Tracking Five Millennia of Horse Management with Extensive Ancient Genome Time Series.

Horse domestication revolutionized warfare and accelerated travel, trade, and the geographic expansion of languages. Here, we present the largest DNA time series for a non-human organism to date, including genome-scale data from 149 ancient animals and 129 ancient genomes (≥1-fold coverage), 87 of which are new. This extensive dataset allows us to assess the modern legacy of past equestrian civilizations. We find that two extinct horse lineages existed during early domestication, one at the far western (Iberia) and the other at the far eastern range (Siberia) of Eurasia. None of these contributed significantly to modern diversity. We show that the influence of Persian-related horse lineages increased following the Islamic conquests in Europe and Asia. Multiple alleles associated with elite-racing, including at the MSTN "speed gene," only rose in popularity within the last millennium. Finally, the development of modern breeding impacted genetic diversity more dramatically than the previous millennia of human management.

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.

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.

A method to investigate muscle target-specific transcriptional signatures of single motor neurons.

BACKGROUND: Motor neurons in the vertebrate spinal cord have long served as a paradigm to study the transcriptional logic of cell type specification and differentiation. At limb levels, pool-specific transcriptional signatures first restrict innervation to only one particular muscle in the periphery, and get refined, once muscle connection has been established. Accordingly, to study the transcriptional dynamics and specificity of the system, a method for establishing muscle target-specific motor neuron transcriptomes would be required. RESULTS: To investigate target-specific transcriptional signatures of single motor neurons, here we combine ex-ovo retrograde axonal labeling in mid-gestation chicken embryos with manual isolation of individual fluorescent cells and Smart-seq2 single-cell RNA-sequencing. We validate our method by injecting the dorsal extensor metacarpi radialis and ventral flexor digiti quarti wing muscles and harvesting a total of 50 fluorescently labeled cells, in which we detect up to 12,000 transcribed genes. Additionally, we present visual cues and cDNA metrics predictive of sequencing success. CONCLUSIONS: Our method provides a unique approach to study muscle target-specific motor neuron transcriptomes at a single-cell resolution. We anticipate that our method will provide key insights into the transcriptional logic underlying motor neuron pool specialization and proper neuromuscular circuit assembly and refinement.

The genetics of niche-specific behavioral tendencies in an adaptive radiation of cichlid fishes.

Behavior is critical for animal survival and reproduction, and possibly for diversification and evolutionary radiation. However, the genetics behind adaptive variation in behavior are poorly understood. In this work, we examined a fundamental and widespread behavioral trait, exploratory behavior, in one of the largest adaptive radiations on Earth, the cichlid fishes of Lake Tanganyika. By integrating quantitative behavioral data from 57 cichlid species (702 wild-caught individuals) with high-resolution ecomorphological and genomic information, we show that exploratory behavior is linked to macrohabitat niche adaptations in Tanganyikan cichlids. Furthermore, we uncovered a correlation between the genotypes at a single-nucleotide polymorphism upstream of the AMPA glutamate-receptor regulatory gene cacng5b and variation in exploratory tendency. We validated this association using behavioral predictions with a neural network approach and CRISPR-Cas9 genome editing.

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.

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.

Early pastoral economies along the Ancient Silk Road: Biomolecular evidence from the Alay Valley, Kyrgyzstan.

The Silk Road was an important trade route that channeled trade goods, people, plants, animals, and ideas across the continental interior of Eurasia, fueling biotic exchange and key social developments across the Old World. Nestled between the Pamir and Alay ranges at a baseline elevation of nearly 3000m, Kyrgyzstan's high Alay Valley forms a wide geographic corridor that comprised one of the primary channels of the ancient Silk Road. Recent archaeological survey reveals a millennia-long history of pastoral occupation of Alay from the early Bronze Age through the Medieval period, and a stratified Holocene sequence at the site of Chegirtke Cave. Faunal remains were recovered from test excavations as well as surface collection of material from recent marmot activity. Although recovered specimens were highly fragmented and mostly unidentifiable using traditional zooarchaeological methods, species identification via collagen mass fingerprinting (ZooMS) coupled with sex and first-generation hybrid identification through ancient DNA enabled preliminary characterization of the animal economy of Alay herders. Our new results indicate primary reliance on sheep at Chegirtke Cave (ca. 2200 BCE), with cattle and goat also present. The discovery of a large grinding stone at a spatially associated Bronze or Iron Age habitation structure suggests a mixed agropastoral economic strategy, rather than a unique reliance on domestic animals. Radiocarbon-dated faunal assemblages from habitation structures at nearby localities in the Alay Valley demonstrate the presence of domestic horse, as well as Bactrian camel during later periods. The current study reveals that agropastoral occupation of the high-mountain Alay corridor started millennia before the formal establishment of the Silk Road, and posits that ZooMS, when paired with radiocarbon dates and ancient DNA, is a powerful and cost-effective tool for investigating shifts in the use of animal domesticates in early pastoral economies.

Ancient genomes revisit the ancestry of domestic and Przewalski's horses.

The Eneolithic Botai culture of the Central Asian steppes provides the earliest archaeological evidence for horse husbandry, ~5500 years ago, but the exact nature of early horse domestication remains controversial. We generated 42 ancient-horse genomes, including 20 from Botai. Compared to 46 published ancient- and modern-horse genomes, our data indicate that Przewalski's horses are the feral descendants of horses herded at Botai and not truly wild horses. All domestic horses dated from ~4000 years ago to present only show ~2.7% of Botai-related ancestry. This indicates that a massive genomic turnover underpins the expansion of the horse stock that gave rise to modern domesticates, which coincides with large-scale human population expansions during the Early Bronze Age.

Experimental conditions improving in-solution target enrichment for ancient DNA.

High-throughput sequencing has dramatically fostered ancient DNA research in recent years. Shotgun sequencing, however, does not necessarily appear as the best-suited approach due to the extensive contamination of samples with exogenous environmental microbial DNA. DNA capture-enrichment methods represent cost-effective alternatives that increase the sequencing focus on the endogenous fraction, whether it is from mitochondrial or nuclear genomes, or parts thereof. Here, we explored experimental parameters that could impact the efficacy of MYbaits in-solution capture assays of ~5000 nuclear loci or the whole genome. We found that varying quantities of the starting probes had only moderate effect on capture outcomes. Starting DNA, probe tiling, the hybridization temperature and the proportion of endogenous DNA all affected the assay, however. Additionally, probe features such as their GC content, number of CpG dinucleotides, sequence complexity and entropy and self-annealing properties need to be carefully addressed during the design stage of the capture assay. The experimental conditions and probe molecular features identified in this study will improve the recovery of genetic information extracted from degraded and ancient remains.

Ancient genomic changes associated with domestication of the horse.

The genomic changes underlying both early and late stages of horse domestication remain largely unknown. We examined the genomes of 14 early domestic horses from the Bronze and Iron Ages, dating to between ~4.1 and 2.3 thousand years before present. We find early domestication selection patterns supporting the neural crest hypothesis, which provides a unified developmental origin for common domestic traits. Within the past 2.3 thousand years, horses lost genetic diversity and archaic DNA tracts introgressed from a now-extinct lineage. They accumulated deleterious mutations later than expected under the cost-of-domestication hypothesis, probably because of breeding from limited numbers of stallions. We also reveal that Iron Age Scythian steppe nomads implemented breeding strategies involving no detectable inbreeding and selection for coat-color variation and robust forelimbs.

The Evolutionary Origin and Genetic Makeup of Domestic Horses.

The horse was domesticated only 5.5 KYA, thousands of years after dogs, cattle, pigs, sheep, and goats. The horse nonetheless represents the domestic animal that most impacted human history; providing us with rapid transportation, which has considerably changed the speed and magnitude of the circulation of goods and people, as well as their cultures and diseases. By revolutionizing warfare and agriculture, horses also deeply influenced the politico-economic trajectory of human societies. Reciprocally, human activities have circled back on the recent evolution of the horse, by creating hundreds of domestic breeds through selective programs, while leading all wild populations to near extinction. Despite being tightly associated with humans, several aspects in the evolution of the domestic horse remain controversial. Here, we review recent advances in comparative genomics and paleogenomics that helped advance our understanding of the genetic foundation of domestic horses.

Evolutionary Genomics and Conservation of the Endangered Przewalski's Horse.

Przewalski's horses (PHs, Equus ferus ssp. przewalskii) were discovered in the Asian steppes in the 1870s and represent the last remaining true wild horses. PHs became extinct in the wild in the 1960s but survived in captivity, thanks to major conservation efforts. The current population is still endangered, with just 2,109 individuals, one-quarter of which are in Chinese and Mongolian reintroduction reserves [1]. These horses descend from a founding population of 12 wild-caught PHs and possibly up to four domesticated individuals [2-4]. With a stocky build, an erect mane, and stripped and short legs, they are phenotypically and behaviorally distinct from domesticated horses (DHs, Equus caballus). Here, we sequenced the complete genomes of 11 PHs, representing all founding lineages, and five historical specimens dated to 1878-1929 CE, including the Holotype. These were compared to the hitherto-most-extensive genome dataset characterized for horses, comprising 21 new genomes. We found that loci showing the most genetic differentiation with DHs were enriched in genes involved in metabolism, cardiac disorders, muscle contraction, reproduction, behavior, and signaling pathways. We also show that DH and PH populations split ∼45,000 years ago and have remained connected by gene-flow thereafter. Finally, we monitor the genomic impact of ∼110 years of captivity, revealing reduced heterozygosity, increased inbreeding, and variable introgression of domestic alleles, ranging from non-detectable to as much as 31.1%. This, together with the identification of ancestry informative markers and corrections to the International Studbook, establishes a framework for evaluating the persistence of genetic variation in future reintroduced populations.