Widespread horse-based mobility arose around 2,200 BCE in Eurasia.

Horses revolutionized human history with fast mobility1. However, the timeline between their domestication and widespread integration as a means of transportation remains contentious2-4. Here we assemble a large collection of 475 ancient horse genomes to assess the period when these animals were first reshaped by human agency in Eurasia. We find that reproductive control of the modern domestic lineage emerged ~2,200 BCE (Before Common Era), through close kin mating and shortened generation times. Reproductive control emerged following a severe domestication bottleneck starting no earlier than ~2,700 BCE, and coincided with a sudden expansion across Eurasia that ultimately resulted in the replacement of nearly every local horse lineage. This expansion marked the rise of widespread horse-based mobility in human history, which refutes the commonly-held narrative of large horse herds accompanying the massive migration of steppe peoples across Europe ~3,000 BCE and earlier3,5. Finally, we detect significantly shortened generation times at Botai ~3,500 BCE, a settlement from Central Asia associated with corrals and a subsistence economy centered on horses6,7. This supports local horse husbandry before the rise of modern domestic bloodlines.

Isotopic biographies reveal horse rearing and trading networks in medieval London.

This paper reports a high-resolution isotopic study of medieval horse mobility, revealing their origins and in-life mobility both regionally and internationally. The animals were found in an unusual horse cemetery site found within the City of Westminster, London, England. Enamel strontium, oxygen, and carbon isotope analysis of 15 individuals provides information about likely place of birth, diet, and mobility during the first approximately 5 years of life. Results show that at least seven horses originated outside of Britain in relatively cold climates, potentially in Scandinavia or the Western Alps. Ancient DNA sexing data indicate no consistent sex-specific mobility patterning, although three of the five females came from exceptionally highly radiogenic regions. Another female with low mobility is suggested to be a sedentary broodmare. Our results provide direct and unprecedented evidence for a variety of horse movement and trading practices in the Middle Ages and highlight the importance of international trade in securing high-quality horses for medieval London elites.

Interdisciplinary evidence for early domestic horse exploitation in southern Patagonia.

The introduction of domestic horses transformed Indigenous societies across the grasslands of Argentina, leading to the emergence of specialized horse cultures across the Southern Cone. However, the dynamics of this introduction are poorly chronicled by historic records. Here, we apply archaeozoological and biomolecular techniques to horse remains from the site of Chorrillo Grande 1 in southern Argentina. Osteological and taphonomic analyses suggest that horses were pastorally managed and used for food by Aónikenk/Tehuelche hunter-gatherers before the onset of permanent European settlement, as early as the mid-17th century. DNA-based sex identifications suggest consumption of both male and female horses, while ceramic residue also shows use of guanaco products. Sequential isotope analyses on horse dentition reveal an origin in southern Patagonia and movement of these animals between the Río Coig and Río Gallegos basins. These results reinforce emerging evidence for rapid Indigenous-mediated dispersal of horses in the Americas and demonstrate that horses catalyzed rapid economic and social transformations.

Improving the extraction of ancient Yersinia pestis genomes from the dental pulp.

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

Early dispersal of domestic horses into the Great Plains and northern Rockies.

The horse is central to many Indigenous cultures across the American Southwest and the Great Plains. However, when and how horses were first integrated into Indigenous lifeways remain contentious, with extant models derived largely from colonial records. We conducted an interdisciplinary study of an assemblage of historic archaeological horse remains, integrating genomic, isotopic, radiocarbon, and paleopathological evidence. Archaeological and modern North American horses show strong Iberian genetic affinities, with later influx from British sources, but no Viking proximity. Horses rapidly spread from the south into the northern Rockies and central plains by the first half of the 17th century CE, likely through Indigenous exchange networks. They were deeply integrated into Indigenous societies before the arrival of 18th-century European observers, as reflected in herd management, ceremonial practices, and culture.

DNA methylation-based profiling of horse archaeological remains for age-at-death and castration.

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

The genomic history and global expansion of domestic donkeys.

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

Assessing the impact of USER-treatment on hyRAD capture applied to ancient DNA.

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.

Performance and automation of ancient DNA capture with RNA hyRAD probes.

DNA hybridization-capture techniques allow researchers to focus their sequencing efforts on preselected genomic regions. This feature is especially useful when analysing ancient DNA (aDNA) extracts, which are often dominated by exogenous environmental 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 restriction of fresh DNA extracts to produce RNA probes that cover only a fraction of the genome and can serve as baits for capturing homologous fragments from aDNA libraries. 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 extremely low endogenous content (<1%). Performance was, however, more limited for those samples already characterized by good DNA preservation (>20%-30%), while the fraction of endogenous reads mapping on- and off-target was relatively insensitive 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 hypomethylated regions, which improved data quality by reducing post-mortem DNA damage and mapping within multicopy regions. Finally, we developed a fully automated hyRAD protocol utilizing inexpensive robotic platforms to facilitate capture processing. Overall, our work establishes hyRAD as a cost-effective strategy to recover a set of shared orthologous variants across multiple ancient samples.

The origins and spread of domestic horses from the Western Eurasian steppes.

Domestication of horses fundamentally transformed long-range mobility and warfare1. However, modern domesticated breeds do not descend from the earliest domestic horse lineage associated with archaeological evidence of bridling, milking and corralling2-4 at Botai, Central Asia around 3500 BC3. Other longstanding candidate regions for horse domestication, such as Iberia5 and Anatolia6, 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 association7 between horseback riding and the massive expansion of Yamnaya steppe pastoralists into Europe around 3000 BC8,9 driving the spread of Indo-European languages10. This contrasts with the scenario in Asia where Indo-Iranian languages, chariots and horses spread together, following the early second millennium BC Sintashta culture11,12.

Evidence for early dispersal of domestic sheep into Central Asia.

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

Predicting haplogroups using a versatile machine learning program (PredYMaLe) on a new mutationally balanced 32 Y-STR multiplex (CombYplex): Unlocking the full potential of the human STR mutation rate spectrum to estimate forensic parameters.

We developed a new mutationally well-balanced 32 Y-STR multiplex (CombYplex) together with a machine learning (ML) program PredYMaLe to assess the impact of STR mutability on haplogourp prediction, while respecting forensic community criteria (high DC/HD). We designed CombYplex around two sub-panels M1 and M2 characterized by average and high-mutation STR panels. Using these two sub-panels, we tested how our program PredYmale reacts to mutability when considering basal branches and, moving down, terminal branches. We tested first the discrimination capacity of CombYplex on 996 human samples using various forensic and statistical parameters and showed that its resolution is sufficient to separate haplogroup classes. In parallel, PredYMaLe was designed and used to test whether a ML approach can predict haplogroup classes from Y-STR profiles. Applied to our kit, SVM and Random Forest classifiers perform very well (average 97 %), better than Neural Network (average 91 %) and Bayesian methods (< 90 %). We observe heterogeneity in haplogroup assignation accuracy among classes, with most haplogroups having high prediction scores (99-100 %) and two (E1b1b and G) having lower scores (67 %). The small sample sizes of these classes explain the high tendency to misclassify the Y-profiles of these haplogroups; results were measurably improved as soon as more training data were added. We provide evidence that our ML approach is a robust method to accurately predict haplogroups when it is combined with a sufficient number of markers, well-balanced mutation rate Y-STR panels, and large ML training sets. Further research on confounding factors (such as CNV-STR or gene conversion) and ideal STR panels in regard to the branches analysed can be developed to help classifiers further optimize prediction scores.

Genomic landscape of human diversity across Madagascar.

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.

Genetic Admixture and Flavor Preferences: Androstenone Sensitivity in Malagasy Populations.

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.

Genetic population study of Y-chromosome markers in Benin and Ivory Coast ethnic groups.

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 (FST>0.05; P<0.01). The Y-chromosome dataset presented here provides new valuable data to understand the complex genetic diversity and human male demographic events in West Africa.

Determined about sex: sex-testing in 45 primate species using a 2Y/1X sex-typing assay.

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.