Estimating the genetic diversity of Pacific salmon and trout using multigene eDNA metabarcoding

Genetic diversity underpins species conservation and management goals, and ultimately determines a species' ability to adapt. Using freshwater environmental DNA (eDNA) samples, we examined mitochondrial genetic diversity using multigene metabarcode sequence data from four Oncorhynchus species across 16 sites in Oregon and northern California. Our multigene metabarcode panel included targets commonly used in population genetic NADH dehydrogenase 2 (ND2), phylogenetic cytochrome c oxidase subunit 1 (COI) and eDNA (12S ribosomal DNA) screening. The ND2 locus showed the greatest within-species haplotype diversity for all species, followed by COI and then 12S rDNA for all species except Oncorhynchus kisutch. Sequences recovered for O. clarkii clarkii were either identical to, or one mutation different from, previously characterized haplotypes (95.3% and 4.5% of reads, respectively). The greatest diversity in O. c. clarkii was among coastal watersheds, and subsets of this diversity were shared with fish in inland watersheds. However, coastal streams and the Umpqua River watershed appear to harbour unique haplotypes. Sequences from O. mykiss revealed a disjunction between the Willamette watershed and southern watersheds suggesting divergent histories. We also identified similarities between populations in the northern Deschutes and southern Klamath watersheds, consistent with previously hypothesized connections between the two via inland basins. Oncorhynchus kisutch was only identified in coastal streams and the Klamath River watershed, with most diversity concentrated in the coastal Coquille watershed. Oncorhynchus tshawytscha was only observed at one site, but contained multiple haplotypes at each locus. The characterization of genetic diversity at multiple loci expands the knowledge gained from eDNA sampling and provides crucial information for conservation actions and genetic management.