Metabarcoding options to study eukaryotic endoparasites of birds.

There is growing interest in the study of avian endoparasite communities, and metabarcoding is a promising approach to complement more conventional or targeted methods. In the case of eukaryotic endoparasites, phylogenetic diversity is extreme, with parasites from 4 kingdoms and 11 phyla documented in birds. We addressed this challenge by comparing different primer sets across 16 samples from 5 bird species. Samples consisted of blood, feces, and controlled mixes with known proportions of bird and nematode DNA. Illumina sequencing revealed that a 28S primer set used in combination with a custom blocking primer allowed detection of various plasmodiid parasites and filarioid nematodes in the blood, coccidia in the feces, as well as two potentially pathogenic fungal groups. When tested on the controlled DNA mixes, these primers also increased the proportion of nematode DNA by over an order of magnitude. An 18S primer set, originally designed to exclude metazoan sequences, was the most effective at reducing the relative number of avian DNA sequences and was the only one to detect

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© 2021 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.

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The authors declare that there is no conflict of interest.