Archaeal diversity in the microbiomes of four wild bird species

Archaea are generally low-abundance members of the vertebrate microbiota that require specific PCR primers to be detected in metabarcoding studies, and the robust intraspecific sample size is necessary for well-supported conclusions about archaeal diversity. Using 16S rRNA gene amplicons generated using both Archaea-Specific and Universal primers, we investigated prokaryotic diversity in 110 fecal samples from four wild bird species from four different orders: Anna's Hummingbird (Calypte anna), Saltmarsh Sparrow (Ammospiza caudacuta), Ruddy Turnstone (Arenaria interpres), and Canada Goose (Branta canadensis). Our aim was to test the hypotheses that Archaea-Specific primers would offer higher resolution of archaeal diversity and that the four ecologically distinct host species would have distinct archaeal communities. Archaea-Specific primers resulted in increases in archaeal richness and detection of Archaea in all four birds compared to the Universal primers. The ammonia-oxidizing archaeal order Nitrososphaerales was detected in all four host species, and methanogenic orders were enriched in samples from Canada Geese. In Bacteria-Archaea co-occurrence networks, Archaea-Specific primers found many more significant interactions than the Universal primers alone. Methanogenic archaeal orders dominated the microbiota in Canada Geese and were found to a lesser extent in the other host species, suggesting an important functional role of methanogens in Canada Geese. Overall, this study advances our knowledge of the archaeal component of the microbiome in wild birds and provides insight into the potential functional roles Archaea play in studies of avian gastrointestinal microbiota. IMPORTANCE Archaea may be persistent members of host-associated microbiomes across diverse host taxa; their detection has been limited due to their low abundance and the inadequacy of Universal primers. Large-scale studies of Archaea in vertebrate microbiomes have historically had low intraspecific sample sizes for bird species and had conflicting results. This study demonstrates the improved capability of the Archaea-Specific primers to detect archaeal diversity in diverse avian host species compared to the widely used Universal primers. We also identified both shared and species-specific archaeal taxa across four ecologically distinct avian host species from four different orders with implications for functional importance. Future studies interested in comprehensively cataloging prokaryotic diversity in avian microbiomes using amplicon-based sequencing methods should include Archaea-Specific primers to adequately probe archaeal diversity.