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Combined use of sedimentary DNA (sedDNA) and Indigenous Knowledge in reconstructing historical fish records. [Display omitted] • Indigenous peoples have a long-term link with Cowpar Lake in the Canadian Oil Sands. • Sedimentary DNA (sedDNA) from sediment core acts as an archive of lake history. • qPCR-based environmental DNA methods reconstructed historical fish occupation. • SedDNA results aligned with Indigenous Knowledge (IK) on fish communities. • Combined sedDNA and IK give powerful insight into natural and human-mediated impact. Lake sediment serves as a natural archive of historical biological information. The use of sedimentary DNA (sedDNA), a form of environmental DNA (eDNA) shed by aquatic organisms and preserved in sediment, has been instrumental in reconstructing past faunal composition in aquatic communities. However, the low abundance of fish sedDNA and the often humic-rich nature of lake sediments create methodological challenges for the accurate detection of target sedDNA using quantitative polymerase chain reaction (qPCR)-based approaches. Herein, we present a consolidated qPCR-based eDNA workflow to reconstruct past and current fish fauna in Cowpar Lake located in the Oil Sands region in Alberta (Canada), which were then validated using Indigenous Knowledge from Chipewyan Prairie First Nation community members. The present study highlights the importance of combining column- and precipitation-based PCR inhibitor clean-up, nucleic acid concentration, incorporating endogenous chloroplast DNA as a sample integrity control. Robust qPCR-based eDNA assays were also useful in preventing the false-negative detection of low copies of target fish DNA. The presence of Northern pike (1905 to 2019) and Cisco (1919 to 1942) in Cowpar Lake was confirmed based on detected sedDNA from sediment core. The reconstructed fish records from sedDNA-inferred data aligned with the Indigenous accounts of natural and human-mediated changes in land use around the lake. Overall, the present study addresses common methodological concerns in processing lake sediment samples for fish eDNA detection and demonstrates the great potential of combined eDNA-inferred data and Indigenous Knowledge in reconstructing historical fish records in aquatic communities. [ABSTRACT FROM AUTHOR]