Detection of Multiple Fish Species Using Environmental DNA (eDNA), Alaska 2018

A value of "Salvelinus sp.3" as a species in the nativeFisheDNAReferenceList_alaska_Menning_2020.csv references a sequence of DNA found in multiple Salvelinus species and was used only to differentiate all Salvelinus sequences from others. Accession number KR733409.1 is listed in Genbank and here as Coregonus clupeaformis, but referred to as C. pidschian in Menning et al 2018 following Brown et al 2012. Some sequences included in nativeFisheDNAReferenceList_alaska_Menning_2020.csv are not known to be in Alaskan waters, but were considered best proxies for known species lacking genetic data in Genbank at the time of reference list preparation (GQ267192.1,GQ267193.1,GQ267194.1,EU186785.1,EU186787.1,EU186792.1,EU186798.1,EU186800.1,AY886762.1,EU186799.1,KP013117.1,and EU186801.1). Unexpected detection of some species given known range, habitat preference and/or documented presence for our sampling locations occurred in some miSeq runs. Specifically, Oncorhynchus mykiss and Salvelinus alpinus were not documented to occur in the UAF Aquarium control site but were only detected using Blast+ and not the Exact match algorithm and while Cottus cognatus was present in the aquarium, a 12S sequence matching C. aleuticus was detected indicating 12S primers may only be specific to the Genus level for Cottus without more specific sampling location reference sequences; unexpected detections were observed, including C. clupeaformis in Hogan Creek, Esox lucius and Pungitius pungitius in Igloo Creek, and O. mykiss in Lake INI-003. Based upon their habitat preferences and known species ranges, it is unlikely that eDNA from these species was actually present in these systems. In every case, these unexpected species were only detected in a single sample or MiSeq run. Conversely, some species detected, though undocumented in traditional species surveys for the locations, are believed to be valid given detection across multiple samples and/or sequencing runs and known habitat preferences. These presumed probable detections include: For the Arctic coastal plain lakes, C. cognatus, Lota lota, C. nasus, Dalia pectoralis, S. namaycush, S. malma, S. alpinus, P. cylindraceum, Catostomus catostomus, and T. arcticus have all been reported either in other nearby lakes from the current study (Matthew Whitman, Bureau of Land Management, pers. comm.) or from other studies conducted in the area (Mecklenburg et al 2002; Haynes et al 2014; Laske et al 2016). This amplicon based environmental DNA (eDNA) study was designed with a targeted list of species of interest and was not intended to identify all species present at each sampling location. Given design parameters and method limitations, it is possible unassigned DNA sequences represent previously undocumented variants of expected/detected species, represent species previously undocumented, or represent sequencing errors intrinsic in the laboratory method. It is expected that not all species present in the eDNA sample were detected.