As one of the first domestic livestock species, sheep have played a fundamental role in human societies since the Neolithic. However, their demographic history remains poorly understood. To shed light on the demographic dynamics of sheep at the western edge of the Mediterranean, we sequenced 22 ancient sheep genomes from Iberia (dating from 7,270 to 1,615 calBP, sequenced up to 8.74X coverage) along with one modern European mouflon from Corsica. We provide evidence for an initial maritime introduction into Iberia, and show that European mouflons are descendants of feralized Neolithic sheep. Furthermore, we identify a secondary influx of "Eastern" genetic ancestry coinciding with the arrival of human Steppe ancestry in Iberia -- an event that likely aligns with the spread of woolly sheep across Europe. A third population expansion is observed during the Roman period, a time when historical sources reference the trade of fine-wool sheep. This Roman-era expansion appears to have significantly influenced the genetic makeup of modern European sheep, contributing to the development of popular modern-day breeds such as Merino. In addition to these major events, we see indications of additional, minor episodes of prehistoric gene flow into the Iberian population, suggesting that western European sheep experienced more dynamic demographic changes than other domestic animals, humans, or sheep populations elsewhere. Together, these results highlight the dynamic history of Iberian sheep populations and demonstrate how human cultural and demographic shifts have left their hoofprints in the sheep gene pool, marking them as a valuable proxy for understanding the human past. ### Competing Interest Statement The authors have declared no competing interest. Swedish Research Council, 2017–05267 Helge Ax:son Johnsons Stiftelse, F20-0274 Ministerio de Ciencia, Innovación y Universidades, PID2024-156477NB C-33 Comunidad de Madrid, PHS-2024/PH-HUM-469 Ministerio de Ciencia, Innovación y Universidades, https://ror.org/05r0vyz12, RYC2018-025223-I, BGS220-461AA-69201

The petrous bone is considered the most efficient source of endogenous DNA across skeletal tissues in ancient DNA (aDNA) research as well as in forensic work. Recently, aDNA in auditory ossicle bones was shown to be comparably well preserved as in the petrous, although no attempt was made to distinguish among the three ossicle bones. In this study, we compare aDNA profiles across matched ossicle- and petrous-derived sequencing libraries prepared from 29 human skeletons from Neolithic Anatolia and Medieval Iberia. We find that the stapes and incus provide higher human endogenous aDNA than the petrous bone, with >2× higher median rates of endogenous aDNA recovery, whereas the malleus performs similarly to the petrous. Human aDNA fragments retrieved from the stapes were 8% longer than those from the petrous, whereas postmortem damage, clonality, and contamination rates were comparable among the studied bone types. These observations are corroborated by data from nonmatched ossicle or petrous libraries from 81 individuals from the same contexts, with the highest endogenous aDNA content observed in the stapes. Despite being the smallest bone in the human skeleton, the stapes, along with the incus, may be among the most optimal aDNA sources yet identified.

Mouflons are flagship species of the Mediterranean islands where they persist. Once thought to be the remnants of a European wild sheep population, archaeology suggests they were introduced by humans to the islands of Cyprus in the Early Neolithic (~10,000 years ago) and later to Corsica and Sardinia. Their status as truly wild animals remains a subject of debate. To investigate the phylogenetic relationship between these island populations and other domestic and wild sheep from the Mediterranean region, we sequenced 50 mitogenomes of mouflons from Sardinia and Corsica, and modern and ancient Sardinian domestic sheep. A total of 68 additional publicly available mitogenomes were included in the comparative analysis and used to reconstruct the phylogeny of sheep and its closest wild relative, the mouflon (Ovis gmelini). Our study analyzed the evolutionary relationships within the C-E-X and haplogroup B clusters, showing that: a) Cyprus mouflons are more related to Anatolian and Iranian mouflons belonging to the wild haplogroup X, which seems to be basal to the domestic C and E haplogroups; b) Corsican and Sardinian mouflon arise from basal lineages associated with the early European expansion of domestic sheep. These results highlight the phylogenetic distinctiveness of the mouflon populations from the Mediterranean islands, suggesting a revision of their systematic classification and an update of the nomenclature for Sardinian and Corsican mouflons from the current status of subspecies of domestic sheep (Ovis aries musimon) to subspecies of their wild relatives (Ovis gmelini musimon) which would facilitate conservation efforts.

West Anatolia has been a crucial yet elusive element in the Neolithic expansion from the Fertile Crescent to Europe. In this work, we describe the changing genetic and cultural landscapes of early Holocene West Anatolia using 30 new paleogenomes. We show that Neolithization in West Anatolia was a multifaceted process, characterized by the assimilation of Neolithic practices by local foragers, the influx of eastern populations, and their admixture, with their descendants subsequently establishing Neolithic Southeast Europe. We then coanalyzed genetic and cultural similarities across early Holocene Anatolian and Aegean Neolithic villages using 58 material culture elements. Cultural distances among villages correlate with their spatial distances but not with their genetic distances after controlling for geography. This suggests that cultural change was often decoupled from genetically visible mobility.

Combining 131 paleogenomes with bioarchaeological and archaeological data, we studied social organization and gendered practices in Çatalhöyük East Mound (7100 to 5950 BCE), a major Neolithic settlement in Central Anatolia. In early Çatalhöyük, burials in the same building were frequently close genetic relatives, suggesting that houses were used by biological family members. In later periods, however, individuals buried in the same building were often genetically unrelated, despite sharing similar diets. We found no indication of sex-biased mobility into Çatalhöyük. Meanwhile, in all periods, within-building genetic connections were predominantly maternal rather than paternal. Burials of female subadults also received a higher frequency of gifts than male subadults. Our results reveal how kinship practices changed while specific practices prioritizing female lines persisted for 1000 years at Neolithic Çatalhöyük.

This protocol describes how to sample the tiny auditory ossicle bones — the stapes (stirrup), malleus (hammer), and incus (anvil). It is particularly recommended for sampling ossicles from uncleaned whole skulls with the temporal bone present, or from isolated temporal bones with burial or depositional soil still filling the ear canal. These bones can be used to extract authentic human DNA fragments in both ancient DNA and forensic contexts.

The petrous bone is considered the most efficient source of endogenous DNA across skeletal tissues in ancient DNA research as well as in forensic work. Recently, ancient DNA (aDNA) in auditory ossicle bones was shown to be comparably well-preserved as in the petrous, although no attempt was made to distinguish among the three ossicle bones. In this study, we used a total of 114 human ossicle- and petrous-derived sequencing libraries from similar contexts (c.10,000 BP - 7,000 BP Anatolia), including 34 matched libraries prepared from the same individuals’ ossicle and petrous bones. Our results suggest that endogenous human aDNA preservation in the stapes is on average two times higher than in the petrous bone; it also tends to be higher than in the malleus and incus. Similarly, aDNA fragment lengths were higher in the stapes than in the petrous, whereas postmortem damage, clonality and contamination rates were comparable. Despite being the smallest bone in the human skeleton, the stapes may be the most optimal aDNA source yet identified. ### Competing Interest Statement The authors have declared no competing interest.

Ancient DNA (aDNA) obtained from human remains is typically fragmented and present in relatively low amounts. Here we investigate a set of optimal methods for producing aDNA data by comparing silica-based DNA extraction and aDNA library preparation protocols. We also test the efficiency of whole-genome enrichment (WGC) on ancient human samples by modifying a number of parameter combinations. We find that the Dabney extraction protocol performs significantly better than alternatives. We further observed a positive trend with the BEST library protocol indicating lower clonality. Notably, our results suggest that WGC is effective at retrieving endogenous DNA, particularly from poorly-preserved human samples, by increasing human endogenous proportions by 5x. Thus, aDNA studies will be most likely to benefit from our results. We studied a set of methods used to generate ancient genome data from archaeological human samples from different sites and periods, and with varying levels of preservation. In these experiments we compared two sample preparation techniques, two silica-based DNA extraction protocols, and two aDNA library preparation protocols. Finally, we explored the efficiency of whole-genome capture (WGC) on aDNA libraries with different characteristics by comparing a set of protocol modifications.

In this chapter, we investigate the genetics of two Early Chalcolithic Catalhoyuk individuals, U.18333 and U.16835, the only two burials yet recovered from the West Mound. These were two neonates buried within the same building. Using shotgun-sequenced partial genomes (0.06x and 0.02x coverage) we identify both as females. Despite being recovered from the same building, we find no close genetic kinship between them, in line with previously published kinship results from Catalhoyuk. We also find that the two West Mound neonates shared the same gene pool with Neolithic Catalhoyuk and other Central and West Anatolian Neolithic populations, and they did not carry the Caucasus-related U+201CeasternU+201D gene flow signature observed in later-coming Chalcolithic Anatolian genomes. This indicates no large-scale admixture between East and West Mound Catalhoyuk, and possibly that the post-Neolithic U+201CeasternU+201D gene flow event into Anatolia may have initiated only by the mid-6th millennium BCE. ### Competing Interest Statement The authors have declared no competing interest.

Arguments have long suggested that the advent of early farming in the Near East and Anatolia was linked to a ‘Mother Goddess’ cult. However, evidence for a dominant female role in these societies has been scarce. We studied social organisation, mobility patterns and gendered practices in Neolithic Southwest Asia using 131 paleogenomes from Çatalhöyük East Mound (7100-5950 BCE), a major settlement in Central Anatolia with an uninterrupted occupation and an apparent egalitarian structure. In contrast to widespread genetic evidence for patrilocality in Neolithic Europe, the Çatalhöyük individuals revealed no indication of patrilocal mobility. Analysing genetic kin ties among individuals buried in the same house (co-burials) across 35 Çatalhöyük buildings, we identified close ties concentrated within buildings and among neighbours in Çatalhöyük’s Early period, akin to those in the preceding Pre-Pottery Neolithic in Southwest Asia. This pattern weakened over time: by the late 7th millennium BCE, subadults buried in the same building were rarely closely genetically related, despite sharing similar diets. Still, throughout the site’s occupation, genetic connections within Çatalhöyük buildings were much more frequently connected via the maternal than the paternal line. We also identified differential funerary treatment of female subadults compared to those of males, with a higher frequency of grave goods associated with females. Our results reveal how kinship practices changed while key female roles persisted over one thousand years in a large Neolithic community in western Eurasia. ### Competing Interest Statement The authors have declared no competing interest.

Sheep was one of the first domesticated animals in Neolithic West Eurasia. The zooarchaeological record suggests that domestication first took place in Southwest Asia, although much remains unresolved about the precise location(s) and timing(s) of earliest domestication, or the post-domestication history of sheep. Here, we present 24 new partial sheep paleogenomes, including a 13,000-year-old Epipaleolithic Central Anatolian wild sheep, as well as 14 domestic sheep from Neolithic Anatolia, two from Neolithic Iran, two from Neolithic Iberia, three from Neolithic France, and one each from Late Neolithic/Bronze Age Baltic and South Russia, in addition to five present-day Central Anatolian Mouflons and two present-day Cyprian Mouflons. We find that Neolithic European, as well as domestic sheep breeds, are genetically closer to the Anatolian Epipaleolithic sheep and the present-day Anatolian and Cyprian Mouflon than to the Iranian Mouflon. This supports a Central Anatolian source for domestication, presenting strong evidence for a domestication event in SW Asia outside the Fertile Crescent, although we cannot rule out multiple domestication events also within the Neolithic Fertile Crescent. We further find evidence for multiple admixture and replacement events, including one that parallels the Pontic Steppe-related ancestry expansion in Europe, as well as a post-Bronze Age event that appears to have further spread Asia-related alleles across global sheep breeds. Our findings mark the dynamism of past domestic sheep populations in their potential for dispersal and admixture, sometimes being paralleled by their shepherds and in other cases not.

Selective funerary practices can inform about social relationships in prehistoric societies but are often difficult to discern. Here we present evidence for an age-specific practice at the Neolithic site of Çatalhöyük in Anatolia, dating to the 7th millennium BCE. Among ancient DNA libraries produced from 362 petrous bone samples, those of subadults contained three times higher average human DNA than those of adults. This difference in organic preservation was also confirmed by FTIR analysis. Studying similar datasets from seven prehistoric and historical sites, we found a similar age-related difference in only one cemetery. We propose that the organic preservation difference with age was caused by the special treatment of chosen corpses before interment, such as defleshing or drying, which was more frequently applied to Çatalhöyük adults and promoted organic decay. ### Competing Interest Statement The authors have declared no competing interest.

Western Anatolia has been a crucial yet elusive element in the Neolithic expansion from the Fertile Crescent to Europe. Using 30 new palaeogenomes from Anatolia c.8000-6000 BCE we describe the early Holocene genetic landscape of Western Anatolia, which reveals population continuity since the late Upper Pleistocene. Our findings indicate that the Neolithisation of Western Anatolia in the 7th millennium BCE was a multifaceted process, characterised by the assimilation of Neolithic practices by indigenous groups and the influx of populations from the east, their admixed descendants eventually laying the foundations of Neolithic Southeast Europe. Intriguingly, the observed diversity in material culture among Aegean Early Neolithic communities correlates with their geographical distances but not their genetic differences, signifying a decoupling between cultural developments and genetic admixture processes. ### Competing Interest Statement The authors have declared no competing interest.

Once widespread in their homelands, the Anatolian mouflon (Ovis gmelini anatolica) and the Cyprian mouflon (Ovis gmelini ophion) were driven to near extinction during the 20th century and are currently listed as endangered populations by the International Union for Conservation of Nature. While the exact origins of these lineages remain unclear, they have been suggested to be close relatives of domestic sheep or remnants of proto-domestic sheep. Here, we study whole genome sequences of n = 5 Anatolian mouflons and n = 10 Cyprian mouflons in terms of population history and diversity, comparing them with eight other extant sheep lineages. We find reciprocal genetic affinity between Anatolian and Cyprian mouflons and domestic sheep, higher than all other studied wild sheep genomes, including the Iranian mouflon (O. gmelini). Studying diversity indices, we detect a considerable load of short runs of homozygosity blocks (<2 Mb) in both Anatolian and Cyprian mouflons, reflecting small effective population size (N-e). Meanwhile, N-e and mutation load estimates are lower in Cyprian compared with Anatolian mouflons, suggesting the purging of recessive deleterious variants in Cyprian sheep under a small long-term N-e, possibly attributable to founder effects, island isolation, introgression from domestic lineages, or differences in their bottleneck dynamics. Expanding our analyses to worldwide wild and feral Ovis genomes, we observe varying viability metrics among different lineages and a limited consistency between viability metrics and International Union for Conservation of Nature conservation status. Factors such as recent inbreeding, introgression, and unique population dynamics may have contributed to the observed disparities.

We present palaeogenomes of three morphologically unidentified Anatolian equids dating to the first millennium BCE, sequenced to a coverage 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, secular name 'hydruntine'. Further, the Anatolian wild ass whole genome profiles fell outside the genomic diversity of other extant and past Asiatic wild ass (E. hemionus) lineages. These observations suggest that the three Anatolian wild asses represent hydruntines, making them the latest recorded survivors of this lineage, about a millennium later than the latest observations in the zooarchaeological record. Our mitogenomic and genomic analyses indicate that E. h. hydruntinus was a clade belonging to ancient and present-day E. hemionus lineages that radiated possibly between 0.6 and 0.8 Mya. We also find evidence consistent with recent gene flow between hydruntines and Middle Eastern wild asses. Analyses of genome-wide heterozygosity and runs of homozygosity suggest that the Anatolian wild ass population may have lost genetic diversity by the mid-first millennium BCE, a possible sign of its eventual demise.

Sheep are among the earliest domesticated livestock species, with a wide variety of breeds present today. However, it remains unclear how far back this diversity goes, with formal documentation only dating back a few centuries. North European short-tailed (NEST) breeds are often assumed to be among the oldest domestic sheep populations, even thought to represent relicts of the earliest sheep expansions during the Neolithic period reaching Scandinavia <6,000 years ago. This study sequenced the genomes (up to 11.6X) of five sheep remains from the Baltic islands of Gotland and Åland, dating from the Late Neolithic (∼4,100 cal BP) to historical times (∼1,600 CE). Our findings indicate that these ancient sheep largely possessed the genetic characteristics of modern NEST breeds, suggesting a substantial degree of long-term continuity of this sheep type in the Baltic Sea region. Despite the wide temporal spread, population genetic analyses show high levels of affinity between the ancient genomes and they also exhibit relatively high genetic diversity when compared to modern NEST breeds, implying a loss of diversity in most breeds during the last centuries associated with breed formation and recent bottlenecks. Our results shed light on the development of breeds in Northern Europe specifically as well as the development of genetic diversity in sheep breeds, and their expansion from the domestication center in general.

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.

We present a spatiotemporal picture of human genetic diversity in Anatolia, Iran, Levant, South Caucasus, and the Aegean, a broad region that experienced the earliest Neolithic transition and the emergence of complex hierarchical societies. Combining 35 new ancient shotgun genomes with 382 ancient and 23 present-day published genomes, we found that genetic diversity within each region steadily increased through the Holocene. We further observed that the inferred sources of gene flow shifted in time. In the first half of the Holocene, Southwest Asian and the East Mediterranean populations homogenized among themselves. Starting with the Bronze Age, however, regional populations diverged from each other, most likely driven by gene flow from external sources, which we term "the expanding mobility model." Interestingly, this increase in inter-regional divergence can be captured by outgroup-f3-based genetic distances, but not by the commonly used FST statistic, due to the sensitivity of FST, but not outgroup-f3, to within-population diversity. Finally, we report a temporal trend of increasing male bias in admixture events through the Holocene.