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 ITS3/4 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 PCRs
conducted with different primer tags. The combined replicates produced 727
fungal Operational Taxonomic Units (OTUs) for the specimen of Hylastes ater,
435 OTUs for Tomicus piniperda, and 294 OTUs for Trypodendron lineatum,
while individual PCR reactions produced on average only 229, 54, and 31
OTUs 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 ITS2 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.