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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. Key words: biomonitoring, high-throughput sequencing, COI metabarcoding, young-of-year, ichthyoplankton, fish identification, Introduction Early life stages of fishes (egg, larval, fry, and juvenile life stages) generally occur in greater abundances than adults, making them excellent candidates for biomonitoring (Lasker 1987). Ichthyoplankton surveys [...]

Environmental DNA (eDNA) metabarcoding has the potential to significantly improve surveys of biodiversity in freshwater systems. However, this methodology is still infrequently used in global hotspots of endemic species, in part because of two major barriers to the success of eDNA metabarcoding: (1) insufficient regional taxonomic representation in public reference sequence databases; and (2) inconsistent species incidence and abundance estimates when compared to conventional surveys.We sampled eDNA and conducted visual surveys in the headwaters of two rivers in the Cape Fold aquatic ecoregion, South Africa. A reference sequence database was generated for the regional diversity of fishes to improve taxonomic classification of endemic species. We also compared the consistency of incidence data and relative abundance estimates of fishes from eDNA metabarcoding sequencing results and visual surveys (snorkel and underwater cameras) at each site sampled.Only 1% of eDNA metabarcoding reads could be classified to fish species without the supplementation of reference sequence databases for local endemic species. Once regional reference sequences were added, a total of nine species were detected and >99% reads classified. A strong positive relationship (Φ = 0.75) was observed between the patterns of detections using eDNA and visual approaches. However, eDNA metabarcoding detected more species than visual methods. In addition, the relative read frequency and abundance observed in visual surveys was significantly correlated (R2 = 0.85–0.88, p < 0.001) in the majority of the small pools surveyed.Incomplete public reference sequence databases hinder the use of eDNA metabarcoding in regions of high endemicity. Local reference sequence libraries can overcome these challenges. When appropriately implemented, eDNA metabarcoding shows promise, producing results consistent or better than the more frequently used and labour‐intensive visual surveys.Efficient survey methods like eDNA metabarcoding are urgently needed to improve aquatic monitoring efforts globally. Understanding how eDNA metabarcoding sequencing results relate to conventional survey methods is a key step in its implementation in ecoregions with high endemicity. [ABSTRACT FROM AUTHOR]