Detection of early life stages of fishes is important for understanding life history patterns and critical spawning habitats. When feasible, identifying early life stages of fishes using morphology requires taxonomic expertise and can be challenging, time consuming, and imprecise. In this study, we used DNA metabarcoding to identify egg and larval batch samples from two sites in the species-rich East Sydenham River, Ontario, Canada. We used a two-step PCR metabarcoding approach to amplify a highly variable region of the mitochondrial COI gene from 1075 mixed species batch samples. Amplicon libraries were sequenced with Illumina Mi-seq and the sequencing reads were filtered and assembled using the software package mothur. Barcodes were then classified using a reference library comprised of Great Lakes fishes and potential invaders. In total, 34 species, including three at-risk species and three invasive species, were detected at the two sampling sites. This study shows the potential utility of metabarcoding for detection and identification of early life stage Great Lake fishes.

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