GoKawiilRadiocarbon and contextual dating
We generated eleven new radiocarbon dates (Supplementary Data 2). Dates were calibrated using the IntCal20 calibration curve, using the OxCal v.4.4 software. For samples where δ¹³C values were obtained independently of accelerator mass spectrometry using high-precision stable-isotope mass spectrometry—providing dietary information—no high values indicative of potential marine diets were observed, and therefore no corrections for marine reservoir effect were applied. The previously published C88 dog from the site of Frälsegården, Gökhem in southwestern Sweden had been attributed to a Neolithic cultural context and an approximate age of 5,000 years bp13, but the new radiocarbon date of 1,154 calibrated years bp instead places it in the Late Iron Age or Viking Age.
For remains that did not have radiocarbon dates, we assigned dates usable in analyses on the basis of available archaeological context. The ‘Analysis date’ column in Supplementary Data 3 holds the best available single number date estimate, with a value of ‘NA’ if we were not able to confidently assign a date.
Sampling, DNA extractions and library preparation
When sampling skeletal remains for DNA, in most instances we aimed to take one sample per biological individual. In some instances, samples were obtained from different remains that possibly could derive from the same individual, based on the zooarchaeological context (in particular this was the case for the Kesslerloch site). In a few cases in which it was unambiguous that data from multiple remains came from the same individual, we merged those data. But in most cases, we treated data from different remains separately, to err on the side of caution and not incorrectly merge data. The true number of sampled individuals is thus not precisely known, but we provide a table listing the groups of remains where archaeological information suggest they might derive from the same individual (Supplementary Data 5).
University of Tübingen
A number of samples had already been processed in the cleanroom facilities of the University of Tübingen and analysed for DNA preservation, as previously described47. In brief, samples were subjected to UV radiation for 30 min, followed by DNA extraction48 and double-stranded library preparation49. For the current project, these double-stranded libraries were transferred to the Max Planck Institute for Evolutionary Anthropology, where they were enriched for targeted canid single nucleotide polymorphisms using an in-solution capture as described below.
Max Planck Institute
Material from the Kesslerloch site was sampled and processed into powder at the cleanroom facilities of the Institute of Evolutionary Medicine, University of Zurich. All bone and teeth were decontaminated via UV irradiation for 30 min in a cross-linker on at least two sides before drilling. For some specimens, the external surface was lightly ablated by hand with a dentist drill to access more sterile tissue. Samples were then drilled using a dentist drill with sterilized bits to produce powder. The powders were then transferred to the Max Planck Institute for Evolutionary Anthropology for further processing. Three samples of the Senckenberg dog (a vertebra, a foot phalanx and a rib), and 25 new specimens from Gnirshöhle, were sampled in the designated clean room laboratories at the Max Planck Institute for Evolutionary Anthropology, in which they were decontaminated under ultraviolet irradiation for 30 min on each side and drilled with a sterile dentist drill to produce bone powder. DNA extractions for all of the aforementioned samples were then performed at the Max Planck Institute.
We extracted DNA from between 6 mg and 104 mg of the powdered sample applying a silica-based method optimized to recover short DNA fragments48,50. In brief, lysates were prepared by adding 1 ml lysis buffer (0.45 M EDTA at pH 8.0, 0.25 mg ml–1 proteinase K, 0.05% Tween-20) to the sample material in a sterile 2.0 ml BioPure Eppendorf tube, which was rotated at 37 °C overnight. DNA was purified from 125 µl of the lysate using silica-coated magnetic beads and the binding buffer D (ref. 50) on an automated liquid-handling system Bravo NGS Workstation B (Agilent Technologies). Purified DNA extracts were eluted in 30 µl Tris–EDTA–Tween buffer (TET). Extraction blanks without sample material were processed with the samples.
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