Metabarcoding of fungal communities associated with bark beetles.

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

doi:10.1002/ece3.1925

Kirsten E Miller, Alfried P Vogler + Show 2 more

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https://doi.org/10.1002/ece3.1925

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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.

Highlights

Bark beetles (Scolytinae) are among the most destructive of forest pests, but much of the influence they exert is only made possible by the associations they form with fungi (Paine et al 1997)
Phytopathogenic fungi may assist in overwhelming the defenses of a host tree and facilitating a successful attack by the beetle (Paine et al 1997), but pathogenicity may provide the fungal associates with their own competitive advantage within the host tree (Six & Wingfield 2011)
These sequences were no longer seen after the ITSx filtering step, except for a single Operational Taxonomic Units (OTUs) that was classified as non-fungal in the Lowest Common Ancestor (LCA) analysis and removed

Summary

Introduction

Bark beetles (Scolytinae) are among the most destructive of forest pests, but much of the influence they exert is only made possible by the associations they form with fungi (Paine et al 1997). Phytopathogenic fungi may assist in overwhelming the defenses of a host tree and facilitating a successful attack by the beetle (Paine et al 1997), but pathogenicity may provide the fungal associates with their own competitive advantage within the host tree (Six & Wingfield 2011) Current knowledge of this system is biased towards a relatively small subset of economically important species. Reports are accruing of unforeseen levels of damage caused by both native and invasive bark beetle species across the globe (Hulcr & Dunn 2011; Humble & Allen 2006) The causes of these occurrences are not well understood but there is evidence to suggest they result from a combination of environmental change, active and passive movement of beetle hosts and fungal associates, and changes in pathogenicity (Hulcr & Dunn 2011; Ploetz et al 2013; Six 2013). It is clear that we need to take a more holistic approach towards understanding these interactions in order to better predict how environmental change might affect a r native community and how an invasive beetle might integrate into a new ecosystem

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