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.

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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 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.

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.

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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.

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