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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. [ABSTRACT FROM AUTHOR]

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. • Two now-extinct horse lineages lived in Iberia and Siberia some 5,000 years ago • Iberian and Siberian horses contributed limited ancestry to modern domesticates • Oriental horses have had a strong genetic influence within the last millennium • Modern breeding practices were accompanied by a significant drop in genetic diversity Genome-wide data from 278 ancient equids provide insights into how ancient equestrian civilizations managed, exchanged, and bred horses and indicate vast loss of genetic diversity as well as the existence of two extinct lineages of horses that failed to contribute to modern domestic animals. [ABSTRACT FROM AUTHOR]

Horses, asses and zebras, can produce first-generation F1-hybrids, despite their striking karyotypic and phenotypic differences. Such F1-hybrids are mostly infertile, but often present characters of considerable interest to breeders. They were extremely valued in antiquity, and commonly represented in art and on coinage. However, hybrids appear relatively rarely in archaeological faunal assemblages, mostly because identification based on morphometric data alone is extremely difficult. Here, we developed a methodological framework that exploits high-throughput sequencing data retrieved from archaeological material to identify F1-equine hybrids. Our computational methodology is distributed in the open-source Zonkey pipeline, now part of PALEOMIX ( https://github.com/MikkelSchubert/paleomix ), together with full documentation and examples. Using both synthetic and real sequence datasets, from living and ancient F1-hybrids, we find that Zonkey shows high sensitivity and specificity, even with limited sequencing efforts. Zonkey is thus well suited to the identification of equine F1-hybrids in the archaeological record, even in cases where DNA preservation is limited. Zonkey can also help determine the sex of ancient animals, and allows species identification, which advantageously complements morphological data in cases where material is fragmentary and/or multiple candidate equine species coexisted in sympatry. [ABSTRACT FROM AUTHOR]

Mules (Equus asinus x Equus caballus) represent first-generation hybrids between a female horse (mare) and a male donkey (jack). They are generally considered to have first appeared north of the Alps with Roman influence, a time period in which written and iconographic sources support their key role for transport and traction, both in farming and the military. The archaeozoological evidence for mules is, however, contentious as faunal assemblages are difficult to assign to either parental species or hybrids based on morphometric data alone. Here we leverage low-coverage DNA sequence data and Zonkey computational analyses to assess the occurrence of mules within Roman equid faunal assemblages in the alpine foreland. While morphological data previously assigned 17 remains to mules, successful DNA analysis of 12 remains revealed that 11 were in fact horses, one female and ten males. Eight mtDNA d-loop haplogroups were identified and genetic diversity within Roman equids corresponds to non-threatened modern local breeds. Two remains genetically identified as mules belonged to haplogroups F and I. Our results suggest that the importance of mules in the Roman archaeological record of the alpine foreland, and probably elsewhere, may have been previously over-estimated. Whether this is true for other regions of the Roman Empire needs to be evaluated. Further genomic testing for equid species and their hybrids and molecular sexing will improve our knowledge on this important issue. • Classical texts, art and archaeozoological evidence support the large socio-economic impact of equids in the Roman Empire. • Archaeozoological evidence is ambiguous due to overlapping morphology amongst horses, donkeys and their hybrids. • 10 out of 12 remains identified as mules on the basis of their morphology were reassigned to male horses genetically. • We propose that the occurrence of mules at Roman sites may be more limited than previously claimed. [ABSTRACT FROM AUTHOR]