Finding flies in the mushroom soup: Host specificity of fungus‐associated communities revisited with a novel molecular method

Abstract

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...

Table for five, please: Dietary partitioning in boreal bats

“…We used a single primer pair (SFF‐145f: 5′‐GTHACHGCYCAYGCHTTYGTAATAAT‐3′ and SFF‐351r: 5′‐CTCCWGCRTGDGCWAGRTTTCC‐3′; primers and PCR setup from Walker, Williamson, Sanchez, Sobek, & Chambers, ) to test the DNA extraction success in the pooled samples and confirm the bat species by molecular analysis and another primer pair to amplify the potential prey (ZBJ‐ArtF1c: 5′‐AGATATTGGAACWTTATATTTTATTTTTGG‐3′ and ZBJ‐ArtR2c: 5′‐WACTAATCAATTWCCAAATCCTCC‐3′; primers and PCR setup from Zeale, Butlin, Barker, Lees, & Jones, ). Despite the proposed bias in Zeale primers toward Diptera and Lepidoptera (Clarke, Soubrier, Weyrich, & Cooper, ), we chose these for several reasons: (a) These are the most widely applied markers, (b) many species have been detected using exactly the same primers, even though claimed to be nonamplifiable in the earlier criticism, and (c) we wanted to allow comparison of our results with those of other studies using the same primers (Clare et al, ; Kaunisto, Roslin, Sääksjärvi, & Vesterinen, ; Koskinen et al, ; Krüger, Clare, Greif, et al, ; Krüger, Clare, Symondson, et al, ; Vesterinen et al, ; Wirta et al, ; Eitzinger et al, ). The PCR and library construction closely followed Kaunisto et al (), except we used MyTaq HS Red Mix (product nr BIO‐25048, Bioline, UK) polymerase throughout the protocol.…”

“…We then combined ZBJ (90% of the final pool volume) and SFF (10%) pools into one. See Vesterinen et al () and Koskinen et al () for further instructions for how to prepare and use SPRI. The pool included a smaller set of samples (approximately one‐third of the input DNA in the pool) to be used in another study.…”

“…We focused sampling on roosts inhabited by a single species, and likewise, we intended to pool pellets from a single species into a single pooled sample. In total, we initially sampled 1,252 fecal pellets from the five bat species in this study ( we wanted to allow comparison of our results with those of other studies using the same primers Kaunisto, Roslin, Sääksjärvi, & Vesterinen, 2017;Koskinen et al, 2018;Krüger, Clare, Greif, et al, 2014;Vesterinen et al, 2013Vesterinen et al, , 2016Wirta et al, 2015;Eitzinger et al, 2018). The PCR and library construction closely followed Kaunisto et al (2017), TA B L E 1 Information on the sampling details and characteristics of the field and molecular data.…”

Promises and pitfalls of using high‐throughput sequencing for diet analysis

“…Similar issues can be found when samples incorporate large amounts of acidic polysaccharides such as pectin or xylan that are well‐known enzymatic inhibitors (Pandey, Adams, & Flournoy, ). When this occurs, custom extraction protocols might be required (Juen & Traugott, ; Koskinen et al, ; Oehm et al, ). The tissular characteristics of prey taxa can also distort the eDNA signal during extraction, since for instance the extraction from schlerotized animals (e.g., chitinized insects) is more difficult than soft‐body species, thereby affecting the relative DNA abundances obtained in the extracts.…”

Introduction: Special issue on species interactions, ecological networks and community dynamics – Untangling the entangled bank using molecular techniques

“…The findings regarding particular interactions range from insight into emergent features such as levels of specialization in ecological interaction networks (Clare et al, ; Doña, Proctor, et al, ; Doña, Serrano, et al, ; Koskinen et al, ; Sepp et al, ) to the role of individual seed dispersers in seed dispersal networks. As a case in point, González‐Varo, Arroyo, and Jordano () are able to show how much of an ecological function (seed dispersal) is concentrated to particular species at particular points in time.…”