Dire wolves were the last of an ancient New World canid lineage.

Dire wolves are considered to be one of the most common and widespread large carnivores in Pleistocene America1, yet relatively little is known about their evolution or extinction. Here, to reconstruct the evolutionary history of dire wolves, we sequenced five genomes from sub-fossil remains dating from 13,000 to more than 50,000 years ago. Our results indicate that although they were similar morphologically to the extant grey wolf, dire wolves were a highly divergent lineage that split from living canids around 5.7 million years ago. In contrast to numerous examples of hybridization across Canidae2,3, there is no evidence for gene flow between dire wolves and either North American grey wolves or coyotes. This suggests that dire wolves evolved in isolation from the Pleistocene ancestors of these species. Our results also support an early New World origin of dire wolves, while the ancestors of grey wolves, coyotes and dholes evolved in Eurasia and colonized North America only relatively recently.

Dogs accompanied humans during the Neolithic expansion into Europe.

Near Eastern Neolithic farmers introduced several species of domestic plants and animals as they dispersed into Europe. Dogs were the only domestic species present in both Europe and the Near East prior to the Neolithic. Here, we assessed whether early Near Eastern dogs possessed a unique mitochondrial lineage that differentiated them from Mesolithic European populations. We then analysed mitochondrial DNA sequences from 99 ancient European and Near Eastern dogs spanning the Upper Palaeolithic to the Bronze Age to assess if incoming farmers brought Near Eastern dogs with them, or instead primarily adopted indigenous European dogs after they arrived. Our results show that European pre-Neolithic dogs all possessed the mitochondrial haplogroup C, and that the Neolithic and Post-Neolithic dogs associated with farmers from Southeastern Europe mainly possessed haplogroup D. Thus, the appearance of haplogroup D most probably resulted from the dissemination of dogs from the Near East into Europe. In Western and Northern Europe, the turnover is incomplete and haplogroup C persists well into the Chalcolithic at least. These results suggest that dogs were an integral component of the Neolithic farming package and a mitochondrial lineage associated with the Near East was introduced into Europe alongside pigs, cows, sheep and goats. It got diluted into the native dog population when reaching the Western and Northern margins of Europe.

Natural and human-driven selection of a single non-coding body size variant in ancient and modern canids.

Domestic dogs (Canis lupus familiaris) are the most variable-sized mammalian species on Earth, displaying a 40-fold size difference between breeds.1 Although dogs of variable size are found in the archeological record,2-4 the most dramatic shifts in body size are the result of selection over the last two centuries, as dog breeders selected and propagated phenotypic extremes within closed breeding populations.5 Analyses of over 200 domestic breeds have identified approximately 20 body size genes regulating insulin processing, fatty acid metabolism, TGFß signaling, and skeletal formation.6-10 Of these, insulin-like growth factor 1 (IGF1) predominates, controlling approximately 15% of body size variation between breeds.8 The identification of a functional mutation associated with IGF1 has thus far proven elusive.6,10,11 Here, to identify and elucidate the role of an ancestral IGF1 allele in the propagation of modern canids, we analyzed 1,431 genome sequences from 13 species, including both ancient and modern canids, thus allowing us to define the evolutionary history of both ancestral and derived alleles at this locus. We identified a single variant in an antisense long non-coding RNA (IGF1-AS) that interacts with the IGF1 gene, creating a duplex. While the derived mutation predominates in both modern gray wolves and large domestic breeds, the ancestral allele, which predisposes to small size, was common in small-sized breeds and smaller wild canids. Our analyses demonstrate that this major regulator of canid body size nearly vanished in Pleistocene wolves, before its recent resurgence resulting from human-imposed selection for small-sized breed dogs.

The evolution of dog diet and foraging: Insights from archaeological canids in Siberia.

Research on the evolution of dog foraging and diet has largely focused on scavenging during their initial domestication and genetic adaptations to starch-rich food environments following the advent of agriculture. The Siberian archaeological record evidences other critical shifts in dog foraging and diet that likely characterize Holocene dogs globally. By the Middle Holocene, body size reconstruction for Siberia dogs indicates that most were far smaller than Pleistocene wolves. This contributed to dogs' tendencies to scavenge, feed on small prey, and reduce social foraging. Stable carbon and nitrogen isotope analysis of Siberian dogs reveals that their diets were more diverse than those of Pleistocene wolves. This included habitual consumption of marine and freshwater foods by the Middle Holocene and reliance on C4 foods by the Late Holocene. Feeding on such foods and anthropogenic waste increased dogs' exposure to microbes, affected their gut microbiomes, and shaped long-term dog population history.

Stable isotopes reveal diet shift from pre-extinction to reintroduced Przewalski's horses.

The Przewalski's horse (Equus ferus przewalskii), the only remaining wild horse within the equid family, is one of only a handful of species worldwide that went extinct in the wild, was saved by captive breeding, and has been successfully returned to the wild. However, concerns remain that after multiple generations in captivity the ecology of the Przewalski's horse and / or the ecological conditions in its former range have changed in a way compromising the species' long term survival. We analyzed stable isotope chronologies from tail hair of pre-extinction and reintroduced Przewalski's horses from the Dzungarian Gobi and detected a clear difference in the isotopic dietary composition. The direction of the dietary shift from being a mixed feeder in winter and a grazer in summer in the past, to a year-round grazer nowadays, is best explained by a release from human hunting pressure. A changed, positive societal attitude towards the species allows reintroduced Przewalski's horses to utilize the scarce, grass-dominated pastures of the Gobi alongside local people and their livestock whereas their historic conspecifics were forced into less productive habitats dominated by browse.

Spondylosis deformans in three large canids from the Gravettian Predmostí site: Comparison with other canid populations.

Spondylosis deformans is a common degenerative condition of the spinal column, especially in modern domestic dogs. The presence and severity of lesions are related to age and physical activity, but they can be influenced by genetics, with some modern breeds being particularly predisposed. Spondylosis deformans also has been reported in prehistoric dogs. Here, we describe three affected vertebrae, likely from three individuals, in a large canid assemblage from the Gravettian Predmostí site, Czech Republic. We compare the proportion of affected individuals from the Predmostí assemblage with that of affected individuals among recent wild Northern wolves, inbred wolves, captive wolves, and recent Northern dogs. The proportion of affected individuals among the captive wolves differs significantly from the other wolf groups. The proportion in the Predmostí assemblage does not differ significantly from that of the wild wolf groups.

Amy2B copy number variation reveals starch diet adaptations in ancient European dogs.

Extant dog and wolf DNA indicates that dog domestication was accompanied by the selection of a series of duplications on the Amy2B gene coding for pancreatic amylase. In this study, we used a palaeogenetic approach to investigate the timing and expansion of the Amy2B gene in the ancient dog populations of Western and Eastern Europe and Southwest Asia. Quantitative polymerase chain reaction was used to estimate the copy numbers of this gene for 13 ancient dog samples, dated to between 15 000 and 4000 years before present (cal. BP). This evidenced an increase of Amy2B copies in ancient dogs from as early as the 7th millennium cal. BP in Southeastern Europe. We found that the gene expansion was not fixed across all dogs within this early farming context, with ancient dogs bearing between 2 and 20 diploid copies of the gene. The results also suggested that selection for the increased Amy2B copy number started 7000 years cal. BP, at the latest. This expansion reflects a local adaptation that allowed dogs to thrive on a starch rich diet, especially within early farming societies, and suggests a biocultural coevolution of dog genes and human culture.

Genomic and archaeological evidence suggest a dual origin of domestic dogs.

The geographic and temporal origins of dogs remain controversial. We generated genetic sequences from 59 ancient dogs and a complete (28x) genome of a late Neolithic dog (dated to ~4800 calendar years before the present) from Ireland. Our analyses revealed a deep split separating modern East Asian and Western Eurasian dogs. Surprisingly, the date of this divergence (~14,000 to 6400 years ago) occurs commensurate with, or several millennia after, the first appearance of dogs in Europe and East Asia. Additional analyses of ancient and modern mitochondrial DNA revealed a sharp discontinuity in haplotype frequencies in Europe. Combined, these results suggest that dogs may have been domesticated independently in Eastern and Western Eurasia from distinct wolf populations. East Eurasian dogs were then possibly transported to Europe with people, where they partially replaced European Paleolithic dogs.

Burying dogs in ancient Cis-Baikal, Siberia: temporal trends and relationships with human diet and subsistence practices.

The first objective of this study is to examine temporal patterns in ancient dog burials in the Lake Baikal region of Eastern Siberia. The second objective is to determine if the practice of dog burial here can be correlated with patterns in human subsistence practices, in particular a reliance on terrestrial mammals. Direct radiocarbon dating of a suite of the region's dog remains indicates that these animals were given burial only during periods in which human burials were common. Dog burials of any kind were most common during the Early Neolithic (∼7-8000 B.P.), and rare during all other time periods. Further, only foraging groups seem to have buried canids in this region, as pastoralist habitation sites and cemeteries generally lack dog interments, with the exception of sacrificed animals. Stable carbon and nitrogen isotope data indicate that dogs were only buried where and when human diets were relatively rich in aquatic foods, which here most likely included river and lake fish and Baikal seal (Phoca sibirica). Generally, human and dog diets appear to have been similar across the study subregions, and this is important for interpreting their radiocarbon dates, and comparing them to those obtained on the region's human remains, both of which likely carry a freshwater old carbon bias. Slight offsets were observed in the isotope values of dogs and humans in our samples, particularly where both have diets rich in aquatic fauna. This may result from dietary differences between people and their dogs, perhaps due to consuming fish of different sizes, or even different tissues from the same aquatic fauna. This paper also provides a first glimpse of the DNA of ancient canids in Northeast Asia.