18S metabarcoding of AM fungi: the SYNERSYS experiment

The diversity of root-associated arbuscular mycorrhizal (AM) fungal communities is a crucial driver of plant and ecosystem functioning. However, most research exploring the factors that shape AM fungal diversity, particularly climate change stressors like drought, tends to focus on plant diversity and soil properties, often overlooking interactions between factors. In this study, we focus on these interactions among plant biodiversity, soil biodiversity, and drought, aiming to disentangle how they influence each other and how this links to ecosystem functioning. We employed an experimental setup examining the impacts of plant species diversity, soil biodiversity, and drought on root-associated AM fungal diversity through three partly overlapping mesocosm experiments.In the first experiment, we utilized eight AM herbaceous plant species in a full-factorial design with two levels of plant diversity (monocultures and six-species mixtures), two soil biodiversity conditions (an unsterilized but naturally low-diversity sub-surface soil mixture inoculated with either sterile or live, naturally high-diversity surface soil), and two watering conditions (ambient and drought). The second experiment analyzed mycorrhizal diversity at the plant species level, using the same eight species in two-species mixtures under high and low soil biodiversity conditions (see experiment one). The third experiment involved 16 herbaceous plant species and addressed temporal variability by assessing AM fungal diversity in plant monocultures at two distinct stages of vegetation maturity.Summary: DNA Extraction and Sequencing of Root Samples1. DNA ExtractionRoot samples were flash-frozen in liquid nitrogen, ground, and sub-sampled to a dry weight of 200 mg. DNA was extracted separately for each plant species in mesocosms using the Qiagen DNeasy 96 Plant Kit. Spectrophotometry was used to ensure quality and yield. For plant mixtures, DNA extracts were pooled at the mesocosm level with equal DNA contribution from each species.2. AM Fungal DNA AnalysisAM fungi were characterized using 18S rDNA metabarcoding with Glom01 primers (216 bp fragment, forward sequence: ACTATCCCTATTAATCATTAC; reverse sequence: CTCGTAGTTGAATTTCG, Taberlet et al., unpublished). These primers, specifically designed for AM fungi, underwent stringent quality control for specificity, taxonomic resolution, and mismatch reduction. Amplification was repeated four times for reliability.3. Sequencing and BioinformaticsShort sequences (1,276 purified and tagged amplicons) allowed for large sample analysis but reduced taxonomic and ecological resolution compared to long-read sequencing. Evolutionary placement algorithms were used to mitigate biases from short reads. Rigorous quality controls, including PCR and bioinformatic controls, were applied to detect errors like tag-jumping.4. Library Preparation and SequencingPurified amplicons were pooled, verified via capillary electrophoresis, and sent to Fasteris (Geneva, Switzerland) for library preparation using the MetaFast protocol. Sequencing was performed with Illumina MiSeq paired-end runs (2x150 V2 and V3).