Metabarcoding of insect-associated fungal communities: a comparison of internal transcribed spacer (ITS) and large-subunit (LSU) rRNA markers

Abstract

Full taxonomic characterisation of fungal communities is necessary for establishing ecological associations and early detection of pathogens and invasive species. Complex communities of fungi are regularly characterised by metabarcoding using the Internal Transcribed Spacer (ITS) and the Large-Subunit (LSU) gene of the rRNA locus, but reliance on a single short sequence fragment limits the confidence of identification. Here we link metabarcoding from the ITS2 and LSU D1-D2 regions to characterise fungal communities associated with bark beetles (Scolytinae), the likely vectors of several tree pathogens. Both markers revealed similar patterns of overall species richness and response to key variables (beetle species, forest type), but identification against the respective reference databases using various taxonomic classifiers revealed poor resolution towards lower taxonomic levels, especially the species level. Thus, Operational Taxonomic Units (OTUs) could not be linked via taxonomic classifiers across ITS and LSU fragments. However, using phylogenetic trees (focused on the epidemiologically important Sordariomycetes) we placed OTUs obtained with either marker relative to reference sequences of the entire rRNA cistron that includes both loci and demonstrated the largely similar phylogenetic distribution of ITS and LSU-derived OTUs. Sensitivity analysis of congruence in both markers suggested the biologically most defensible threshold values for OTU delimitation in Sordariomycetes to be 98% for ITS2 and 99% for LSU D1-D2. Studies of fungal communities using the canonical ITS barcode require corroboration across additional loci. Phylogenetic analysis of OTU sequences aligned to the full rRNA cistron shows higher success rate and greater accuracy of species identification compared to probabilistic taxonomic classifiers.

First experimental evidence for active farming in ambrosia beetles and strong heredity of garden microbiomes

“…Amplicon sequencing does not provide information on absolute abundances of phylotypes, it sets the amplified taxa in relation [ 85 ] which is important to keep in mind especially when some taxa could not be amplified as their appearance may shift relative abundance pictures. For future studies we propose to carefully choose metabarcoding primers that amplify the fungal groups of main interest for testing the key hypothesis, while acknowledging their limitations [ 47 , 86 ].…”

Sapwood mycobiome varies across host, plant compartment and environments in Nothofagus forests from Northern Patagonia

“…In other words, fungal inoculum can be there and does not trigger any decay because of other factors acting at the individual level as health promoters (i.e., cohabiting mycobiota, plant genetics, host defence mechanisms). Nevertheless, many of these potentially pathogenic species pointed out by culture prospecting were taxa difficult to detect in HTS approaches targeting partial ITS regions, such as species from the order Ophiostomatales and the family Hymenochaetaceae (Ceballos‐Escalera et al, 2022; Skelton et al, 2019).…”

Modular, multi‐barcode amplicon sequencing for improved species‐level detection of fungal phytopathogens: A case study of pipeline establishment targeting the Ophiostomatales

“…While multi‐barcode metabarcoding offers a suitable platform to rapidly survey and monitor fungal communities, a challenge remains in linking independently generated barcodes from a complex mixture of taxa back to an origin taxon or organism (Ceballos‐Escalera et al, 2022; Cobo‐Díaz et al, 2019a). A problem exemplified here by the unsuccessful attempts to establish an association between two short barcodes, which are genetically linked within the rDNA operon (ITS2 and LSU), despite using a phylogenetic approach to connect the short fragments through alignment against full‐length sequences spanning the entire region (Ceballos‐Escalera et al, 2022). While ecologically this may leave some uncertainty about the biological relevance of the two independently generated amplicons (Ceballos‐Escalera et al, 2022), from a diagnostic and surveillance perspective, it remains to be determined whether there is a need to ascertain whether two independent barcodes are obtained from the same individual or not.…”

“…A problem exemplified here by the unsuccessful attempts to establish an association between two short barcodes, which are genetically linked within the rDNA operon (ITS2 and LSU), despite using a phylogenetic approach to connect the short fragments through alignment against full‐length sequences spanning the entire region (Ceballos‐Escalera et al, 2022). While ecologically this may leave some uncertainty about the biological relevance of the two independently generated amplicons (Ceballos‐Escalera et al, 2022), from a diagnostic and surveillance perspective, it remains to be determined whether there is a need to ascertain whether two independent barcodes are obtained from the same individual or not. The ability to amplify independent barcodes simultaneously adds to the power with which organisms can be detected, as is evident in the opportunity of recovering more taxa and retrieving complementary sequence data that allows for distinction of closely related species or even haplotypes (Figure 3a).…”

“…While ecologically this may leave some uncertainty about the biological relevance of the two independently generated amplicons (Ceballos-Escalera et al, 2022), from a diagnostic and surveillance perspective, it remains to be determined whether there is a need to ascertain whether two independent barcodes are obtained from the same individual or not. The ability to amplify independent barcodes simultaneously adds to the power with which organisms can be detected, as is evident in the opportunity of recovering more taxa and retrieving complementary sequence data that allows for distinction of closely related species or even haplotypes (Figure 3a).…”