Sheep were among the first domesticated animals, but their demographic history is little understood. Here we analyzed nuclear polymorphism and mitochondrial data (mtDNA) from ancient central and west Anatolian sheep dating from Epipaleolithic to late Neolithic, comparatively with modern-day breeds and central Asian Neolithic/Bronze Age sheep (OBI). Analyzing ancient nuclear data, we found that Anatolian Neolithic sheep (ANS) are genetically closest to present-day European breeds relative to Asian breeds, a conclusion supported by mtDNA haplogroup frequencies. In contrast, OBI showed higher genetic affinity to present-day Asian breeds. These results suggest that the east-west genetic structure observed in present-day breeds had already emerged by 6000 BCE, hinting at multiple sheep domestication episodes or early wild introgression in southwest Asia. Furthermore, we found that ANS are genetically distinct from all modern breeds. Our results suggest that European and Anatolian domestic sheep gene pools have been strongly remolded since the Neolithic.

22Sheep was among the first domesticated animals, but its demographic history is little 23 understood. Here we present combined analyses of mitochondrial and nuclear polymorphism 24 data from ancient central and west Anatolian sheep dating to the Late Glacial and early 25 Holocene. We observe loss of mitochondrial haplotype diversity around 7500 BCE during the 26 early Neolithic, consistent with a domestication-related bottleneck. Post-7000 BCE, 27 mitochondrial haplogroup diversity increases, compatible with admixture from other 28 domestication centres and/or from wild populations. Analysing archaeogenomic data, we 29 further find that Anatolian Neolithic sheep (ANS) are genetically closest to present-day 30 European breeds, and especially those from central and north Europe. Our results indicate that 31 Asian contribution to south European breeds in the post-Neolithic era, possibly during the 32 Bronze Age, may explain this pattern.33 34 35 36 65 traits across the globe, especially within the last 5 millennia, as part of the secondary products 66 revolution 18,19 . Although the first domesticated sheep were likely used for their meat and 67 possibly their milk 20 , they started to be increasingly exploited for their wool in Bronze Age SW 68 Asia, during the 3rd millennium BCE 21 . Intriguingly, a comparison of DNA retroelements 69 across modern breeds implies an expansion of SW Asian lineages, estimated to date back to the 70 Bronze Age; according to this model, SW Asian sheep with desired traits, such as fine wool, 71 were introduced into local breeds across the globe 22 . A recent ancient DNA study reports 72 evidence consistent with novel breeds being introduced to Bronze Age Europe, coinciding with 73 archaeological evidence for the introduction of wool to this continent 21 . In later periods, export 74 and admixture of selected sheep breeds into local stocks continued 11 . Indeed, the most recent 75 common ancestor of domestic sheep breeds has been inferred to date back only 800 generations 76 ago 11 -an unexpectedly recent estimate. 77 4We currently lack a solid demographic history model to explain these observations: high 78 diversity, clear genetic structure, and recent coalescence times. What is missing is genetic data 79 on the initial steps of domestication and characterisation of the early domesticated sheep gene 80 pool. Here we present a first attempt to bridge this gap, studying ancient DNA from Neolithic 81 period sheep remains from Anatolia, one of the possible domestication centres. Analysing both 82 mitochondrial DNA (mtDNA) sequences and nuclear polymorphism data, we find support for 83 the notions that the present-day domestic sheep population has multiple origins, and also that 84 the sheep gene pool changed considerably since the Neolithic period. 85 86 Results 87 We analysed DNA from c.200 archaeological sheep bone and tooth samples from early 88 Holocene Anatolia, originating from six different sites from central and west Anatolia and 89spanning the Epipaleolithic, Neolithic, and Ch...

We present paleogenomes of three morphologically-unidentified Anatolian equids dating to the 1st millennium BCE, sequenced to coverages of 0.6-6.4X. Mitochondrial DNA haplotypes of the Anatolian individuals clustered with those of Equus hydruntinus (or Equus hemionus hydruntinus), the extinct European wild ass. The Anatolian wild ass whole genome profiles fall outside the genomic diversity of other extant and past Asiatic wild ass (E.hemionus) lineages. These observations strongly suggest that the three Anatolian wild asses represent E.hydruntinus, making them the latest recorded survivors of this lineage, about a millennium later than the latest observations in the zooarchaeological record. Comparative genomic analyses suggest that E.hydruntinus was a sister clade to all ancient and present-day E.hemionus lineages, representing an early split. We also find indication of gene flow between hydruntines and Middle Eastern wild asses. Analyses of genome-wide heterozygosity and runs of homozygosity reveal that the Anatolian wild ass population had severely lost genetic diversity by the mid-1st millennium BCE, a likely omen of its eventual demise.

A major challenge in zooarchaeology is to morphologically distinguish closely related species’ remains, especially using small bone fragments. Shotgun sequencing aDNA from archeological remains and comparative alignment to the candidate species’ reference genomes will only apply when reference nuclear genomes of comparable quality are available, and may still fail when coverages are low. Here, we propose an alternative method, MTaxi, that uses highly accessible mitochondrial DNA (mtDNA) to distinguish between pairs of closely related species from ancient DNA sequences. MTaxi utilises mtDNA transversion-type substitutions between pairs of candidate species, assigns reads to either species, and performs a binomial test to determine the sample taxon. We tested MTaxi on sheep/goat and horse/donkey data, between which zooarchaeological classification can be challenging in ways that epitomise our case. The method performed efficiently on simulated ancient genomes down to 0.3x mitochondrial coverage for both sheep/goat and horse/donkey, with no false positives. Trials on n=18 ancient sheep/goat samples and n=10 horse/donkey samples of known species identity also yielded 100% accuracy. Overall, MTaxi provides a straightforward approach to classify closely related species that are difficult to distinguish through zooarchaeological methods using low coverage aDNA data, especially when similar quality reference genomes are unavailable. MTaxi is freely available at https://github.com/goztag/MTaxi.

A major challenge in zooarchaeology is to morphologically distinguish closely related species’ remains, especially using small bone fragments. Shotgun sequencing aDNA from archeological remains and comparative alignment to the candidate species’ reference genomes will only apply when reference nuclear genomes of comparable quality are available, and may still fail when coverages are low. Here, we propose an alternative method, MTaxi, that uses highly accessible mitochondrial DNA (mtDNA) to distinguish between pairs of closely related species from ancient DNA sequences. MTaxi utilises mtDNA transversion-type substitutions between pairs of candidate species, assigns reads to either species, and performs a binomial test to determine the sample taxon. We tested MTaxi on sheep/goat and horse/donkey data, between which zooarchaeological classification can be challenging in ways that epitomise our case. The method performed efficiently on simulated ancient genomes down to 0.5x mitochondrial coverage for both sheep/goat and horse/donkey, with no false positives. Trials on n=18 ancient sheep/goat samples and n=10 horse/donkey samples of known species identity with mtDNA coverages 0.1x - 12x  also yielded 100% accuracy. Overall, MTaxi provides a straightforward approach to classify closely related species that are compelling to distinguish through zooarchaeological methods using low coverage aDNA data, especially when similar quality reference genomes are unavailable. MTaxi is freely available at https://github.com/goztag/MTaxi.