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.

Grazing high and low: Can we detect horse altitudinal mobility using high-resolution isotope (δ13 C and δ15 N values) time series in tail hair? A case study in the Mongolian Altai.

RATIONALE: Carbon and nitrogen stable isotope time series performed in continuously growing tissues (hair, tooth enamel) are commonly used to reconstruct the dietary history of modern and ancient animals. Predicting the effects of altitudinal mobility on animal δ13 C and δ15 N values remains difficult as several variables such as temperature, water availability or soil type can contribute to the isotope composition. Modern references adapted to the region of interest are therefore essential. METHODS: Between June 2015 and July 2018, six free-ranging domestic horses living in the Mongolian Altaï were fitted with GPS collars. Tail hairs were sampled each year, prepared for sequential C and N isotope analysis using EA-IRMS. Isotopic variations were compared with altitudinal mobility, and Generalized Additive Mixed (GAMMs) models were used to model the effect of geographic and environmental factors on δ13 C and δ15 N values. RESULTS: Less than half of the pasture changes were linked with a significant isotopic shift while numerous isotopic shifts did not correspond to any altitudinal mobility. Similar patterns of δ13 C and δ15 N variations were observed between the different horses, despite differences in mobility patterns. We propose that water availability as well as seasonal availability of N2 fixing type plants primarily controlled horse hair δ13 C and δ15 N values, overprinting the influence of altitude. CONCLUSIONS: Our study shows that altitudinal mobility is not the main factor that drives the variations in horse tail hair δ13 C and δ15 N values and that seasonal change in the animal dietary preference also plays an important role. It is therefore risky to interpret variations in δ13 C and δ15 N values of animal tissues in terms of altitudinal mobility alone, at least in C3 -dominated environments.

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.

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 horse pinworm (Oxyuris equi) in archaeology during the Holocene: Review of past records and new data.

This paper focuses on the horse pinworm, Oxyuris equi, in archaeology during the Holocene period, and presents an overview of past published occurrences, early mentions in texts, and new data from our paleoparasitology research. This original compilation shows that the most ancient record of the horse pinworm dates to ca. 2500 years before present (ybp) in Central Asia and to ca. 2020 ybp in Western Europe. It also shows that the parasite is not detected on the American continent until contemporary periods. The role of European migrations from 1492 (Christopher Columbus) is discussed to explain the transfer of the horse pinworm from the Old World to the Americas. The absence of any record of this parasite before ca. 2500 ybp in Eurasia could be explained by parasite ecology, unfavorable sampling and scarcity of horse archeological remains. For the Americas, the absence of horse for long periods can be an additional explanation for the absence of the parasite.

Molecular detection of Bartonella henselae DNA in the dental pulp of 800-year-old French cats.

Bartonella species are responsible for chronic bacteremia in domestic cats, which raises a question about the antiquity of the relationship between Bartonella species and cats that act as reservoirs for the organism. The sequencing of Bartonella pap31 and groEL genes from the dental pulp of cats dating from the 13th to 16th centuries identified the presence of B. henselae genotype Houston; the observation of a unique mutation in the results of PCR assays for Bartonella species ruled out modern DNA contamination of the dental pulp samples. We conclude that cats had bacteremia due to B. henselae 800 years ago.