revealed in this study does not necessarily imply far-away importation.

The Gallia Belgica overlapped the area where most Roman mules were identified in this study. This region had the agro-pastoral capacity to develop such a largescale production [START_REF] Lepetz | Agro-pastoral systems during the late Iron Age and Roman period in Northern Gaul[END_REF]. In fact, mule production does not require large donkey stocks as only a limited number of jacks can mate with many mares. More than the mules themselves, import strategies may, thus, have focused on acquiring good donkey breeders, such as those described by Varo [START_REF] Orlando | Ancient DNA analysis[END_REF][START_REF] Levine | Dereivka and the problem of horse domestication[END_REF], which could apparently be sold for 40,000

(3, 2) and even 60,000 [START_REF] Orlando | Ancient DNA analysis[END_REF][START_REF] Levine | Dereivka and the problem of horse domestication[END_REF] sesterces, an amount that is approximately worth 3.2-4.8 kilograms of gold. Interestingly, the donkeys identified at Boinville-en-Woëvre during the 3 rd -5 th centuries CE showed an exceptional size (Fig. 3) and may reflect such animals, or Saint-Riquier (Fossier, 1968). Our work, thus, highlights the archaeological record as an essential source to reconstruct a comprehensive history of animal management.

Furthermore, our dataset reveals that donkeys were primary used in rural elite contexts between the 7 th and 10 th centuries CE (Fig. 5B). Such contexts most often consist of seigneurial farms or Church-controlled establishments. For example, excavations at Boves respectively, in which complete skeletons could be excavated. However, most of the material available in other contexts has remained incompatible with sexual determination [START_REF] Brunaux | Le sanctuaire de Gournay-sur-Aronde (Oise) : structures et rites, les animaux du sacrifice[END_REF][START_REF] Méniel | Les sacrifices d'animaux chez les Gaulois[END_REF]. A full characterization of the entire range of sacrificial practices at the time is, thus, still pending. Similarly, the material present in Gallic food wastes has helped depicting butchery activities evenly balanced between males and females, but preferentially oriented toward old animals [START_REF] Méniel | Les chevaux découpés du village d'Acy-Romance et l'hippophagie en Gaule septentrionale[END_REF]). The consumption of horse meat was, however, not widespread at the time, and was generally depreciated during the Roman Period, from the first century CE [START_REF] Arbogast | Archéologie du cheval. Des origines à la période moderne en France[END_REF].

Apart from cities, where considerable amounts of equid carcasses were evacuated and often accumulated (Lepetz et Textual evidence has remained especially elusive regarding sex-specific horse husbandry techniques in early Medieval France. Grégoire de Tours's 'Historia Francorum' reported that special care was paid to mares in studs, which hints at differentiated horse husbandry strategies between males and females at least from the 6 th century CE [START_REF] Bautier | Contribution à l'histoire du cheval au Moyen Âge[END_REF]. Strict legal rules governed livestock production, including for horses, and property at the time but the overall nature and quality of such production remains poorly documented, apart from abbey charters, where access to horse mounts was ruled (Bautier 1981). Although horse husbandry was limited both in range and numbers (Fossier 1968), textual sources provide important details on the composition of equine herds during the Carolingian Period (8 th -10 th century CE) [START_REF] Prévot | Le cheval en France au Moyen Âge ; sa place dans le monde médiéval[END_REF]. For example, 'L'inventaire d'Annape' reported that an average of one stallion every 20 mares was present in villae rural establishments, from the early 9 th century CE. Outside abbeys, textual evidence remains, however, insufficient to characterize the broad range of horse production modes until the end of the Middle ages, when regional production, royal studs and trading sustained an increasing horse demand for transportation and war [START_REF] Bautier | Contribution à l'histoire du cheval au Moyen Âge[END_REF]).

Overall, our current understanding of past management strategies of equine resources remains limited for France across space, time and social contexts, especially regarding the respective roles of males and females. In this study, we used state-of-the-art approaches in ancient DNA research to complement the dataset gener ated by Lepetz S1; Supplementary Information), one Azilian habitat (Le Closeau, N=5)

and one natural site from Southwestern France, at Igue du Gral, Sauliac-sur-Célé (N=13).

The latter site corresponds to a natural trap, which provides a baseline for assessing sex ratios in natural horse populations and testing for potential differences with habitat sites.

Sex-ratio were determined using the Zonkey pipeline (Schubert et The Medieval and Modern Period were subdivided into the four main time periods described below.

The first period spanned the 7 th to 10 th centuries CE and included a total of 113 samples, mostly from rural farms and villages ('civil rural' thereafter; 67/113~59.3%), castral sites (38/113~33.6%, including rural castles but also the Offin horse (excavated from an elite burial) and urban elites (8/133~7.1%). The Urban elite sites comprise remains from the Château-Thierry castle and the episcopal palace at Beauvais, but also the 'Rue Barrabin' site at Surgères, which had strong connection with the city castle at the time [START_REF] Vacher | Surgères (Charente-Maritime). Rue Barrabin et rue de la Binnetterie[END_REF]. This was also the case of the 'Vismaret' site at La Calotterie, which was located in the ancient harbor of Quentovic, a city of rich merchands [START_REF] Wood | Quentovic et le sud-est Britannique (VIe-IXe siècle)[END_REF] Chi-squared statistical tests were carried out in the R programming language while the factorial analysis was done in the PAST software (version 3).

Results

Upper

Gallic sites

Considering all horse remains from archaeological sites belonging to the Hallstatt and La Tène A periods together suggested balanced sex ratios (4 vs 4), but the effective size is too limited to be meaningful. However, an over-representation of horse males was statistically supported when La Tène sites were considered altogether (103 vs 24; Chi-squared test, pvalue=2.381x10 -12 ). This signal was mainly driven by sacrificial sites (82 vs 15; Chisquared test, p-value=1.026x10 -11 ), as the sex-ratio was not as strongly unbalanced and the test was considerably less statistically significant when conditioning on habitat sites only ( 21 vs 9; Chisquared test, p-value=0.02846). The selection of males in sacrificial rituals was especially pronounced at Vertault (N=9), Le Cendre (Gondole; N=8) and Orcet (N=36), where no females were identified (Chi-squared test, p-value=3.335x10 -13 ). The bone assemblages were also sex-biased at Pech-Maho, despite the presence of females on site (18 vs 7; Chi-squared test, p-value= 0.02781). The over-representation of male horses was, however, not a universal rule, as balanced sex-ratios characterized the Saint-Just-en-Chaussée sanctuary (4 vs 6; Chisquared test, p-value=0.5271).

Gallo-Roman sites

The horse was no longer the only species identified in our dataset from the Roman Period (Lepetz et al. submitted) (Fig. 2). Regardless of the taxonomic group considered, sex ratios were found to be biased towards males, as 157 horse males were identified for only 46 A factorial analysis of the 436 samples belonging to the Medieval and Modern Periods recapitulated the strong difference observed between rural and urban contexts along the second factorial axis (Fig. 4). This difference was maintained throughout the four time periods considered (i.e. Combined, all these factors may underpin the strong bias towards males reported here for both Roman and Medieval cities, and the marked difference in the management of equine resources observed between rural and urban contexts. These depict 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.

Finally, it should be stressed that urban environments were also extremely stressful for males. In addition to the considerable numbers of skeletal pathologies found in the archaeological record ( information provides important insights into the impact of the sex-biased process such as migration, the unequal contribution of different sexes to the breeding process. For example, recent phylogenetic research on wolves found different demographic histories between males and females [START_REF] Smeds | The evolutionary history of grey wolf Y chromosomes[END_REF], [START_REF] Phung | Complex patterns of sex-biased demography in canines[END_REF]. They identify in both dogs and wolves, there were larger numbers of reproducing males than females by comparing effective population size on the X chromosome and the autosomes.

One aim of this Ph.D. is to demonstrate maternal and paternal demographic changes in horse history. My findings have clearly suggested that both maternal and paternal DOM2 lineages had significant, parallel population expansions beginning in the late third millennium BCE, showing that there was no sex bias in reproduction at the period.

Previously, a mitochondrial Bayesian skyline demographic profile was created by Gaunitz et al 2018 by using 123 modern and 88 ancient mitochondrial genomes. This study proved mtDNA demographic expansion ~4500 years ago in line with the possible ancestors of DOM2 expansion [START_REF] Gaunitz | Ancient genomes revisit the ancestry of domestic and Przewalski's horses[END_REF]. Similar to this study, I found that only ancient mitochondrial genomes belonging to modern domestic lineage (DOM2) unwent a bottleneck at this time, so were likely used in the early stages of domestication. By using the Bayesian skyline method with 266 ancient samples across Eurasia, the sex specific changes in the genetic makeup of horses during this time period in this region could be uncovered. Y chromosomal Bayesian skyline construction required careful attention due to reasons mentioned before, but ultimately showed a demographic expansion specific to DOM2 lineage, with the same pattern as seen in the mtDNA. Accordingly, the regional barriers that had shaped ancestral geographic lines before the early third millennium BC disappeared by the end of this millennium, as both maternal and paternal DOM2 lineages experienced massive demographic expansions [START_REF] Librado | Genomics and the Evolutionary History of Equids[END_REF] .Both matrilineal and patrilineal effective population sizes increased through time until this point, when they started to expand rapidly, due to the increased practice of horse breeding around this time. Also,until now the standard model of horse domestication supposedly consisted of local introgression through mares. Because stallions may be difficult to control, it is probable that mares contributed significantly more to the establishment of the domestic horse throughout history than stallions. Furthermore, most selection is focused on male since, at the individual level, they may generate a greater number of offspring than females. (In other words, a limited number of relatively cooperative stallions are likely to have been utilized

to impregnate a huge number of mares.) However, with comparing maternal and paternal demographic patterns, we cannot support that idea anymore.

It has already been demonstrated that Y chromosomal diversity is low in modern horses as all stallions are carrying the same Y-chromosome haplotype [START_REF] Lindgren | Limited number of patrilines in horse domestication[END_REF] but it was larger in the past [START_REF] Librado | Ancient genetic changes associated with domestication of the horse[END_REF], [START_REF] Lippold | Whole mitochondrial genome sequencing of domestic horses reveals incorporation of extensive wild horse diversity during domestication[END_REF] indicating that particular male lineages have become increasingly important.

The extremely low Y chromosomal variety in existing horses has been attributed mostly to intense breeding techniques following domestication, and Y chromosome diversity has fallen substantially due to positive selection [START_REF] Lippold | Whole mitochondrial genome sequencing of domestic horses reveals incorporation of extensive wild horse diversity during domestication[END_REF], [START_REF] Levine | Botai and the Origins of Horse Domestication[END_REF], [START_REF] Cunningham | Microsatellite diversity, pedigree relatedness and the contributions of founder lineages to thoroughbred horses[END_REF]. The low degree of variety in horse Y chromosome sequences makes it difficult to distinguish between stallions and leads to low phylogenetic resolution of horse patrilineal lines, limiting their use in assessing patrilineal diversity in horses.

This loss in diversity started around 2,000 years ago and half of the diversity was lost in the past 1,000 years [START_REF] Fages | Tracking Five Millennia of Horse Management with Extensive Ancient Genome Time Series[END_REF] suggesting that the extreme lack of genetic diversity in modern horses was only recent. In the light of this recent loss of the genetic diversity in the Y chromosome, we would expect to see a decline in Y chromosomal skylines through modern times. We generated the most plausible polymorphic positions to use for demographic analysis for the Y chromosome with the widest sample set and improved Y chromosomal reference. Unfortunately, it is not possible to state the recent decline confidentially with this study. Even though the median of the Y chromosomal skyline shows a slight decrease after the huge expansion, we cannot make a precise assumption owing to 95% confidence intervals. The main obstacle with Y chromosomal analysis is the difficulty in obtaining good levels of coverage for the Y chromosome, which is more difficult than for autosomes. In order to assess whether or not there is power to detect this recent bottleneck both in terms of samples and sequence length simulations are needed. For example by using a demographic modelling software such as Bayesian Serial SimCoal [START_REF] Excoffier | SIMCOAL: a general coalescent program for the simulation of molecular data in interconnected populations with arbitrary demography[END_REF], it is possible to create the demographic pattern we are expecting to see and create ~=3.981072 and at the time 0, after domestication; (10^6)/(10^5)~=10. Evidently, during and immediately after domestication, there was more variation in female lineages used by humans for breeding than males. In order to have precise assumptions about this, the whole posterior distribution should be used to draw a continuous ratio over time.

Interpretation of shallow DNA sequences from zooarchaeological data

Taxonomic identifications are a highly important part of archeological research. Due to their high resemblance, taxonomic identification in equids is highly challenging.

Furthermore, morphological variation in usually incomplete osseous assemblages is frequently insufficient to differentiate hybrids and sex [121].

Standard morphometric techniques, particularly for hybrids and genders, are prone to significant misidentification rates [121], [START_REF] Granado | The mules that are not mules -metrics, morphology, archaeogenomics and mtDNA d-loop diversity in equids from Roman Switzerland[END_REF] unless based on the integration of numerous skeletal parts [START_REF] Hanot | Identifying domestic horses, donkeys and hybrids from archaeological deposits: A 3D morphological investigation on skeletons[END_REF] However, horse archaeological remains are frequently fragmented, making multi-proxy analyses difficult. More complex approaches, such as Geometric MorphoMetrics applied to teeth [START_REF] Owen | The zooarchaeological application of quantifying cranial shape differences in wild boar and domestic pigs (Sus scrofa) using 3D geometric morphometrics[END_REF] or petrosal bones [START_REF] Clavel | Assessing the predictive taxonomic power of the bony labyrinth 3D shape in horses, donkeys and their F1-hybrids[END_REF] can yield high, but not optimal, true positive assignment rates. This difficulty has reduced our ability to follow the commencement of mule breeding and differential male and female management across areas and time [START_REF] Schubert | Zonkey: A simple, accurate and sensitive pipeline to genetically identify equine F1-hybrids in archaeological assemblages[END_REF] [START_REF] Schubert | Zonkey : a simple, accurate and sen-sitive pipeline to genetically identify equine F1-hybrids in archaeological assemblages[END_REF]. The only method that yields precisely accurate taxonomy identification employs shallow DNA data and the Zonkey statistical program [START_REF] Schubert | Zonkey: A simple, accurate and sensitive pipeline to genetically identify equine F1-hybrids in archaeological assemblages[END_REF].

In both projects which included in this PhD thesis, vast collection of bones and teeth from the end of the Iron Age to the Modern Period distributed over France are analysed with the help of Zonkey statistical program. Firstly, we showed altering patterns of species across time, space and social contexts from the Iron Age in France. Secondly, we focused on sex proportion in the same area and extended the time period until the Upper Paleolithic. This is the first time that DNA analysis has been performed on such a large animal collection from a single country. Because textual and iconographic sources already have details of mule and donkey management as early as the Roman periods, it was expected to see changing patterns of equine management choice in the studied historical period. Our research shows that horses have been the most commonly used animal throughout history.

Additionally, the Roman Period appears to be the golden period of mule breeding, with mule bone assemblages accounting for up to 30% of all bone assemblages. Furthermore, mules were supplanted by donkeys in the 5 th c. CE and remained negligible after that.

Donkeys followed numerous human activities throughout the medieval period. They functioned in diverse social settings across time, with rural and urban elites reflecting the biggest demand during the 7th-10th c. CE and the 11th-13th c. CE, respectively. The significance of this research is to fill historical gaps in horse resource management in

France and shows substantial temporal, regional, and social preferences. Moreover, we discovered mules as important economic assets during the Roman Period. In that period it was crucial the physiological features of the animal due to extraordinarily high demand for long-distance transportation. However, their production is costly because they are sterile and hence cannot self-propagate. Demand for the mules declines with the collapse of Roman Empire and breeders shifted their focus to donkeys as short-distance transit supported more local economies. Our findings pave the way for additional research throughout the Roman Empire to better understand how such global economic needs were translated locally, as well as to localize mule breeding centers.

In addition to equid species differentiation, the follow up study provides new insights on the diverse roles that males and females performed in a variety of human activities, as hunting, herding, and sacrifice rituals. Previously, it was demonstrated that temporally sexbiased distribution of equine bone assemblages across Eurasia [START_REF] Fages | Tracking Five Millennia of Horse Management with Extensive Ancient Genome Time Series[END_REF]. They also reported that horse males and females have balanced representation in the early stages of domestication unlike bison [START_REF] Gower | Widespread male sex bias in mammal fossil and museum collections[END_REF] and woolly mammoth [START_REF] Pečnerová | Genome-Based Sexing Provides Clues about Behavior and Social Structure in the Woolly Mammoth[END_REF] which have strong representation of males during the late Pleistocene. In our study we also take in account of context of different archeological sites. We discovered modest sex distribution trends at the more local scale, which change depending on the archaeological setting. Firstly, we show that Magdalenian horse hunts did not target single bachelor groups, but rather single-family groups with a harem-like structure in accordance with the morphometric archaeological data. Then, we report on ceremonial behaviors significantly biased towards male sacrifice in the Iron Age and show that such practices continued until the Roman Period. Finally, our most remarkable discovery is that there is a significant variation in the distribution of men and females across urban and rural situations, with horse males dominating cities. Based on textual evidence, we believe that this represents rural contexts centered on horse production and cities in need of both renewable and disposable labor. The finding of this study fills critical historical gaps in our understanding of the management of equine male and female resources in France. It also paves the way for future research question about whether males in the city geldings or animals from more docile breeds were predominantly.

Figure 1

 1 Figure 1 Consensify approach. Numbers are indicating counts of specific bass at specific positions. 3 bases are randomly selected and common one is recorded as final.

Figure 3a :

 3a Figure 3a: mtDNA and Y chromosome Bayesian Skylines

France Article 1 Title:

 1 Historical management of equine resources in France from the Iron Age to the Modern Period Authors: Sébastien Lepetz a,$, Benoît Clavel a,$, Duha Alioğlu b,$, Lorelei Chauvey b, Stéphanie Schiavinato b, Laure Tonasso-Calvière b, Xuexue Liub b, Antoine Fages b, Naveed Khan b,c, Andaine Seguin-Orlando b,d, Clio Der Sarkissian b, Pierre Clavel b, Oscar Estrada b, Charleen Gaunitz

  (e.g.[START_REF] Putelat | Maultiere in der römischen Armee zur Zeit der Eroberungsfeldzüge in Germanien[END_REF], as well as on the animal individual life history (e.g.[START_REF] Lignereux | Un cheval gallo-romain inhumé dans le cimetière du site de Notre-Dame du Bon Accueil (IIe-IIIe siècle après J.-C., Rodez, Aveyron)[END_REF]) and more. In France, no site reports indicate the presence of mules during the Iron Age. In contrast, donkeys are known along the Mediterranean coast at Béziers[START_REF] Columeau | Nouveau regard sur la production et la consommation de la viande dans le Languedoc occidental[END_REF] Ugulini et al., 1991) from the 5 th century BCE (Before Common Era), at Ambrussum(Columeau, 1991) and Pech Maho (Boulbes & Gardeisen, 2014) from the 3 rd century BCE, and further North only during the Roman Period. They apparently remained relatively rare there (Lepetz & Yvinec, 2002), representing ~4.7% and ~6.7% of equine assemblages in Gaul and Rhineland, respectively (Johnstone, 2004), and only represented sporadic identifications in Eastern regions during Antiquity (Lepetz & Morand, 2017). Mules are believed to have been more common then, reaching up to ~30% and over 40% of equine bone assemblages in Gaul and Rhineland, respectively (Johnstone, 2004). Such an apparent ubiquity of mules contrasts with the scarcity of donkeys and raises the possibility of import from other provinces, especially as isotopic analyses have identified non-local mules in Bavarian sites at the time (Berger et al., 2010). Both the existence and the location of underlying production centers remain, however, elusive and the evolution of the status of mules into the Medieval Period is largely unknown. The Medieval donkey has received more zooarchaeological attention, and is generally considered to have become common, and possibly even dominant, from the 6 th -8 th century CE (Lepetz & Yvinec, 2002). No data are, however, available across the full temporal transect, from the 2 nd Middle Ages (12 th -15 th centuries CE) into the Modern Period. In addition to being lacunar, equine zooarchaeological data suffer from important limitations, as the morphological identification of horses, donkeys and hybrids from fragmentary material remains problematic (Peters, 1998, Uerpmann & Uerpmann, 1994, Johnstone, 2004 & 2006; Chuang 2016; Hanot et al., 2017). Although some recent applications of geometric morphometrics to whole tooth rows (Cucchi et al., 2017) or the bony labyrinth (Clavel et al., 2021) have shown great performance, including in hybrids, mule identification based on standard morphological criteria can still be unreliable (Chuang, 2016). In fact, recent DNA work has provided striking examples of species mis-specification in Roman Swiss bone assemblages (Granado et al., 2020). DNA also identified donkeys in Chalcolithic Portugal, contradicting archaeological models associating the first arrival of the animal in Iberia with Phoenician trade, some 1500 years later (Cardoso et al., 2013). Combined, these studies raise important concerns on the robustness of previous equine identifications, and consequently, challenge the overall validity of our current understanding of past equine management both in France, and more generally around the world.In order to start to remediate this, we have gathered the largest collection of equine archaeological remains in France, representing a total of 874 specimens from 128 sites spread across the country, and have applied state-of-the-art DNA-based taxonomical tools[START_REF] Schubert | Zonkey : a simple, accurate and sen-sitive pipeline to genetically identify equine F1-hybrids in archaeological assemblages[END_REF] Fages et al., 2020). The methodology relies on the Zonkey pipeline that we previously developed, which can accurately identify all equid members and their hybrids from minimal amounts of high-throughput DNA sequencing data[START_REF] Schubert | Zonkey: A simple, accurate and sensitive pipeline to genetically identify equine F1-hybrids in archaeological assemblages[END_REF]. Our approach provides the first in-depth characterization of temporal and social changes in equine management for a single country, from the Iron Age to the Modern Period. While horses remained the most prevalent equine species across the whole period investigated, our dataset reveals the importance of mule breeding in Northern France during the Roman Period, especially between the 1 st and the 3 rd centuries CE. It also unveils their almost disappearance from the early Medieval Period at the benefit of donkeys, which became closely associated with important rural and urban commercial and economical centers. Finally, our work uncovers donkeys of exceptionally large sizes, which calls for a reconsideration of the true morphospace of past equine species, even in the most recent time periods.

( 53 /

 53 55 ~ 96.4%) while the vast majority of donkeys were associated with Medieval and Modern contexts (85/100 ~ 85.0%). Therefore, our data indicate shifting temporal preferences in the management of donkey and mule resources, with horses remaining dominant and essential throughout the whole period investigated and hinnies exceptional.

1

  st century CE (G'VA-1057) was identified at Meaux (Rue Alfred Maury) and consisted of a complete fetus found in an urban dump deposit. In fact, while the size of our dataset is limited for the 1 st century CE (N=25), mules represented ~20.0% of the taxonomic assignments (5/25). Their relative importance significantly increased to ~24.7%(22/89) in the first half of 2 nd century CE and to ~34.2% (13/38) from the second half of 2 nd century CE (Fig.2), then decreased in the first half of 3 rd century CE to ~12.9% (12/93) until it suddenly vanished from the mid-3 rd century CE (Chi-squared test, p-value = 0.039). Sample G'VA-555, excavated from the rural context of Saint-Pathus, represents the latest Roman sample identified during the Late Antiquity. Strikingly, no mules were identified amongst the 297 Medieval samples present in our dataset, apart from one individual from Aix-en-Provence, Southern France, from the 15 th century CE. In fact, the number of mule identifications remained also extremely limited during the Modern Period, with only one individual (1/163 ~ 0.6%) detected at Evreux (Parking de l'Hôtel de Ville), a site from the Northern region dated to the 17 th century CE. The under-representation of Southern France assemblages in our dataset prevents rigorous comparisons with the situation observed in

  work. Alongside horses, mules have represented an important resource during the Roman Period in France, especially between the end of the 1 st and the beginning of the 3 rd centuries CE where they contributed between ~20.0% and 34.2% of the equine skeletal assemblages. They suddenly vanished almost completely from the archaeological record by the Late Antiquity and were largely absent during the Middle Ages, a time period when they were superseded by donkeys. That our work uncovers the Roman Period as the Golden Age of mule breeding reflects the important socio-economic changes at the time. During the Roman Period, the magnitude of trade exchange indeed reached unprecedented levels (Reddé et al., 2018). The need for transportation was ubiquitous and equally fit private and public interests, both for trade, transportation and military supply (Roth, 1999; Porte, 2015), and the Imperial post service (the so-called cursus publicus) (Pflaum, 1940). Long-distance exchange between civitates and provinces were mainly carried out by sea and rivers. However, wherever waterways were impracticable or lacking, and for short distances, including between ports, cities and the countryside, transportation was by land (Deman, 2002). An important road network was, thus, developed across the Roman Gauls (Duval, 1989). Our work reveals that the physiological qualities of mules, which are more sure-footed and show stronger work capacities than horses (Tegetmeier et al., 1895), largely contributed to fuel this economic expansion and land transportation demands. Our findings mirror the countless numbers of Antique figured reliefs representing carriages often composed of two mules led by the muliones, literally the mule drivers (Raepsaet, 2002).

  their local descent. With current evidence, this remains, however, speculative. In any case, as donkeys remained uncommon in the Northern regions at the time, their main purpose may have been to sire mules. Now that archaeological sites characterized with large proportions of mules or donkeys have been identified, future work shall be aimed at investigating the geographic and evolutionary origins of such equine resources. This will ideally be achieved by coupling the power of strontium isotope[START_REF] Bendrey | Patterns of Iron Age Horse Supply: An Analysis of Strontium Isotope Ratios in Teeth*[END_REF] together with high-resolution genetic data informing on local population affinities[START_REF] Lawson | Inference of population structure using dense haplotype data[END_REF]. Investigating the case of the mule fetus found at Meaux (1 st century CE) with such technologies will also be essential to address whether import was also oriented toward pregnant horse females carrying mules.The Late Antiquity and early Medieval Period mark the rise of the donkey in our dataset. This largely coincides with new economic systems involving a reorganization of trade and transportation(Devroey, 1992;[START_REF] Lebecq | Les marchands au long cours et les formes de leur organisation dans l'Europe du nord et du nord-ouest aux VIIe-XIe siècles[END_REF] Devroey & Nissen, 2015), which noticeably no longer favored the production of sterile mules. Redirecting equine management toward donkeys, which represent strong pack animals, particularly suited to farming activities and easier to produce, may, thus, reflect pragmatic decisions adapting to the new economic reality of the time. The substantial proportion of donkeys identified in this study contrasts with their relative scarcity in the textual record. This is especially true in rural elite contexts during the Carolingian Empire, a time when horses are exceedingly mentioned in textual sources from large rural establishments, such as at Saint-Bertin and

FiguresFigure 1

 1 Figures

Figure 2

 2 Figure 2 Sample dataset. A: Geographic sample distribution. The size of the different piecharts is proportionate to the number of samples analyzed locally (717 horses, 100 donkeys, 55 mules, and; 1 hinny). B: Temporal distribution of samples (N=873). C: Sample distribution across the various archaeological contexts (N=873).

Figure 3

 3 Figure 3 Temporal patterns of horse, donkey, and mule representation within our collection of equine remains.

Figure 4

 4 Figure 4 Donkey identification and morphological characteristics. A: In situ photograph of the donkey identified along Structure 3144 at Boinville-en-Woëvre. B: Distribution of measurements (greatest length and smallest breadth of the diaphysis) of equid metacarpals from the Roman period (1 st -5 th centuries CE; Lepetz, 1996 and unpublished data).Estimates of height at the withers are proposed for 4 individuals for illustration purpsose (following May, 1985; the function for metacarpal bones is Gl x 0.6102). The specimens genetically identified as donkeys at Boinville appear amongst the largest equids measured, and are closely followed by the donkey characterized at Woippy. Conversely, a very small horse was also identified in Limoges (Villa Brachaud) during the Roman period.

Figure 5 3 . 2 1 Title:

 5321 Figure 5 Temporal proportions of horse (green) and donkey (blue) identifications. A: Overall proportions from the Middle Ages (7th century CE) to the Modern period (18th century CE). B: Temporal dynamics within four individual socio-economic contexts.

2

  and colleagues (submitted) with 27 additional ancient horse remains from 6 Upper Paleolithic sites. We applied the Zonkey statistical package to shallow next -generation DNA sequence data in order to determine the sex of a total of 897 individuals. This represented 134 archaeological sites spanning the Upper Paleolithic (Upper Magdalenian and Azilian; N=27 samples) and a time period extending from the Iron Age to the Modern Period (N=870 samples). The Upper Paleolithic data provided a baseline for natural patterns of equine sex ratio and in relation to hunting, while the remaining material offered a first glimpse at the respective role of males and females in equine production, consumption and sacrifice. Material and methods Archaeological Samples A total of 897 equine remains were collected from 134 archaeological sites and subjected to the DNA analysis fully described by Lepetz and colleagues (submitted) and following the same sequence quality, alignment and statistical requirements . The vast majority of the underlying sequence data was previously reported, in studies aimed at taxonomic identification (Gaunitz et al. 2018 ; Fages et al. 2019; Fages et al. 2020; Lepetz et al. submitted). Novel DNA sequence data were generated for the present study, representing a total of 25444516 sequence reads from 27 samples obtained from six Upper Paleolithic sites, all of which confirmed as horses (Fig 1). These include four Magdalenian habitats from the Paris Basin (N=9; Le Closeau, Étiolles, Tureau-des-Gardes, Grand-Canton and Pincevent; Table

  Paleolithic sites Our dataset combines a total of 27 Upper Paleolithic horse remains, including 12 males and 15 females. This provides evidence for balanced sex ratios in this time period (Chi squared test, p-value=0.5637), in line with earlier reports based on other Upper Paleolithic sites from across Belgium to Siberia (Fages et al. 2020). Interestingly, the presence of balanced sex ratios is not statistically rejected when conditioning the analyses to those Magdalenian habitat sites (Chi-squared test, p-value=1. 0) or the natural trap at Igue du Gral (Chi -squared test, p-value=0.4054). This finding rules out horse prey hunting strategies focused on one specific sex during the time period investigated.

Figure 1 .

 1 Figure 1. Geographic location of the archaeological sites investigated in this study. Panel a: Paleolithic Period. Panel b: Iron Age (5 th -1 st century BCE). Panel c: Roman Period (1 st -5 th centuries CE). Panel d: Medieval & Modern Period (5 -18 centuries CE). The size of the circles is commensurate with the number of samples amenable to ancient DNA sequencing. Only the sites mentioned in this article are located on the map. The complete site projections are presented in Lepetz et al. submitted, Fig. 1.

Figure 2 Figure 3 .

 23 Figure 2 Temporal changes of mule (top), donkey (middle) and horse (bottom) sex-ratios. Sex-ratios are provided as the proportion of males (blue) identified in each category. The proportion of females is shown in orange. The total numbers of remains included in the analysis are also reported. *: Statistical significance

Figure 4 . 4 Discussion, Conclusion and Perspectives 4 . 1

 4441 Figure 4. Factorial analysis of horse and donkey sex-ratios within various social contexts.

  sequences and samples with desired length. Secondly, even if the Y chromosomal reference genome is much improved in terms of covering MSY sequence variation of horses, it lacks diversity found in ancient samples. When we focus on modern diversity with the existing Y chromosomal reference genome, it is possible that we are missing the signals from past generations. Including ancient genome diversity into reference genomes could help to improve this aspect. On the other hand, we observe a decrease just before the dramatic increase around 3000 BC in effective population size. This could indicate through the time to domestication, selection towards breeding males with specific traits of interest, but the Bayesian skyline with Y chromosome itself is not enough to make a conclusion. Also, in the phylogenetic tree, all the Dom2 samples are clustered together which indicates little differentiation in the population at this time. Thus, even if there is a selection towards specific males, this selection pattern probably exists in autosomal variants, rather than Y chromosomal.Phylogenetic reconstructions showed highly divergent mtDNA and Y chromosome lineages. Previously in Fages 2019[START_REF] Fages | Tracking Five Millennia of Horse Management with Extensive Ancient Genome Time Series[END_REF] it is shown that the extremely divergent IBE Y chromosome suggest that a divergent but yet unidentified ghost population could have contributed to the IBE genetic makeup. In the Y chromosome phylogenetic tree, we observe the same pattern for IBE samples and also one Neolithic sample from Köşk Höyük(KSK16232).It has Botay-related ancestry and suggests connections between Central Asia and Anatolia prior to the fifth to seventh mill BCE, thus, pre-domestication.In the Y chromosomal phylogenetic tree. The ghost population should not only contribute to IBE genetic makeup but also pre domestication Anatolian horses.Although mtDNA and Y chromosomal bayesian skylines are following similar patterns, which indicates that there was no sex bias in breeding practices at this time, I observed a huge change in the mtDNA to Y-chromosome effective population size ratio before and after domestication. This change in the mtDNA/Y-chromosome effective population size ratio change is as follows: at the time 18,000 BCE, before domestication; 10^(5.4)/10^(4.8)

Table of contents

 of

	1 INTRODUCTION	-1 -
	1.1	A primer to horse domestication	--
	1.2	A primer on ancient DNA research	--

  The third milestone did not come in fact from ancient DNA research first, but came from the development of

next-generation DNA sequencing (NGS) platforms, which provided unprecedented sequencing capacity. NGS shotgun-sequencing could originally characterize hundreds of thousand sequences overnight in parallel (now, over several billions routinely)[30]. It, thus, provided an opportunity to convert any microlitre of ancient DNA extract into hundreds of thousand sequences, a feat that no PCR approaches, including in their multi-round multiplex versions (Rompler et al. 2005), nor any other DNA sequencing technologies then available (and based on Sanger Sequencing; Noonan et al. 2005) could achieve. The first NGS application to ancient DNA research retrieved 13 Mb of sequence information from a mammoth specimen, which represented more sequence data than what the whole of ancient

2 Demographic Analysis with Uniparental Markers and Bayesian Skyline Method

	Populations are defined as groups of individuals of the same species living and
	interbreeding within a given area. Members of a population often rely on the same
	resources, are subject to similar environmental constraints, and may depend on the
	availability of other members to persist over time. Scientists study populations by
	examining how individuals interact with each other and how the population as a whole
	interacts with its environment.
	Demographic analysis are studies that are conducted to examine the past dynamics of a
	population and include the analysis of genetic data with various population genetics
	methods. Migrations, when populations diverge from each other, population sizes, cultural
	and genetic interactions of different populations with each other, kinship, mechanisms of
	gene flow from one population to another are among the most important questions
	answered by demographic analyzes. With these analyses, we can make inferences about
	the past history of a population, such as population growth, collapse shrinkage, migration
	dynamics and even comment on what might happen in the future.

  In the 1 st century CE, Pline also reported that donkeys were mainly used for breeding mules (H.N. 8, 68, 1), an animal Both mules and donkeys seem common in Central Poitou or Limousin, while horses seem to have largely dominated the Northern part of the country. This is indeed in this region that donkeys, but no mules, are mentioned as peasant auxiliaries in the 11 th -12 th centuries CE (Brunel, 1999) and by the 13 th -14 th centuries CE around the Abbeys from Bur forests (Normandy)(Delisle, 1903). Likewise, while the acts of the ecclesiastical province of Reims (Champagne) abundantly refer

	Modern		period.			Our collection includes a	total
	of 874 ancient specimens spanning 128 sites and comprising 718 horses, 100 donkeys, 55 familiar to all authors and for which ample information	is
	mules and a single hinny individual. While horses were ubiquitous and the most dominant provided about breeding techniques and situations in which they can outperform other
	species identified, our dataset reveals the importance of mule breeding during Roman equids (Columelle, 6, 36; Columelle, 7, 1-3; Moulé, 1919; Johnstone 2004; Chuang
	times, especially between the 1 st and 3 rd centuries CE (Common Era), where they 2016). Mules appear in various mosaics and reliefs, e.g. turning the grinding wheel,
	represented between 20.0% and 34.2% of equine assemblages. In contrast, donkeys were or pulling Roman military chariots or the so-called vallus used at the time for harvesting
	almost (Toynbee, 1973; Raepsaet, 2002). Hinnies (the offspring of a donkey jenny and a horse absent from northern France assemblages during
	the whole Roman period, but replaced mules in rural and urban commercial and economic stallion) remain, however, more rarely mentioned and somewhat discordantly. They are for
	centers from the early Middle Ages. Our work also identified donkeys of exceptional instance described as equal to donkeys for Varro (2, 8) but lazier and more impulsive
	size during Late Antiquity, which calls for a deep reassessment of the true morphological for Pline (8, 69).
	space of	past	equine		species. This	study	confirmed the
	general preference toward horses throughout	all time	periods	investigated
	but revealed dynamic management strategies leveraging the whole breadth of equine
	resources in various social, geographic and temporal contexts.
	uninformative	regarding the	exact	species	designated. Combined,	available
	sources nonetheless portray a highly heterogenous society, with strong regional and
	temporal specificities. For example, mules are more frequently mentioned than horses in
	the	Conques Abbey charters (Aveyron,		Southwestern	France) and	those	of
	2007; Roche, Cluny (Burgundy) where counter-gifts concern twice as often mules than horses (Bautier, 2008-2015; Mitchell, 2018). Horses (Equus
	caballus) undoubtedly represent the equine species that is the most accounted for. This is 1981).
	not surprising given their high symbolic status and their suitability for a broad range of
	equestrian activities, spanning from horseback riding to chariot pulling and plowing.
	Donkeys (Equus asinus) and mules (the offspring of a horse mare and a donkey jack) are
	considerably less documented, but are also known to have played important roles.

b, Jean-Marc Aury e, Maude Barme f, Nicolas Boulbes g, Alice Bourgois f, Franck Decanter h, Sylvain Foucras i, Stéphane Frère a,h, Armelle Gardeisen j, Gaëtan Jouanin a,f, Charlotte Méla k, Nicolas Morand l, Ariadna Nieto Espinet mn, Aude Perdereau e, Olivier Putelat o, Julie Rivière a,p, Opale Robinf h, Marilyne Salin q, Silvia Valenzuela-Lamas nm, Christian Vallet r, Jean-Hervé Yvinec a,h, Patrick Wincker e, Ludovic Orlando b,* Affiliations: a Archéozoologie, Archéobotanique: sociétés, pratiqueset environnements (AASPE), Muséum national d'histoire naturelle, CNRS, CP 56, 55 rue Buffon 75005 Paris, France. b Centre d'Anthropobiologie et de Génomique de Toulouse, CNRS UMR 5288, Université de Toulouse, Université Paul Sabatier, 31000 Toulouse, France. c Department of Biotechnology, Abdul Wali Khan University, Mardan, 23200, Pakistan. d Institute for Advanced Study in Toulouse, University of Toulouse Capitole, 31080 Toulouse, France. e Génomique Métabolique, Genoscope, Institut de biologie François Jacob, CEA, CNRS, Université d'Evry, Université Paris-Saclay, Evry, France. f Centre de Recherche Archéologique de la Vallée de l'Oise, 17 Rue James de Rothschild, 60200 Compiègne, France. g Université de Perpignan, Histoire naturelle de l'Homme préhistorique (HNHP), CNRS, MNHN, Centre Européen de Recherche Préhistorique de Tautavel, 3 Avenue Léon Jean Grégory, 66720 Tautavel, France. h Institut national de recherches archéologiques préventives (Inrap), 121 rue d'Alésia CS 20007 75685 Paris cédex 14, France. i Archéodunum SAS, UMR 5138 -ArAr, 500 rue Juliette Récamier 69970 Chaponnay. j Archéologie des Sociétés Méditéranéennes (ASM), CNRS, Université Paul Valéry -Site Saint-Charles 2 -Rue du Professeur Henri Serre 34090 Montpellier, France. k Direction Archéologie et Muséum Ville d'Aix-en-Provence, Route des Milles, 13290 Aixen-Provence, France. l Institut royal des Sciences naturelles de Belgique (Bioarchéologie), Rue Vautier 29, 1000 Bruxelles, Belgium. m Archéologie Alsace-11 rue Jean-François Champollion -67600 Sélestat. UMR 7044 Archéologie et Histoire ancienne: Méditerranée-Europe, équipe Archéologie médioeuropéenne et rhénane. Maison Interuniversitaire des Sciences de l'Homme Alsace, Strasbourg, France. n Direction de l'Archéologie de la ville de Chartres, 2 Rue Georges Brassens, 28000 Chartres o Service d'archéologie preventive -Communauté d'agglomération Bourges Plus, 23-31 boulevard Foch CS 20321 18023 Bourges cédex, France. p 11 rue de l'Énéide 87280 Limoges, France. $ These authors equally contributed to this work * To whom correspondence should be sent Abstract Alongside horses, donkeys and their first-generation hybrids represent members of the Equidae family known for their social, economic and symbolic importance in protohistoric and historical France. However, their relative importance and their respective roles in different regions and time periods are difficult to assess based on textual, iconographic and archaeological evidence. This is both due to incomplete, partial and scattered historical sources and difficulties to accurately assign fragmentary archaeological remains at the proper taxonomic level. DNA-based methods, however, allow for a robust identification of the taxonomic status of ancient equine osseous material from minimal sequence data. Here, we leveraged shallow ancient DNA sequencing and the dedicated Zonkey computational pipeline to obtain the first baseline distribution for horses, mules and donkeys in France from the Iron Age to the Text:

Introduction

Several members of the horse family (Equidae) are known for their major social and economic impact on proto-historical and historical France. Extensive textual and artistic evidence indeed describe horses, donkeys and their hybrids in a wide breadth of contexts, including not only the battlefield, castles, military and trade roads, but also in cities, villages and farmlands (e.g.,

[START_REF] Vigneron | Le cheval dans l'antiquité gréco-romaine (Des guerres médiques aux grandes invasions)[END_REF] Bautier & Bautier, 1978

; Digard, While they are completely absent from the artistic record during the Iron Age, donkeys appear in various and multiple historical sources during Antiquity, including agronomic treaties of the scriptores rei rusticae (Cato, Varro, Columella and Palladius) where they are acknowledged for their contribution to farming

[START_REF] Bodson | L'utilisation de l'âne dans l'antiquité grécoromaine[END_REF]

.

While such sources provide invaluable information on equine management during Roman times, they leave many questions unanswered regarding the respective role and relative importance of each animal type in both rural and urban contexts. This is especially true for those regions such as the western provinces outside the Mediterranean range that are less documented. The situation is no different in Medieval times, due to abundant, yet ambiguous, textual evidence and iconography. Equine silhouettes, especially in miniatures, indeed often remain too imprecise to recognize mules and donkeys based on their morphology. Texts using words such as summarius (literally, 'beasts of burden') are

  Thoseremains originate from 128 excavation sites and 96 municipalities spread across modern , tarsal bones, tibia, radius, humerus, to maximize the number of sites investigated. At sites characterized by extensive numbers of equine remains, as whole skeletons were deposited (e.g. Boinville-en-Woëvre) and/or large amounts of bones were accumulated (e.g. Chartres (Rue de la Courtille), Longueil-Sainte-Marie (L'Orméon)), we systematically selected one specific anatomical element located on the same body side so as to ensure that a given individual was sampled only once.Our sample set encompasses different archaeological contexts, including both rural and urban settings (Fig.2C). Nearly half (424/874 ~ 48.5%, from 82/128 ~ 64.1% sites) consist of domestic dumps and occupation refills, with diverse elements indicative of food waste, craftwork, and garbage of all kinds. In this type of dump, Iron Age samples were obtained from parts of the body that were consumed, while those from the Roman, accumulated, and sometimes even exploited for their skin, hair, tendons and bones. During the Roman Period, such sites were located nearby cities and close to necropolis(Lepetz et al., 2013), e.g. at Evreux (Clos-au-Duc) and Louvres (ZAC du Parc). In the Medieval Period, these sites could be located within non-

	collaborators (2021). In brief, 40-1,110 mg of powder is first partially digested within a filtered for minimal mapping quality of 25 and PCR duplicates, and assessed for signatures
	3.85mL solution consisting of EDTA (0.45M), N-lauryl Sarcosine (0,5%) and proteinase K of post-mortem DNA damage, including nucleotide mis-incorporation and base
	(0,25mg/mL) for 1 hour at 37°C. The remaining undigested pellets are then collected compositional patterns, using mapDamage2 (Jonsson et al., 2013). Such patterns
	France (Fig. following 2 min centrifugation at 3,000 rpm and fully digested overnight at 42°C and 1). Sampling was originally focused on petrosal bones to conformed to what expected considering the USER-treatment of DNA extracts and the
	increase the chances of ancient DNA preservation (Pinhasi et al., 2015) but was adding 3.85mL mL of a fresh extraction buffer. Some of the samples were subjected to type of DNA library constructed. More precisely, the genomic position preceding the first
	successfully extended to teeth and post-cranial skeletal elements, including e.g., Medieval and Ancien Régime periods were generally not. Around ~18.9% of the samples bleach pretreatment as described by Boessenkool and collaborators (2017) before being alignment position was enriched in Cytosine residues, as a result of USER treatment
	material (165/874, 17/128 ~ 13.3% sites) were collected from city relegation zones where animals analyzed spans the whole temporal range between were gathered as they died, samples), the animal bones collected were used as part of processed for DNA extraction. DNA libraries were constructed from double stranded DNA templates that were treated with USER enzymes (New England Biolab), following Fages et index). Amplified DNA libraries were then purified through MinElute columns (QIAgen) or Agencourt AMPure XP beads (Beckman Coulter), with a 1.4 beads/DNA solution volume ratio and eluted in 25uL EB buffer supplemented with 0.05% Tween. Purified DNA libraries were quantified using the TapeStation 4200 instrument (High Sensitivity D1000 Assay; Agilent) or BioAnalyzer 2100 instrument (High Sensitivity DNA Labchip Kit; Agilent) and on a QuBit HS dsDNA assay (Invitrogen), before being pooled together with 30-50 other libraries for sequencing on the MiniSeq Illumina platform (80 Paired-End mode) at CAGT, or on the HiSeq4000 Illumina platform (150 Paired-End mode) at Genoscope, Evry. cleaving DNA at those Cytosine residues that were deaminated after death (Briggs et al., 2010). The magnitude of CàT mis-incorporation rates was limited due to USER treatment phalanges, metapodsThe 2.1% al. (2019), and included two 7-bp internal indices (Rohland et al. 2015) as well as one 6-bp external index (Fages et al. 2019) so as to allow sequence demultiplexing robust to index hopping (van der Valk et al., 2020). DNA libraries amplification were carried out for 4-15 cycles in 25uL reactions of the 3µL of DNA in solution with EB-Tween 0.05%, 2.5µL of of AccuPrime Pfx DNA polymerase (Thermo Fisher Scientific), 0,8mg/mL of BSA, and 200nM of each PCR primer (inPE1 and one custom primer including a 6-bp Illumina but decreased consistently from read starts, as expected (Briggs et al., 2007). Finally, taxonomic and sex identification were carried out by subjecting BAM alignments files to the Zonkey pipeline (Schubert et al., 2017), and considering a minimal of 1,018 alignments in order to achieve full specificity (median=115,469, maximum=959,339). Final and carcasses were /874 10X AccuPrime Pfx Reaction Mix (Thermo Fisher Scientific) supplemented with 1U assignments are provided as Supplemental Table
	the Hallstatt and the Modern were buried in gardens or near rural habitats (35/874 ~ 4.0% samples, 18/128 ~ 14.1% period, with Gallo-Roman and Contemporary Both those sequences previously published and those generated here were subjected to the
	sites representing the two time periods the most (271/874 ~ 31.0% samples) and the sites), within single, e.g. at Longueil-Annel (Le same downstream analyses. Fastq sequences were demultiplexed, trimmed for low-quality
	least (19/874 ~ 2.2% samples) sampled, respectively (Fig. 2B). Additionally, the material Village), Noyon (Rue d'Orroire), Brachaud or collective burials (Boinville-en-Woëvre). ends and collapsed if showing sufficient overlap using AdapterRemoval2 (Schubert et al.,
	shows a clear over-representation of sites located in the Northern/Northeastern part of 2016) with default parameters, except that at best one sequence mismatch was tolerated in
	France (100/128 ~ 78/1% sites, 703/874 ~ 80.4% samples). Three other regions were also Ancient DNA Methods each internal index. Sequences were further processed for mapping against the EquCab2
	investigated, including Charente-Maritime (7/128 ~ 5.5% sites, 73/874 ~ 8.3% samples), A total number of 68 samples were studied in previous work (Gaunitz et al. 2018, Fages et horse reference genome (Wade et al., 2009), to match the Zonkey pipeline expectations
	Auvergne (2/128 ~ 1.6% sites, 44/874 ~ 5.0% samples) and the Mediterranean coast al. 2019, Clavel et al. 2021) using methodologies similar to those applied to (Schubert et al., 2017), supplemented with the Y-chromosome contigs from Wallner et al.

The assemblages represented in our final dataset cover a range of cultural, socioeconomical and temporal contexts. The archaeological information available for Medieval sites allowed us to identify rural civil, rural elite (i.e. seigneurial or monastic), urban elite (civil or religious) contexts. This admittedly only represents a subset of the socioeconomical spectrum and may reflect a different local social experience from site to site and even within sites, as the complexity of individual social statuses cannot be fully appreciated. The temporal range indicated for each site merges various contextual data, including from ceramics, as provided by archaeologists and radiocarbon dates when available (Supplemental Table

1

).

(10/128 ~ 7.8% sites, 42/874 ~ 4.8% samples). Finally, a total of 12 samples originated from nine sites spread across the country (Fig.

2A

). constructed city sections (e.g. at Beauvais (44 rue des Jacobins), or at Reims (Rue des capucins)) or close to the city walls (e.g. at Beauvais (Le Jeu de Paume)). At Chartres (Rue de la Courtille, 77/874 ~ 8.8% samples) and Beauvais (Abord du Théâtre, 18constructions for draining humid zones, while they provided the raw material for craftwork at Lille (Rue de Tournai) and Vermand (Rue Charles de Gaulle), representing a total of 7/874 ~ 0.8% samples for both sites. Ritual sites also provided around 13.6% of the samples (119/874, 10/128 ~ 7.8% sites), including animals associated within human sepultures (Woippy and Offin (Le-Bois-Monclair)), or sacrificed and concentrated within sanctuaries ritual deposits (Gondole, Orcet, Saint-Just-en-Chaussée (site 94-95), Vertault, Longueil-Sainte-Marie). In our dataset, the latter are restricted to the La Tène and Roman Periods. Finally, the last site category includes animals that died from natural death and the 806 remaining samples that were analyzed here for the first time. Ancient DNA analyses were carried out in the clean lab facilities of the Centre for Anthropobiology and Genomics of Toulouse (CAGT), following the methodology fully described by Clavel and (2017) and the horse mitochondrial reference genome (NC_001640, Xu & Arnason 1994) using Paleomix v1.2.13.2 together with the Bowtie2 aligner (Langmead et al. 2012) and the parameters recommended by Poullet & Orlando (2020). Sequence alignments were

  The only other site in our dataset that delivered donkey remains in Northern France in the Roman Period was Woippy and was located 35

	The situation changed drastically from the beginning of the Medieval Period. Donkeys
	were found within limited proportions (two bones at Moussy-le-Neuf) in the natural region
	of 'Pays de	France' (North of	Paris) during	the	Merovingian	Period	(6 th -
	8 th centuries CE) but became more common thereafter. They represented ~28.2% (20/71)
	of the remains analyzed, during the Carolingian Period (8 th -10 th century CE) and until the
	11 th century CE (Fig. 5A). It reached maximal proportions (43/135 ~ 31.8%) between the
	11 th and the 13 th centuries CE, and decreased to ~17.7% (11/62) in the 14 th -
	15 th centuries CE to only represent 5.5% (9/163) of the remains analyzed in the
	next three centuries.

kilometers east of Boinville-en-Woëvre. It comprised a complete skeleton deposited next to a human burial dated to the 5 th century CE. In the South, only two donkeys were identified in the late Antiquity context of La Bourse in Marseille. Overall, our results portray a picture of limited donkey presence across the northern country, with the exception of Late Antiquity sites such as Boinville-en-Woëvre, where donkeys appear locally, at exceptionally elevated percentages.

  One of the main limitations of the present study pertains to the over-representation of archaeological material from the Northern part of France. Therefore, it remains unknown whether the management of equine resources followed similar temporal and socioeconomic trajectories in the southern half of the country. Future work should, thus, be aimed to extend our focal area to fill this important gap in knowledge. the full predictive power of the new tools now available, such as those based on DNA identification(Schubert et al., 2017), 2D geometric morphometrics applied to teeth (Cucchi et al., 2017) or 3D geometric morphometrics applied to the bony labyrinth (Clavel et al., 2021), and possibly, ancient proteins (Hendy et al. 2018). Boulbes, N., Gardeisen, A., 2018. Equus asinus remains (Mammalia, Perissodactyla) from the protohistoric site of Pech Maho (Sigean, South of France) and variation in donkey size during the Iron Age. Int. J. Osteoarchaeol. 28, 428-438. https://doi.org/10.1002/oa.2670. Columeau, P., 2002. Alimentation carnée en Gaule du Sud (VIIe s. av. J.-C.-XIVe s.), Travaux du centre Camille Jullian 29. Aix-en-Provence. . In: Lorans, E. (Ed.), Le cheval au Moyen-Âge. Tours, pp. 319-342. https://doi.org/10.4000/books.pufr.15021. Alfarhan, A.H., Alquraishi, S.A., Al-Rasheid, K.A.S., Chuang, R., Ermini, L., Gamba, C., Weinstock, J., Vedat, O., Orlando, L., 2017. Zonkey: A simple, accurate and sensitive pipeline to genetically identify equine F1-hybrids in archaeological assemblages. J. Arc.

	characterized. This	calls	for future re-assessment	of	equine	skeletal
	(Le Château -Picardy), a location under Bishop control, uncovered a large number of storage structures used for cereals, which indicated a site of particular Contamine, P., 2008. Le cheval « noble » aux XIVe-Raepsaet, G., 2002. Attelages et techniques de transport dans le monde gréco-assemblages that embrace Briggs, A.W., Stenzel, U., Johnson, P.L.F., Green, R.E., Kelso, J., Prüfer, K., Meyer, M., XVe siècles : une approche européenne. Comptes rendus des séances de l'Académie des romain. Laboratoire d'archéologie classique de l'Université libre de Bruxelles : Édition-
	economic importance. There, 14 out of the 18 equine remains analyzed were identified as Krause, J., Ronan, M.T., Lachmann, M., Pääbo, S., 2007. Patterns of damage in genomic Inscriptions et Belles-Lettres, 152 e année, 4, 1695 -diffusion, Livre Timperman, Bruxelles.
	donkeys (~77.8%). They may thus have been particularly useful for transporting cereals DNA sequences from a Neandertal. Proc. Natl. Acad. Sci. U. S. A 104, 14616-1726. https://doi.org/10.3406/crai.2008.92264.
	(and other farm products) between farmlands and storage facilities, which were located at 14621. https://doi.org/10.1073/pnas.0704665104. Reddé, M., Bernigaud, N., Lepetz, S., Zech-Matterne, V., 2018. Les conditions
	places where elites, political and religious power were concentrated. This would depict Cucchi, T., Mohaseb, A., Peigné, S., Debue, K., Orlando, L., Mashkour, M., 2017. du développement économique. II -marchés, in: Reddé, M. (Ed.), Gallia Rustica 2,
	donkeys as instrumental vectors linking together the different elements composing what Brunel, C., 1999. L'élevage dans le Nord de la France (XIe-Detecting taxonomic and phylogenetic signals in equid cheek teeth : towards Les campagnes du nord-est de la Gaule, de la fin de l'âge du Fer
	was then a new form of territorial organization. A similar phenomenon was characteristic XIIIe siècles). Quelques jalons de recherche. ABPO 106, 41-new palaeontological and archaeological proxies. R. Soc. Open Sci. 4, à l'antiquité Tardive, mémoires, Ausonius Éditions, Bordeaux. pp. 519-584.
	61. https://doi.org/10.3406/abpo.1999.4014. donkeys into the Iberian 4490. B., Lepetz, S., Braga, J., Orlando, L., 2021. Assessing the predictive taxonomic power of Peninsula. J. Arc. Sci. 40, 4483-the bony labyrinth 3D shape in horses, donkeys and their F1-hybrids. J. Arc. Sci. 131, 105383. https ://doi.org/10.1016/j.jas.2021.105383. Columeau, P., 2000. Nouveau regard sur la production et la consommation de 160997. https://doi.org/10.1098/rsos.160997. Delisle, L., 1903. Étude sur la condition de la classe agricole et l'état de l'agriculture en Normandie au Moyen-Age, Honoré Champion. ed. Paris. et Rhin, in: Mercati e Mercanti Nell'alto Medioevo: L'area Euroasiatica e l'area Mediterranea (23-29 Avril 1992: Spoleto). pp. 327-393. Devroey, J.-P., Nissen, A., 2015. Early Middle Ages, 500-1000, in: Thoen, E., Soens, T. (Eds.), Struggling with the Environment: Land Use and Productivity, Rural Economy and 68. https://doi.org/10.1484/M.RES-EB.5.108034. Roche, D., 2008-2015. La culture équestre de l'Occident, XVIe-XIXe siècle, l'ombre du cheval, volume 1 : le cheval moteur ; volume 2 : La gloire et la puissance ; volume 3 : Connaissance et passions. Fayard. of the following time period (11 Finally, our work uncovered considerable size variation amongst Gallo-Roman equine Society in North-Western Europe, 500-2000. Brepols Publishers, Turnhout, pp. 11-Sci. 78, 147-157. https://doi.org/10.1016/j.jas.2016.12.005.
	species, including miniature horses and gigantic donkeys. Therefore, size differences can la viande dans le Languedoc occidental. In : C. Mata Parreño, & G. Pérez Jordà Tegetmeier, W.B., Sutherland, C.L., 1895. Horses, asses, zebras, mules and mule breeding.
	no longer be used to make robust taxonomic assignments from fragmentary (Eds.), Ibers, agricultors, artesans i comerciants ()III Reuniò sobre Economia en el Mòn Ib Digard, J., 2007. Une histoire du cheval. Actes sud, Arles. H. Cox, London.
	èric, Valence, Universitat de Valencia, 167-173.

th to 13 th centuries CE), however, not in rural but urban elite contexts. This is perhaps best illustrated by Surgères (Rue Barabin), a site where skeletal elements were found close to the city castle, but this is also true for other sites accumulating economic and political power, such as Boves (Le Château) and Démuin (Le Château). During the next two centuries, donkey management appears to have shifted again to rural elite contexts, including monasteries and castles, such as at Saint Sornin (Tour de Broue), where donkeys were likely used in relation to saltworks

[START_REF] Périsse | Les marais salants de la prévôté d'Hiers d'après les terrages de 1478[END_REF] Normand & Champagne, 2019)

. Finally, the donkey remained used in rural contexts during the Modern Period but became rather uncommon, if not exceptional, within cities. remains. Likewise, while hinnies never became common, mules appeared to have been only second to horses during Antiquity. Therefore, proper identification of mules is needed before the equine skeletal assemblages of that time period can be accurately Cardoso, J.L., Vilstrup, J.T., Eisenmann, V.,

Orlando

, L., 2013. First evidence of Equus asinus L. in the Chalcolithic disputes the Phoenicians as the first to introduce https://doi.org/10.1016/j.jas.2013.07.010. Chuang, R., 2016. The Acquisition of Domestic Equids in Roman Britain -the identification of domestic equids and case study with isotopic analyses (Thesis for the degree of Doctor of Philosophy). University of Southampton. Clavel, P., Dumoncel, J., Der Sarkissian, C., Seguin-Orlando, A., Calvière-Tonasso, L., Schiavinato, S., Chauvey, L., Perdereau, A., Aury, J.-M., Wincker, P., Onar, V., Clavel, Deman, A., 2002. Avec les utriculaires sur les sentiers muletiers de la Gaule romaine. Cahiers du Centre Gustave Glotz, 13, 233-246. https://doi.org/10.3406/ccgg.2002.1567. Devroey, J.-P., 1992. Courants et réseaux d'échange dans l'économie franque entre Loire Duval, P.-M., 1989. Les voies gallo-romaines. In: Travaux sur la Gaule (1946-1986). Publications de l'École française de Rome, 116, Rome, pp. 739-756. riqueRohland, N., Harney, E., Mallick, S., Nordenfelt, S., Reich, D., 2015. Partial uracil-DNAglycosylase treatment for screening of ancient DNA. Phil. Trans. Roy. Soc. B: Biol. Sci. 370, 20130624. https://doi.org/10.1098/rstb.2013.0624. Roth, J.P., 1999. The logistics of the Roman Army at War (264 B.C. -A.D. 235), Columbia studies in the classical tradition. Brill, Leiden -Boston -Köln. Schubert, M., Mashkour, M., Gaunitz, C., Fages, A., Seguin-Orlando, A., Sheikhi, S., Toynbee, J.M.C., 1973. Animals in Roman life and art. (Aspects of Greek and Roman Life series.). Thames and Hudson, London.

  al. 2013), the archaeological material available for the period is, thus, limited. Equid material has also remained scarce and fragmentary during the Medieval and Modern Period. For instance, it represents only 0.7% (Rhone-Alpes region) to 2.1% (Languedoc) of the assemblages relative to cattle, sheep and pigs between the 6 th and 16 th century CE (Forest 1997/98), despite being present in over two thirds of 140 archaeological sites (Rodet-Belarbi et al. 2017). In these conditions, only rendering deposits have delivered sufficient material for statistical comparisons, with a handful of

sites portraying activities strongly biased towards old males (Parent-Duchâtelet 1827; Arbogast & al. 2002; Barme & Clavel 2015; Rodet-Belarbi et al. 2017).

  The late Middle Ages defined the third time period, with 81 samples dated to between the 14 th and 16 th centuries CE. These samples were distributed almost equally between 'civil rural' contexts (villages: 21/81~25.9 %), rural castles (24/81~29.6%) and rural monasteries (19/81~23.4%) as well as 'civil urban' contexts (17/81~20.9%). It is noteworthy that the 'Rue Barrabin' site at Surgères did belong to the latter category and not any longer to elite contexts at the time.

	Finally, the last time period investigated corresponded to the Modern Period (16 th to18 th
	centuries CE) and was mainly marked by rural trading networks and important economic
	crises until the French Revolution in 1789. Approx imately one fifth of the 148 remains
	belonging to this period (29/148~19.6%) came from 'civil rural' contexts, represented both
	by villages (e.g. 'Impasse du Moulin' at Chambly and 'La plaine d'Herneuse' at Verberie)
	and castles such as at Saint-Sornin and Eaucourt-sur-Somme, which became agricultural
	establishments at the time. In the 17 th century CE, the castle at Boves ('Le Château') was
	dismantled to become a stone quarry, and Châtenois was no longer belonging to elite
	categories. For convenience, they were classified as rural sites. Sites from 'civil urban'
	contexts then provided the major fraction of samples (85/148~57.4%), while 'rural elite'
	and 'urban non-monastic religious' categories comprised the remaining 9 (~6.1%) and 25
	(~16.9%) remains.
	, thus, representing
	the 'urban elite' category.
	The second period extended from the 11 th to 13 th centuries CE and was represented by a
	total of 94 samples, mostly across 'civil rural' (27/94~28.7%) and 'rural castle' categories
	(42/94~44.7%). The remaining samples were obtained from urban contexts, including the
	three elite sites (21/94~22.3 %) from the episcopal palace at Beauvais, the Saint Crépin
	Abbey at Soissons and the 'Rue Barrabin' site at Surgères. They also included other urban
	sites reflecting more modest social contexts (4/94~4.2 %), such as at Pont-Sainte-Maxence
	(14 Rue Bodchon), Saint-Omer (Rue Saint-Sépulcre) and Saint-Denis (Rue Charles
	Michel).

  7 th -10 th centuries CE, 11 th -13 th centuries CE, 14 th -16 th centuries CE, and; 16 th 18 th centuries CE). The first factorial axis mainly highlighted the changing preference to donkey jacks in civil/elite urban contexts during the 11 th -13 th centuries CE. It also revealed equine management strategies varying according to social contexts, with increasing proportions of horse mares and donkey jennies within rural Iron Age and Roman sacrificial rituals Overall, our analyses are in accordance with earlier reports supporting male-biased representation in pan-Eurasian archaeological remains from the early Bronze Age(Fages et al. 2020). The new data analyzed in this study, however, reveals more subtle sex distribution patterns at the more local scale and according to the archaeological context (i.e. habitat vs ritual).A fraction of the Iron Age material examined here comes from well-established sacrificial sites, such as Vertault, Le Cendre-Gondole, Orcet and Saint-Just-en Chaussée 94-95. It also includes sites where the presence of sacrificial ritual practice is consid ered likely (ThézyGlimont), or suggestive (Pech Maho). Combined, and despite archaeological evidence supporting diverse practices across sites and time periods, they illustrate the central religious role that horses played at the time in Gauls. Males in the city By revealing that males were strongly over-represented in cities, our work provides the first archeozoological evidence supporting sex-biased factors shaping the organization of equine husbandry in France during Antiquity, the Middle Ages and the Modern Period .Equine sex-ratios indeed reached approximately ~88.6% in Antique urban contexts versus only ~54.5% in rural contexts. Horse males also appeared central to the economy of Medieval and Modern cities, where they represented approximately ~70.6% of the bone assemblages. In contrast, females were proportionally more abundant in rural contexts, and for donkeys, even balanced in numbers with males.Following the work from historians[START_REF] Bautier | Contribution à l'histoire du cheval au Moyen Âge[END_REF], increased proportions of mares provide a robust marker for breeding. Importantly, many of the rural sites analyzed represent villages that were under the control of abbeys (e.g. 'Rue du bois Tillet' at Crépy-enValois, where the abbey was built during the 10 th century CE[START_REF] Gnat | Le prieuré Saint-Arnoul de Crépy-en-Valois (Oise : Le point sur vingt-cinq ans de recherches archéologiques[END_REF]). The large abbeys and civil elite context sites investigated here were also mostly located into the It is likely that living in such difficult conditions would have compromised the female fitness. Therefore, females may have also been preferentially maintained in rural contexts to decrease the risk of miscarriage and, thus, sustain production demands.Medieval hippiatric treaties time and again urge horse breeders to separate gravid mares from stallions, monitor feeding and pay attention to cold temperatures[START_REF] Rusio | La Mareschalerie de Laurent Ruse, où sont contenuz remèdes très singuliers contre les maladies des chevaux, avec plusieurs figures de mors ; en laquelle y avons adjousté un autre traicté de remèdes[END_REF]. This is also true for later textual documents, such as the 'Théâtre d'agriculture', first published in 1600 CE and re-edited until the 19 th century CE (de Serres 1804-1805), but may have equally applied to Antiquity as Varro already recommended that gravid females are spared from work (Varro 7.2). The fact that weaning and training did not generally start before two and three years after birth, respectively (Rusio 1583), may also have motivated the keeping of females away from cities. Additionally, the presence of females in cities during the breeding season may not be fit to the urban environment, as female heats can cause bouts of tension[START_REF] Marcenac | Encyclopédie du cheval[END_REF] and may have caused diminishing returns for working males.

	monasteries and elite contexts, especially during the 14 th to 18 th centuries CE, although
	never significantly exceeding those of males.
	4 Discussion
	In this study, we applied shallow shotgun DNA sequencing and the Zonkey bioinformatic
	pipeline to identify the sex of 897 ancient equine remains excavated from 134
	archaeological sites in France. Calculation of sex-ratio across sites, time periods and social
	contexts shed new light on the history of equine population management, especially
	regarding the preferential selection of males or females in various situations, including
	hunting, sacrificial rituals and husbandry.
	4.1 Prehistoric hunting
	Previous work on Late Glacial bone assemblages from the Paris Basin have highlighted the
	importance of horses for the subsistence economies of local hunter-gatherers (Bridault et
	al. 2003; Bignon 2006; Bignon 2008). Seasonality and paleo-demographic profiles have
	portrayed hunting episodes focused on family groups, including mares as well as young
	foals and fillies, rather than isolated bachelors, which was further confirmed for other sites
	located in Southwestern France (Feyfant et al. 2015). While the amount of sequence data
	generated in this study precludes any investigation of familial relationships, they support
	balanced sex ratios both in natural (Igue du Gral) and anthropic contexts. This is in line with previous reports applying a similar methodology to Late Pleistocene equine material from Belgium and Siberia (Fages et al. 2020) and confirms hunting strategies not targeting bachelor groups. Interestingly, with four mares and one male identified at the Magdalenian camp of Etiolles, our data also confirm previous contention that the horse assemblage was formed following a single hunting episode of a single-family group showing a harem-like structure (Bignon 2006). sacred natural humid zone, with evidence including miniature ceramics associated with an 4.2 Marie (L'Orméon) is considered to represent a local, rural sanctuary that was linked to a important accumulation of equine bones, derived from at least 40 individuals (Gaudefroy & Lepetz 2000). Remains from a total of 26 of these individuals showed DNA preservations compatible with taxonomic and sex identification. Our data revealed that only horse males were deposited, even though other contemporary archaeological sites not associated with ritual activities showed substantial proportions of mules (Lepetz et al. subm itted). This finding reinforces previous interpretations of the Longueil-Sainte-Marie site as ritual. animal carcasses were evacuated, accumulated and reused for th eir bones, skins and tendons (e.g. Chartres, Evreux 'Clos-aux-Duc'; Lepetz et al. 2013). These accumulations are particularly illustrative of the animals participating to the city life and only consist of adult, often old, animals (Lepetz et al. 2013). Carcass recycling for bones, skins and 4.3 In Gallo-Roman cities, the sites investigated include many relegation zones in which tendons was particularly well organized and effective from the Middle Age, and comprised

Interestingly, our data indicate that horse sacrifice was preferentially, and sometimes even exclusively, oriented towards males. This is reminiscent of practices reported in other Iron Age contexts, such as Pazyryk Scythian burials from the Altay region

[START_REF] Lepetz | The sacrifice of horses in a Pazyryk culture kurgan : the princely tomb of Berel'(Kazakhstan). Criteria for choice and slaughter procedure[END_REF][START_REF] Librado | Ancient genomic changes associated with domestication of the horse[END_REF][START_REF] Lepetz | To accompany and honour the deceased: the horses from the graves of the Pazyryk culture[END_REF]

), but also of more recent contexts, such as Viking tombs in Northwestern Europe

[START_REF] Nistelberger | Sexing Viking Age horses from burial and non-burial sites in Iceland using ancient DNA[END_REF]

). The over-representation of horse males may simply reflect ritual decisions purely oriented according to the sex of the animals. Alternatively, it may reflect the indirect consequences of a generally particular status that males had at the time, e.g. in relation to mounting, prestige and wars.

Regardless, our work reveals that such sacrificial practices were not limited to the Iron Age in France but also extended to the Roman Period. Indeed, the site from Longueil-Sainte-fertile regions of Picardy and Pays-de-France, where the horse was paramount to local economies, both used in agriculture along with the plough since at least 1100 CE (Fossier

19 68)

and in relation to military activities

[START_REF] Prévot | Le cheval en France au Moyen Âge ; sa place dans le monde médiéval[END_REF]

. Although this remains speculative, the increased concentrations of females observed at locations associated with power may reflect intentional control by the social elites over horse reproductive resources at a time when the animal was not only expensive but also a military asset , providing, thus, both a social marker and a commodity for exercising power, reinforcing social dominance. Sites such as Boves and Démuin (Picardy), Saint -Sornin (Charente-Maritime) and Châtenois and Saverne (Alsace) would then offer typical examples of such production centers where control and breeding were organized. all kinds of animals, including reform animals displaying abundant skeletal pathologies characteristic of particularly hard work conditions (Barme & Clavel 2015; Deborde & Bandelli 2017).

  Orlando 2018) in stallions and geldings, should be developed to confirm this hypothesis and determine when and in which contexts, this practice was first applied to horses.

	Barme & Clavel 2015), textual sources insist on the importance of
	getting animals used to ambient noise (e.g. see Pierre de Crescent's 'Opus ruralium
	commodorum' published in 1492, and cited by Prévot & Ribémont 1994 ). Such
	environments may have proved particularly challenging for those most nervous animals
	and breeds. Although this remains to be demonstrated genetically, more tempered animals
	from cold blood breeds may have, thus, been preferred. It is also likely that a large fraction
	of the males identified were castrated, as gelding is a practice known for increasing
	docility that is already reported in Roman sources (Varron, 2, 7, 15 ; Peters 1998; Gitton-
	Rippol 2016), and common to many Medieval hippiatric treaties (Poulle-Drieux 1966;

Lignereux 2005;[START_REF] Prévot | Le cheval en France au Moyen Âge ; sa place dans le monde médiéval[END_REF]

. Methods aimed at the detection of geldings, possibly scrutinizing bone remodeling patterns as a result of increased muscular mass

[START_REF] Luff | The conundrum of castration in the archaeological record: An interpretation of Roman cattle horn-cores from Chelmsford, Essex[END_REF][START_REF] Davis | The effect of castration and age on the development of the Shetland sheep skeleton and a metric comparison between bones of males, females and castrates[END_REF]

), or perhaps leveraging differentially methylated markers

(Hanghøj &

APPENDIX -92 -6 REFERENCES-116 -

Acknowledgements

Acknowledgements

We thank Agnès Orsoni, Michela Leonardi, Tomasz Suchan, and Stefanie Wagner for lab assistance and all members of the AGES research team at CAGT for fruitful discussions.

We also thank all archaeologists, curators and staff in charge of archaeological warehouses, who have facilitated access to the material analyzed in this study. Pierre Clavel's PhD position is funded by the CNRS MITI interdisciplinary programme (Mission pour les Initiatives Transverses et Interdisciplinaires). Xuexue Liu was supported by the European Union's Horizon 2020 research and Innovation programme under the Marie Skłodowska-Curie grant agreement 101027750. Andaine Seguin-Orlando acknowledges IAST for funding from ANR under grant ANR-17-EURE-0010 (Investissements d'Avenir programme). This work was supported by the France Génomique Appel à Grand Projet (ANR-10-INBS-09-08, BUCEPHALE project); the Initiative d'Excellence Chaires d'attractivité, Université de Toulouse (OURASI) and the Villum Fonden miGENEPI research project. This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No. 681605). Acknowledgements We thank Agnès Orsoni, Michela Leonardi, and Stefanie Wagner for lab assistance and all members of the AGES research team at CAGT for fruitful discussions. We also thank all archaeologists, curators and staff in charge of archaeological warehouses, who have facilitated access to the material analyzed in this study. Pierre Clavel's PhD position is funded by the CNRS MITI interdisciplinary programme ('Mission pour les Initiatives Transverses et Interdisciplinaires'). Xuexue Liu was supported by the European Union's Horizon 2020 r esearch and Innovation programme under the Marie Skłodowska-Curie grant agreement 101027750. This work was supported by the France Génomique Appel à Grand Projet (ANR -10-INBS-09-08, BUCEPHALE project); the Initiative d'Excellence Chaires d'attractivité, Un iversité de Toulouse (OURASI) and the Villum Fonden miGENEPI research project. Andaine Seguin-Orlando acknowledges IAST for funding from ANR under grant ANR-17-EURE-0010 ('Investissements d'Avenir' programme). This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No. 681605).

APPENDIX

The Origins and Spread of domestic horses from the Western Eurasian steppes Librado*, et al., Nature, 2021