Received 27 Feb 2017 | Accepted 25 May 2017 | Published 18 Jul 2017
Ancient European dog genomes reveal continuitysince the Early NeolithicLaura R. Botigue1,*, Shiya Song2,*, Amelie Scheu3,4,*, Shyamalika Gopalan1, Amanda L. Pendleton5,
Matthew Oetjens5, Angela M. Taravella5, Timo Seregely6, Andrea Zeeb-Lanz7, Rose-Marie Arbogast8,
Dean Bobo1, Kevin Daly4, Martina Unterlander3, Joachim Burger3, Jeffrey M. Kidd2,5 & Krishna R. Veeramah1
Europe has played a major role in dog evolution, harbouring the oldest uncontested
Palaeolithic remains and having been the centre of modern dog breed creation. Here we
sequence the genomes of an Early and End Neolithic dog from Germany, including a sample
associated with an early European farming community. Both dogs demonstrate continuity
with each other and predominantly share ancestry with modern European dogs, contradicting
a previously suggested Late Neolithic population replacement. We find no genetic evidence to
support the recent hypothesis proposing dual origins of dog domestication. By calibrating
the mutation rate using our oldest dog, we narrow the timing of dog domestication to
20,00040,000 years ago. Interestingly, we do not observe the extreme copy number
expansion of the AMY2B gene characteristic of modern dogs that has previously been
proposed as an adaptation to a starch-rich diet driven by the widespread adoption of
agriculture in the Neolithic.
DOI: 10.1038/ncomms16082 OPEN
1 Department of Ecology and Evolution, Stony Brook University, Stony Brook, New York 11794-5245, USA. 2 Department of Computational Medicine andBioinformatics, University of Michigan, Ann Arbor, Michigan 48109, USA. 3 Palaeogenetics Group, Johannes Gutenberg-University Mainz, 55099 Mainz,Germany. 4 Smurfit Institute of Genetics, Trinity College Dublin, Dublin 2, Ireland. 5 Department of Human Genetics, University of Michigan, Ann Arbor,Michigan 48109, USA. 6 Department of Prehistoric Archaeology, Institute of Archaeology, Heritage Sciences and Art History, University of Bamberg,96045 Bamberg, Germany. 7 Generaldirektion Kulturelles Erbe Rheinland-Pfalz, Direktion Landesarchaologie, Auenstelle Speyer, 67346 Speyer, Germany.8 CNRS UMR 7044-UDS, 5 Allee du General Rouvillois F 67083 Strasbourg, France. * These authors contributed equally to this work. Correspondence andrequests for materials should be addressed to K.R.V. (email: firstname.lastname@example.org).
NATURE COMMUNICATIONS | 8:16082 | DOI: 10.1038/ncomms16082 | www.nature.com/naturecommunications 1
Europe has been a critically important region in the historyand evolution of dogs, with most modern breeds sharingpredominantly European ancestry1. Furthermore, the oldest
remains that can be unequivocally attributed to domestic dogs(Canis lupus familiaris) are found on this continent, includingan Upper Palaeolithic 14,700-year-old jaw-bone from theBonnOberkassel site in Germany2 (older specimens fromSiberia and the Near East that have been proposed remainhighly controversial3,4). While ancient mitochondrial DNA(mtDNA) suggests a European centre of dog domestication5,analyses of mitochondrial and genomic data from modern dogshave suggested East Asia6,7, the Middle East8 and Central Asia9.
The Neolithic period in Central Europe ranges from B7,500 to4,000 BP and can be further subdivided based on specific featuresof human culture10 (Supplementary Table 1). Multiple studies havefound evidence of a prehistoric turnover of canid mtDNA lineagessometime between the Late Neolithic and today, with haplogroupC, which appears in almost all Neolithic dogs but in less than 10%of modern dogs, being replaced by haplogroup A in most ofEurope5,11,12. By analyzing genomic data from modern dogs and aLate Neolithic (B5,000 years old) Irish dog from Newgrange(hereafter referred to as NGD), Frantz et al.12 argue that thismatrilineal turnover was a consequence of a major populationreplacement during the Neolithic. However, NGD primarily sharesancestry with modern European dogs, implying the proposedpopulation replacement had largely already occurred beforethis individual lived. Frantz et al.12 also estimate a relativelyrecent eastwest dog divergence (14,0006,000 years ago), which,placed within the context of existing archaeological data, theyexplain with a dual origin of dog domestication.
The characterization of samples from earlier in the Neolithicand from continental Europe is necessary to examine whetherand to what extent a large-scale demographic replacementoccurred during this period. This would be evidenced by adistinct ancestry absent in modern dog genomes that was moreprominent in dogs from earlier in the Neolithic, as opposed togenomic continuity from the Early Neolithic to today. Therefore,we present analysis of B9 coverage whole genomes of two dogsamples from Germany dating to the Early and End Neolithic(B7,000 years old and B4,700 years old, respectively). Weobserve genetic continuity throughout this era and into thepresent, with our ancient dogs sharing substantial ancestry withmodern European dogs. We find no evidence of a majorpopulation replacement; instead, our results are consistent witha scenario where modern European dogs emerged from astructured Neolithic population. Furthermore, we detect anadditional ancestry component in the End Neolithic sample,consistent with admixture from a population of dogs locatedfurther east that may have migrated concomitant with steppepeople associated with Late Neolithic and Early Bronze agecultures, such as the Yamnaya and Corded Ware culture13. Wealso show that most autosomal haplotypes associated withdomestication were already established in our Neolithic dogs,but that adaptation to a starch-rich diet likely occurred later.Finally, we obtain divergence estimates between Eastern andWestern dogs of 17,00024,000 years ago, consistent with a singlegeographic origin for domestication, the timing of which wenarrow down to between B20,000 and 40,000 years ago.
ResultsArchaeological samples and ancient DNA sequencing.The older specimen, which we refer to hereafter as HXH, wasfound at the Early Neolithic site of Herxheim and is dated to5,2235,040 cal. BCE (B7,000 years old) (Supplementary Fig. 1).The younger specimen, which we refer to hereafter as CTC, was
found in Cherry Tree Cave and is dated to 2,9002,632 cal. BCE(B4,700 years old), which corresponds to the End Neolithicperiod in Central Europe14 (Supplementary Fig. 2 andSupplementary Notes 13).
We generated whole-genome sequence data for the two ancientdogs and mapped over 67% of the reads to the dog referencegenome (CanFam3.1), confirming high endogenous canine DNAcontent for both samples (Supplementary Table 2 andSupplementary Note 4). MapDamage15 analysis demonstratedthat both samples possess damage characteristics typical ofancient DNA16 (Supplementary Fig. 3). The final mean coveragefor both samples was B9 , while coverage on the X and Ychromosome was B5 , indicating they both are male. We alsoreprocessed the NGD data12 using the same pipeline as for CTCand HXH. To call variants, we used a custom genotype callerimplemented in Python (see Supplementary Note 5) thataccounts for DNA damage patterns17. We found that ourapproach eliminated many false positives that are likely due topostmortem damage (Supplementary Fig. 4).
Modern canid reference data sets. We analysed these Neolithicdogs within the context of a comprehensive collection of5,649 canids, including breed dogs, village dogs and wolves thathad been previously genotyped at 128,743 single nucleotidepolymorphisms (SNPs)9,18 (Supplementary Table 3), as well as99 canid whole genomes sequenced at medium to high coverage(645) (Supplementary Table 4). To account for biases in variantcalling that might occur as a result of this variable coverage, weascertained variable sites in an outgroup (such that mutations areknown to have occurred in the root of all the populations beinganalysed). We explored different ascertainment schemes for thewhole-genome data (Supplementary Note 6) and chose to use a callset that includes sites variable in New World wolves (we note,though, that our primary results are robust to changes in theascertainment scheme). This call set contains 1,815,911 variantsthat are likely either private to New World wolves or arose in thegrey wolf (Canis lupus) ancestral population, and thus is the leastbiased with regard to their ascertainment in Old World wolves anddogs.
mtDNA analysis. We examined the phylogenetic relationshipof the entire mitochondrial genomes of HXH and CTC with acomprehensive panel of modern dogs across four major clades(AD), modern wolves and coyotes, and previously reported ancientwolf-like and dog-like whole mitochondrial sequences5,12. Like otherEuropean Neolithic dogs, both HXH and CTC belong to haplogroupC (Fig. 1a, Supplementary Fig. 5 and Supplementary Note 7)together with NGD and the Upper Palaeolithic 12,500-year-oldKartstein Cave dog (also from Germany). We note thatBonnOberkassel also falls in the same haplogroup5 (Suppleme-ntary Figs 6 and 7), although analysis of this sample is complicatedby low mtDNA sequence coverage, pointing to some degree ofmatrilineal continuity in Europe over B10,000 years, ranging fromthe Late Palaeolithic to almost the entire Neolithic. The inclusion of24 additional clade C samples19 in the phylogenetic analysis revealsthe expected C1 and C2 split (100% support) and that HXH, CTC,NGD and the Kartstein Cave dog share a common lineage with C1dogs (Supplementary Fig. 8). This topology suggests that theseancient European dogs belong to an older sub-haplogroup that issister to the progenitor of the C1b and C1a sub-haplogroups andpossibly absent in modern dog