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.