ERP126587 PRJEB42692 6ce94cda-d128-4057-9cdc-cfe6d5f2e4fc Environmental Genomics of Late Pleistocene Black Bears and Giant Short-Faced Bears - eDNA Analysis of ancient environmental DNA (eDNA) has revolutionized our ability to describe biological communities in space and time1–3, by allowing for parallel sequencing of DNA from all trophic levels4–8. However, because environmental samples contain sparse and fragmented data from multiple individuals, and often contain closely related species 9, the field of ancient eDNA has so far been limited to organellar genomes in its contribution to population and phylogenetic studies5,6,10,11. This is in contrast to data from fossils12,13 where full-genome studies are routine, despite these being rare and their destruction for sequencing undesirable14–16. Here, we report the retrieval of three low coverage (0.03x) environmental genomes from American black bear (Ursus americanus) and a 0.04x environmental genome of an extinct giant short-faced bear (Arctodus simus) from cave sediment samples from northern Mexico dated to 16-14 thousand calibrated years before present (cal kyr BP), which we contextualize with a new high coverage (26x) and two lower coverage giant short-faced bear genomes obtained from fossils recovered from Yukon Territory, Canada which date to ~22-50 cal kyr BP. We show that the Late Pleistocene black bear population in Mexico is ancestrally related to the present day eastern American black bear population, and that the extinct giant short-faced bears present in Mexico were deeply divergent from the earlier Beringian population. Our findings demonstrate the ability to separately analyse genomic-scale DNA sequences of closely related species co-preserved in environmental samples, which brings the use of ancient eDNA into the era of population genomics and phylogenetics. Environmental Genomics Analysis of ancient environmental DNA (eDNA) has revolutionized our ability to describe biological communities in space and time1–3, by allowing for parallel sequencing of DNA from all trophic levels4–8. However, because environmental samples contain sparse and fragmented data from multiple individuals, and often contain closely related species 9, the field of ancient eDNA has so far been limited to organellar genomes in its contribution to population and phylogenetic studies5,6,10,11. This is in contrast to data from fossils12,13 where full-genome studies are routine, despite these being rare and their destruction for sequencing undesirable14–16. Here, we report the retrieval of three low coverage (0.03x) environmental genomes from American black bear (Ursus americanus) and a 0.04x environmental genome of an extinct giant short-faced bear (Arctodus simus) from cave sediment samples from northern Mexico dated to 16-14 thousand calibrated years before present (cal kyr BP), which we contextualize with a new high coverage (26x) and two lower coverage giant short-faced bear genomes obtained from fossils recovered from Yukon Territory, Canada which date to ~22-50 cal kyr BP. We show that the Late Pleistocene black bear population in Mexico is ancestrally related to the present day eastern American black bear population, and that the extinct giant short-faced bears present in Mexico were deeply divergent from the earlier Beringian population. Our findings demonstrate the ability to separately analyse genomic-scale DNA sequences of closely related species co-preserved in environmental samples, which brings the use of ancient eDNA into the era of population genomics and phylogenetics. ENA-FIRST-PUBLIC 2022-03-01 ENA-LAST-UPDATE 2022-03-01