A metabarcoding protocol targeting two DNA regions to analyze root‐associated fungal communities in ferns and lycophytes

Abstract

Premise: Detailed studies of the fungi associated with lycophytes and ferns provide crucial insights into the early evolution of land plants. However, most investigations to date have assessed fern-fungus interactions based only on visual root inspection. In the present research, we establish and evaluate a metabarcoding protocol to analyze the fungal communities associated with fern and lycophyte roots. Methods: We used two primer pairs focused on the ITS rRNA region to screen the general fungal communities, and the 18S rRNA to target Glomeromycota fungi (i.e., arbuscular mycorrhizal fungi). To test these approaches, we collected and processed roots from 12 phylogenetically distant fern and lycophyte species. Results: We found marked compositional differences between the ITS and 18S data sets. While the ITS data set demonstrated the dominance of orders Glomerales (phylum Glomeromycota), Pleosporales, and Helotiales (both in phylum Ascomycota), the 18S data set revealed the greatest diversity of Glomeromycota. Non-metric multidimensional scaling (NMDS) ordination suggested an important geographical effect in sample similarities. Discussion: The ITS-based approach is a reliable and effective method to analyze the fungal communities associated with fern and lycophyte roots. The 18S approach is more appropriate for studies focused on the detailed screening of arbuscular mycorrhizal fungi.

Fern mycorrhizae do not respond to fertilization in a tropical montane forest

“…DNA extraction and purification were carried out following the metabarcoding protocol described by Guillen‐Otero et al. (2023) for root‐associated fungi in ferns and lycophytes. To recover an optimal representation of the general fungal community and the arbuscular mycorrhizal fungi (AMF) community we targeted the ITS rRNA region with the universal primers ITS1F (Gardes & Bruns, 1993) and ITS4 (White et al., 1990).…”

“…To recover an optimal representation of the general fungal community and the arbuscular mycorrhizal fungi (AMF) community we targeted the ITS rRNA region with the universal primers ITS1F (Gardes & Bruns, 1993) and ITS4 (White et al., 1990). This metabarcoding approach allowed us to gain a wider perspective of fern‐associated fungal communities and also served as a measure of the intensity of mycorrhization by relating the number of sequences of arbuscular mycorrhizal fungi to that of overall fungi (Guillen‐Otero et al., 2023, 2024). A sequencing approach targeting exclusively Glomeromycota with primers NS31 (Simon et al., 1992) and AML2 (Lee et al., 2008), for example, would likely have resulted in higher resolution for these fungi (Öpik et al., 2010); however, it would have failed in recovering the dominant fungal groups, and the intensity of mycorrhization would not have been be estimated.…”

“…We excluded taxa with relative abundances lower than 1% in each sample to obtain the core microbiome by specimen as described by Guillen‐Otero et al. (2023). A table containing taxonomic classification and ASVs relative abundance for the general core microbiome dataset can be found in Appendix S1.…”

“…For taxonomic nomenclature, we followed the classification of the kingdom fungi proposed by Wijayawardene et al (2022). We excluded taxa with relative abundances lower than 1% in each sample to obtain the core microbiome by specimen as described by Guillen-Otero et al (2023).…”

“…These findings suggest a switch from AMF to DSE as allies during fern evolution (Lehnert et al., 2017), but until now there are no studies exploring fern‐DSE interactions. During the last decade, molecular methods have shed new light into the composition of fungal communities associated with fern (Cook & Taylor, 2023, Guillen‐Otero et al., 2023, 2024; Perez‐Lamarque et al., 2022; Sandoz et al., 2020; Strullu‐Derrien et al., 2018). Based on our current state of knowledge, ferns have a lower incidence of mycorrhizal colonization than angiosperms, with about 34% of all fern species sampled so far lacking mycorrhizae, as opposed to about 18% in angiosperms (Lehnert et al., 2017; Wang & Qiu, 2006).…”