Metabarcoding of fungal communities associated with bark beetles

Many species of fungi are closely allied with bark beetles, including many tree pathogens, but their species richness and patterns of distribution remain largely unknown. We established a protocol for metabarcoding of fungal communities directly from total genomic DNA extracted from individual beetles, showing that the ITS 3/4 primer pair selectively amplifies the fungal ITS . Using three specimens of bark beetle from different species, we assess the fungal diversity associated with these specimens and the repeatability of these estimates in PCR s conducted with different primer tags. The combined replicates produced 727 fungal Operational Taxonomic Units ( OTU s) for the specimen of Hylastes ater , 435 OTU s for Tomicus piniperda , and 294 OTU s for Trypodendron lineatum, while individual PCR reactions produced on average only 229, 54, and 31 OTU s for the three specimens, respectively. Yet, communities from PCR replicates were very similar in pairwise comparisons, in particular when considering species abundance, but differed greatly among the three beetle specimens. Different primer tags or the inclusion of amplicons in separate libraries did not impact the species composition. The ITS 2 sequences were identified with the Lowest Common Ancestor approach and correspond to diverse lineages of fungi, including Ophiostomaceae and Leotiomycetes widely found to be tree pathogens. We conclude that Illumina MiSeq metabarcoding reliably captures fungal diversity associated with bark beetles, although numerous PCR replicates are recommended for an exhaustive sample. Direct PCR from beetle DNA extractions provides a rapid method for future surveys of fungal species diversity and their associations with bark beetles and environmental variables.