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Miller KE, Hopkins K, Inward DJ, et al. Metabarcoding of fungal communities associated with bark beetles. Ecol Evol. 2016;6(6):1590-600doi: 10.1002/ece3.1925.
Miller, K. E., Hopkins, K., Inward, D. J., & Vogler, A. P. (2016). Metabarcoding of fungal communities associated with bark beetles. Ecology and evolution, 6(6), 1590-600. https://doi.org/10.1002/ece3.1925
Miller, Kirsten E, et al. "Metabarcoding of fungal communities associated with bark beetles." Ecology and evolution vol. 6,6 (2016): 1590-600. doi: https://doi.org/10.1002/ece3.1925
Miller KE, Hopkins K, Inward DJ, Vogler AP. Metabarcoding of fungal communities associated with bark beetles. Ecol Evol. 2016 Feb 12;6(6):1590-600. doi: 10.1002/ece3.1925. eCollection 2016 Mar. PMID: 26904186; PMCID: PMC4752364.
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Ecol Evol. 2016 Feb 12;6(6):1590-600. doi: 10.1002/ece3.1925. eCollection 2016 Mar.

Metabarcoding of fungal communities associated with bark beetles.

Ecology and evolution

Kirsten E Miller, Kevin Hopkins, Daegan J G Inward, Alfried P Vogler

Affiliations

  1. Department of Life Sciences Natural History Museum Cromwell Road London SW7 5BD UK; Department of Life Sciences Imperial College London Silwood Park Campus Ascot SL5 7PY UK.
  2. Department of Life Sciences Natural History Museum Cromwell Road London SW7 5BD UK.
  3. Centre for Ecosystems, Society and Biosecurity Forest Research Alice Holt Lodge Farnham Surrey GU10 4LH UK.

PMID: 26904186 PMCID: PMC4752364 DOI: 10.1002/ece3.1925

Abstract

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.

Keywords: ITS2; Illumina; Ophiostomaceae; Scolytinae; primer tags; tree pathogens

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