ABSTRACT Environmental DNA (eDNA) metabarcoding is widely used to detect animals from environmental samples and on the brink of being implemented into routine species monitoring. Surprisingly, birds are among the taxonomic groups which have received comparably little attention in eDNA research regarding primer optimization—particularly reducing the amplification of non‐target taxa, the availability of appropriate reference sequence databases for the targeted fragments, and the evaluation of different filter types for their capability to detect avian eDNA from water samples. Here, we present a novel primer pair (BirT) for metabarcoding avian eDNA. We optimized specificity and fragment length with regard to taxonomic resolution and available sequencing platforms. Additionally, we evaluated the availability of 12S rRNA gene reference sequences for birds and filled database gaps by generating novel barcodes. Finally, we tested the applicability of the BirT primer pair using field‐collected eDNA samples obtained with three different filter types and compared the eDNA metabarcoding results to visual observations uploaded to eBird ( www.eBird.org ). Our results confirm the suitability of the BirT primer pair for avian eDNA metabarcoding with no amplification of key non‐target groups and improved taxonomic resolution. Albeit there are still substantial gaps in the 12S reference sequence database, the analysis of bird eDNA from water samples resulted in species‐level taxonomic resolution for 91% of the detected taxa. All tested filters delivered similar results for total read numbers per sample (mean: 623,990 ± 331,236 SD) and species detected per sample (mean: 5.0 ± 2.0 SD). Ninety‐five percent of the bird detections were highly plausible based on eBird data collected at the time of eDNA sampling and 64% were directly confirmed by visual observations. Most detected species were closely associated with aquatic habitats confirming the suitability of water samples for the detection of waterfowl and species inhabiting similar ecological niches via eDNA.
Thalinger et al. (Thu,) studied this question.