Ground squirrel coprolites preserve complex archives of ancient environmental DNA over 700,000 years

Permafrost-preserved ground squirrel (Urocitellus) burrows in Yukon, Canada contain coprolites (palaeofaeces) that span from the Holocene to at least the Middle Pleistocene (~700 kya). Using shotgun metagenomics and targeted enrichment, we recover a rich, multi-taxon spectrum of ancient environmental DNA from these pellets, including: plants, insects, microbes, and megafauna consistent with eastern Beringian ecosystems. These coprolites consistently preserve an abundance of eukaryotic DNA, enabling the assembly of >18 mitochondrial genomes (ground squirrel, snowshoe hare, steppe bison, horse, and mammoth), and revealing previously unrecognized diversity within Arctic Urocitellus, including a ~700 kya lineage that predates divergence among several extant clades. Characteristic damage patterns, positive/negative controls, and in silico taxon validations strongly support aDNA authenticity, and comparisons with regional permafrost datasets indicate minimal post-depositional leaching. These results show that permafrost coprolites can yield high-resolution records of Quaternary ecosystems and multi-organism population histories, providing a powerful complement to sedimentary and skeletal ancient DNA. Ancient faeces are powerful archives of ancient environmental DNA with the potential to illuminate both ecosystem history and evolution. This study shows that arctic ground squirrel droppings from the Yukon preserve the frozen biomolecular records of entire ecosystems over hundreds of thousands of years.