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Fruiting bodies of fungi constitute an important resource for thousands of other taxa. The structure of these diverse assemblages has traditionally been studied with labour-intensive methods involving cultivation and morphology-based species identification, to which molecular information might offer convenient complements. To overcome challenges in DNA extraction and PCR associated with the complex chemical properties of fruiting bodies, we developed a pipeline applicable for extracting amplifiable total DNA from soft fungal samples of any size. Our protocol purifies DNA in two sequential steps: (a) initial salt-isopropanol extraction of all nucleic acids in the sample is followed by (b) an extra clean-up step using solid-phase reversible immobilization (SPRI) magnetic beads. The protocol proved highly efficient, with practically all of our samples-regardless of biomass or other properties-being successfully PCR-amplified using metabarcoding primers and subsequently sequenced. As a proof of concept, we apply our methods to address a topical ecological question: is host specificity a major characteristic of fungus-associated communities, that is, do different fungus species harbour different communities of associated organisms? Based on an analysis of 312 fungal fruiting bodies representing 10 species in five genera from three orders, we show that molecular methods are suitable for studying this rich natural microcosm. Comparing to previous knowledge based on rearing and morphology-based identifications, we find a species-rich assemblage characterized by a low degree of host specialization. Our method opens up new horizons for molecular analyses of fungus-associated interaction webs and communities. Fruiting bodies of fungi constitute an important resource for thousands of other taxa. The structure of these diverse assemblages has traditionally been studied with labour-intensive methods involving cultivation and morphology-based species identification, to which molecular information might offer convenient complements. To overcome challenges in DNA extraction and PCR associated with the complex chemical properties of fruiting bodies, we developed a pipeline applicable for extracting amplifiable total DNA from soft fungal samples of any size. Our protocol purifies DNA in two sequential steps: (a) initial salt-isopropanol extraction of all nucleic acids in the sample is followed by (b) an extra clean-up step using solid-phase reversible immobilization (SPRI) magnetic beads. The protocol proved highly efficient, with practically all of our samples-regardless of biomass or other properties-being successfully PCR-amplified using metabarcoding primers and subsequently sequenced. As a proof of concept, we apply our methods to address a topical ecological question: is host specificity a major characteristic of fungus-associated communities, that is, do different fungus species harbour different communities of associated organisms? Based on an analysis of 312 fungal fruiting bodies representing 10 species in five genera fro...

Interactions among fungi and insects involve hundreds of thousands of species. While insect communities on plants have formed some of the classic model systems in ecology, fungus-based communities and the forces structuring them remain poorly studied by comparison. We characterize the arthropod communities associated with fruiting bodies of eight mycorrhizal basidiomycete fungus species from three different orders along a 1200-km latitudinal gradient in northern Europe. We hypothesized that, matching the pattern seen for most insect taxa on plants, we would observe a general decrease in fungalassociated species with latitude. Against this backdrop, we expected local communities to be structured by host identity and phylogeny, with more closely related fungal species sharing more similar communities of associated organisms. As a more unique dimension added by the ephemeral nature of fungal fruiting bodies, we expected further imprints generated by successional change, with younger fruiting bodies harboring communities different from older ones. Using DNA metabarcoding to identify arthropod communities from fungal fruiting bodies, we found that latitude left a clear imprint on fungusassociated arthropod community composition, with host phylogeny and decay stage of fruiting bodies leaving lesser but still-detectable effects. The main latitudinal imprint was on a high arthropod species turnover, with no detectable pattern in overall species richness. Overall, these findings paint a new picture of the drivers of fungus-associated arthropod communities, suggesting that latitude will not affect how many arthropod species inhabit a fruiting body but, rather, what species will occur in it and at what relative abundances (as measured by sequence read counts). These patterns upset simplistic predictions regarding latitudinal gradients in species richness and in the strength of biotic interactions.