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Donkeys transformed human history as essential beasts of burden for long-distance movement, especially across semi-arid and upland environments. They remain insufficiently studied despite globally expanding and providing key support to low- to middle-income communities. To elucidate their domestication history, we constructed a comprehensive genome panel of 207 modern and 31 ancient donkeys, as well as 15 wild equids. We found a strong phylogeographic structure in modern donkeys that supports a single domestication in Africa similar to 5000 BCE, followed by further expansions in this continent and Eurasia and ultimately returning to Africa. We uncover a previously unknown genetic lineage in the Levant similar to 200 BCE, which contributed increasing ancestry toward Asia. Donkey management involved inbreeding and the production of giant bloodlines at a time when mules were essential to the Roman economy and military. French National Research Agency (ANR) ANR-10-INBS-09 info:eu-repo/semantics/publishedVersion

DNA hybridization-capture techniques allow researchers to focus their sequencing efforts on preselected genomic regions. This feature is especially useful when analys- ing ancient DNA (aDNA) extracts, which are often dominated by exogenous environ- mental sources. Here, we assessed, for the first time, the performance of hyRAD as an inexpensive and design-free alternative to commercial capture protocols to obtain authentic aDNA data from osseous remains. HyRAD relies on double enzymatic re- striction of fresh DNA extracts to produce RNA probes that cover only a fraction ofthe genome and can serve as baits for capturing homologous fragments from aDNA li- braries. We found that this approach could retrieve sequence data from horse remains coming from a range of preservation environments, including beyond radiocarbon range, yielding up to 146.5-fold on-target enrichment for aDNA extracts showing ex- tremely low endogenous content (20%¿30%), while the fraction of endogenous reads mapping on- and off-target was relatively insensi- tive to the original endogenous DNA content. Procedures based on two instead of a single round of capture increased on-target coverage up to 3.6-fold. Additionally, we used methylation-sensitive restriction enzymes to produce probes targeting hypo- methylated regions, which improved data quality by reducing post-mortem DNA dam- age and mapping within multicopy regions. Finally, we developed a fully automated hyRAD protocol utilizing inexpensive robotic platforms to facilitate capture process- ing. Overall, our work establishes hyRAD as a cost-effective strategy to recover a set of shared orthologous variants across multiple ancient samples., This project received funding from: the University Paul Sabatier IDEX Chaire d’Excellence (OURASI); the CNRS Programme de Recherche Conjoint (PRC); the CNRS International Research Project (IRP AMADEUS); the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 797449; the Russian Foundation for Basic Research, project No. 19-59-15001 “Horses and their importance in the life of the ancient population of Altai and adjacent territories: interdisciplinary research and reconstruction”; and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement 681605).

Sex identification from fragmentary archeozoological assemblages is particularly challenging in the Equid family, including for horses, donkeys and their hybrids. This limitation has precluded in-depth investigations of sex-ratio variation in various temporal, geographic and social contexts. Recently, shallow DNA sequencing has offered an economical solution to equine sex determination, even in environments where DNA preservation conditions is not optimal. In this study, we applied state-of-the-art methods in ancient DNA-based equine sex determination to 897 osseous remains in order to assess whether equal proportions of males and females could be found in a range of archeological contexts in France. We found Magdalenian horse hunt not focused on isolated bachelors, and Upper Paleolithic habitats and natural traps equally balancing sex ratios. In contrast, Iron Age sacrificial rituals appeared to have been preferentially oriented to male horses and this practice extended into the Roman Period. During Antiquity, the Middle Ages and the Modern Period, cities emerged as environments largely dominated by horse males. This strong sex-bias was considerably reduced, and sometimes even absent, in various rural contexts. Combined with previous archaeozoological work and textual evidence, our results portray an urban economy fueled by adult, often old, males, and rural environments where females and subadults of both sexes were maintained to sustain production demands., 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 research and Innovation pro- gramme under the Marie Skłodowska-Curie grant agreement 101027750. This work was supported by the France G ́enomique Appel `a Grand Projet (ANR-10-INBS-09-08, BUCEPHALE project); the Initiative d’Excellence Chaires d’attractivit ́e, Universit ́e de Toulouse (OURASI) and the Villum Fonden miGENEPI research project. Andaine Seguin- Orlando acknowledges IAST for funding from ANR (France) 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).

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 Modern period. Our collection includes a total of 873 ancient specimens spanning 128 sites and comprising 717 horses, 100 donkeys, 55 mules and a single hinny individual. While horses were ubiquitous and the most dominant species identified, our dataset reveals the importance of mule breeding during Roman times, especially between the 1st and 3rd centuries CE (Common Era), where they represented between 20.0% and 34.2% of equine assemblages. In contrast, donkeys were almost absent from northern France as- semblages during the whole Roman period, but replaced mules in rural and urban commercial and economic centers from the early Middle Ages. Our work also identified donkeys of exceptional size during Late Antiquity, which calls for a deep reassessment of the true morphological space of past equine species. This study confirmed the general preference toward horses throughout all time periods investigated but revealed dynamic manage- ment strategies leveraging the whole breadth of equine resources in various social, geographic and temporal contexts., 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 G ́enomique Appel `a Grand Projet (ANR-10-INBS-09-08, BUCEPHALE project); the Initiative d’Excellence Chaires d’attractivit ́e, Universit ́e 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)

Although recognized as one of the most significant cultural transformations in North America, the reintroduction of the horse to the continent after AD 1492 has been rarely addressed by archaeological science. A key contributing factor behind this limited study is the apparent absence of equine skeletal remains from early historic archaeological contexts. Here, we present a multidisciplinary analysis of a horse skeleton recovered in Lehi, Utah, originally attributed to the Pleistocene. Reanalysis of stratigraphic context and radiocarbon dating indicates a historic age for this horse (cal AD 1681–1939), linking it with Ute or other Indigenous groups, whereas osteological features demonstrate its use for mounted horseback riding—perhaps with a nonframe saddle. DNA analysis indicates that the animal was a female domestic horse, which was likely cared for as part of a breeding herd despite outliving its usefulness in transport. Finally, sequentially sampled stable carbon, oxygen, and strontium isotope values from tooth enamel (δ13C, δ18O, and 87Sr/86Sr) suggest that the horse was raised locally. These results show the utility of archaeological science as applied to horse remains in understanding Indigenous horse pastoralism, whereas consideration of the broader archaeological record suggests a pattern of misidentification of horse bones from early historic contexts. The Lehi Horse Materials and Methods - Radiocarbon Dating - Osteological Study - Isotope Analysis - DNA Analysis Results - Radiocarbon Dating - Osteological Study - Isotope Analysis - DNA Analysis Discussion - Early Historic Indigenous Horse Riding and Herding in the Great Basin - Mobility and Movement Conclusion

The development and dispersal of agropastoralism transformed the cultural and ecological landscapes of the Old World, but little is known about when or how this process first impacted Central Asia. Here, we present archaeological and biomolecular evidence from Obishir V in southern Kyrgyzstan, establishing the presence of domesticated sheep by ca. 6,000 BCE. Zooarchaeological and collagen peptide mass fingerprinting show exploitation of Ovis and Capra, while cementum analysis of intact teeth implicates possible pastoral slaughter during the fall season. Most significantly, ancient DNA reveals these directly dated specimens as the domestic O. aries, within the genetic diversity of domesticated sheep lineages. Together, these results provide the earliest evidence for the use of livestock in the mountains of the Ferghana Valley, predating previous evidence by 3,000 years and suggesting that domestic animal economies reached the mountains of interior Central Asia far earlier than previously recognized. Results - Site excavation. - Archaeofaunal remains. - Cementum analysis and dental eruption/wear. - Animal DNA. Discussion Methods - Excavation. - Radiocarbon dating. - Thermoluminescence dating. - Zooarchaeology and ZooMS. - Cementum analysis. - DNA analysis. -- PCR amplification. -- Library construction and sequencing. -- PCA of genome-wide sequences. -- Mitochondrial genome analysis.

Horses and donkeys have had a far-reaching impact on human history, providing mechanical power for agriculture and transportation. Their F1-hybrids, especially mules, have also been of considerable importance due to their exceptional strength, endurance and resistance. The reconstruction of the respective role that horses, donkeys and mules played in past societies requires prior identification of their osseous elements in archaeological assemblages. This, however, remains difficult on the basis of morphological data alone and in the absence of complete skeletal elements. While DNA sequencing provides almost certain identification success, this approach requires dedicated infrastructure and sufficient ancient DNA (aDNA) preservation. Here, we assessed the performance of a cost-effective alternative approach based on geometric morphometric (GMM) analysis of the bony labyrinth, a structure carried within the petrosal bone. This extremely compact osseous structure provides good aDNA preservation and is frequently found in archaeological assemblages. To assess the GMM performance, we first used High-throughput DNA sequencing to identify 41 horses, 24 donkeys, 36 mules and one hinny from 11 archaeological sites from France and Turkey spanning different time periods. This provided a panel of 102 ancient equine remains for micro-computed tomography (microCT) and GMM assessment of the variation of the bony labyrinth shape, including the cochlea and the semicircular canals. Our new method shows good-to-excellent prediction rates (85.7%-95.2%) for the identification of species and hybrids when considering the cochlea and semicircular canals together. It provides a cheap, non-destructive alternative to aDNA for the taxonomic identification of past equine assemblages. France Genomique Appel a Grand Projet (BUCE-PHALE project; the Danish National Research Foundation) [ANR-10-INBS-09-08, DNRF94]; Initiative d'Excellence Chaires d'attractivite, Universite de Toulouse (OURASI); Villum Fonden miGENEPI research project; CNRS MITI interdisciplinary programme (Mission pour les Initiatives Transverses et Interdisciplinaires); ANRFrench National Research Agency (ANR) [ANR-17-EURE-0010]; European Research Council (ERC) under the European UnionEuropean Research Council (ERC) [681605] We thank all members of the AGES (Archaeology, Genomics, Evolution and Societies) and IDEA (IDentification, Environment and Anthropometry) research groups at CAGT (Centre for Anthropobiology and Genomics of Toulouse) for fruitful discussions. The authors would like to thank Alessio Bandelli (Inrap) , Maude Barme (CRAVO) , Alice Bourgois (CRAVO) , Franck Decanter (Inrap) , Julie Rivie`re (Service Archeologique de Chartres) , Nicolas Morand (UMR 7209) and Olivier Putelat (Pole d'Archeologie Interdepartemental RhenanArcheologie Alsace) for their help in sampling, and the archaeologists who provided the dating elements and contexts. This work was supported by the France Genomique Appel a` Grand Projet (ANR10INBS0908, BUCE-PHALE project; the Danish National Research Foundation (DNRF94) ; the Initiative d'Excellence Chaires d'attractivite, Universite de Toulouse (OURASI) and the Villum Fonden miGENEPI research project. Pierre Clavel's PhD position is funded by the CNRS MITI interdisciplinary programme (Mission pour les Initiatives Transverses et Interdisciplinaires) . Andaine SeguinOrlando acknowledges IAST funding from ANR under grant ANR-17-EURE-0010 (Investissements d'Avenir program) . This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement 681605) .

Domestication of horses fundamentally transformed long-range mobility and warfare. However, modern domesticated breeds do not descend from the earliest domestic horse lineage associated with archaeological evidence of bridling, milking and corralling at Botai, Central Asia around 3500 bc3. Other longstanding candidate regions for horse domestication, such as Iberia and Anatolia, have also recently been challenged. Thus, the genetic, geographic and temporal origins of modern domestic horses have remained unknown. Here we pinpoint the Western Eurasian steppes, especially the lower Volga-Don region, as the homeland of modern domestic horses. Furthermore, we map the population changes accompanying domestication from 273 ancient horse genomes. This reveals that modern domestic horses ultimately replaced almost all other local populations as they expanded rapidly across Eurasia from about 2000 bc, synchronously with equestrian material culture, including Sintashta spoke-wheeled chariots. We find that equestrianism involved strong selection for critical locomotor and behavioural adaptations at the GSDMC and ZFPM1 genes. Our results reject the commonly held association between horseback riding and the massive expansion of Yamnaya steppe pastoralists into Europe around 3000 bc driving the spread of Indo-European languages. This contrasts with the scenario in Asia where Indo-Iranian languages, chariots and horses spread together, following the early second millennium bc Sintashta culture., The work by G. Boeskorov is done on state assignment of DPMGI SB RAS. This project was supported by the University Paul Sabatier IDEX Chaire d’Excellence (OURASI); Villum Funden miGENEPI research programme; the CNRS ‘Programme de Recherche Conjoint’ (PRC); the CNRS International Research Project (IRP AMADEUS); the France Génomique Appel à Grand Projet (ANR-10-INBS-09-08, BUCEPHALE project); IB10131 and IB18060, both funded by Junta de Extremadura (Spain) and European Regional Development Fund; Czech Academy of Sciences (RVO:67985912); the Zoological Institute ZIN RAS (АААА-А19-119032590102-7); and King Saud University Researchers Supporting Project (NSRSP–2020/2). The research was carried out with the financial support of the Russian Foundation for Basic Research (19-59-15001 and 20-04-00213), the Russian Science Foundation (16-18-10265, 20-78-10151, and 21-18-00457), the Government of the Russian Federation (FENU-2020-0021), the Estonian Research Council (PRG29), the Estonian Ministry of Education and Research (PRG1209), the Hungarian Scientific Research Fund (Project NF 104792), the Hungarian Academy of Sciences (Momentum Mobility Research Project of the Institute of Archaeology, Research Centre for the Humanities); and the Polish National Science Centre (2013/11/B/HS3/03822). This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie (grant agreement 797449). This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreements 681605, 716732 and 834616).

International audience; The field of ancient genomics has undergone a true revolution during the last decade. Input material, time requirements and processing costs have first limited the number of specimens amenable to genome sequencing. However, the discovery that archeological material such as petrosal bones can show increased ancient DNA preservation rates, combined with advances in sequencing technologies, molecular methods for the recovery of degraded DNA fragments and bioinformatics, has vastly expanded the range of samples compatible with genome-wide investigation. Experimental procedures for DNA extraction, genomic library preparation and target enrichment have become more streamlined, and now also include automation. These procedures have considerably reduced the amount of work necessary for data generation, effectively adapting the processing capacity of individual laboratories to the increasing numbers of analyzable samples. Handling vast amounts of samples, however, comes with logistical challenges. Laboratory capacities, equipment, and people need to be efficiently coordinated, and the progress of each sample through the different experimental stages needs to be fully traceable, especially as archeological remains of animals or plants are often provided and/or handled by many different collaborators. Here we present CASCADE, a laboratory information management system (LIMS) dealing with the specificities of ancient DNA sample processing and tracking, applicable by large and small laboratories alike, and scalable to large projects involving the analysis of thousands of samples and more. By giving an account of the specimen's progress at any given analytical step, CASCADE not only optimizes the collaborative experience, including real-time information sharing with third parties, but also improves the efficacy of data generation and traceability in-house.

International audience; Although situated ∼400 km from the east coast of Africa, Madagascar exhibits cultural, linguistic, and genetic traits from both Southeast Asia and Eastern Africa. The settlement history remains contentious; we therefore used a grid-based approach to sample at high resolution the genomic diversity (including maternal lineages, paternal lineages, and genome-wide data) across 257 villages and 2,704 Malagasy individuals. We find a common Bantu and Austronesian descent for all Malagasy individuals with a limited paternal contribution from Europe and the Middle East. Admixture and demographic growth happened recently, suggesting a rapid settlement of Madagascar during the last millennium. However, the distribution of African and Asian ancestry across the island reveals that the admixture was sex biased and happened heterogeneously across Madagascar, suggesting independent colonization of Madagascar from Africa and Asia rather than settlement by an already admixed population. In addition, there are geographic influences on the present genomic diversity, independent of the admixture, showing that a few centuries is sufficient to produce detectable genetic structure in human populations.

Ninety-six single nucleotide polymorphisms (SNPs) and seventeen short tandem repeat (STRs) were investigated on the Y-chromosome of 288 unrelated healthy individuals from populations in Benin (Bariba, Yoruba, and Fon) and the Ivory Coast (Ahizi and Yacouba). We performed a multidimensional scaling analysis based on FST and RST genetic distances using a large extensive database of sub-Saharan African populations. There is more genetic homogeneity in Ivory Coast populations compared with populations from Benin. Notably, the Beninese Yoruba are significantly differentiated from neighbouring groups, but also from the Yoruba from Nigeria (FST0.05; P0.01). The Y-chromosome dataset presented here provides new valuable data to understand the complex genetic diversity and human male demographic events in West Africa.

Sex-testing using molecular genetic technique is routinely used in the fields of forensics, population genetics and conservation biology. However, none of the assay used so far allows a non-ambiguous and successful sex determination for human and non-human primate species. The most widely used method, AMELY/X, and its alternatives suffer from a set of drawbacks in humans and can rarely be used in New World primate species. Here, we designed a new sex-typing assay using a multiplexed PCR amplification of UTX and UTY-homologous loci and combined male-specific SRY locus. This method was successfully tested on 1048 samples, including 82 non-human primates from 45 Anthropoidea and Lemuriformes species and 966 human samples from 24 populations (Africans, Europeans, and South Americans). This sex-typing method is applicable across all primate species tested from Hominoidea to Indriidae, and also on various populations with different background origins; it represents a robust and cheap sex-typing assay to be used both by the anthropologist and primatologist communities.

The genetic basis of androstenone anosmia has been well studied due to androstenone's putative role as a human sex pheromone and its presence in pork meat. Polymorphisms have been identified on the olfactory receptor gene OR7D4, which significantly affect perception of androstenone pleasantness and intensity in several Western populations. This study aims to investigate androstenone sensitivity and the influence of OR7D4 polymorphisms in non-Western populations. Androstenone perception was tested in 132 individuals from Madagascar using a double three-alternative choice test with two concentrations of androstenone (0.17 and 1.7 µg/ml). We found that Malagasy populations described this molecule in a similar way to European populations, and 21% of the sample was not able to smell androstenone. In contrast to previous studies, there was no significant evidence of the influence of rs61729907: C>T (R88W) and rs5020278: C>T polymorphisms (T133M) on androstenone sensitivity in Malagasy populations. We found, however, a significant effect of the polymorphism rs61732668 (P79L) and a significant difference in androstenone perception between populations in different locations across Madagascar. This study indicates the existence of population-specific factors in androstenone sensitivity, suggesting that population history has a role in shaping an individual's smell and flavor preferences and food preferences in general.

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