Barcoded NS31/AML2 primers for sequencing of arbuscular mycorrhizal communities in environmental samples

Abstract

Premise of the study: Arbuscular mycorrhizal fungi (AMF) are globally important root symbioses that enhance plant growth and nutrition and influence ecosystem structure and function. To better characterize levels of AMF diversity relevant to ecosystem function, deeper sequencing depth in environmental samples is needed. In this study, Illumina barcoded primers and a bioinformatics pipeline were developed and applied to study AMF diversity and community structure in environmental samples.

Methods: Libraries of small subunit ribosomal RNA fragment amplicons were amplified from environmental DNA using a single-step PCR reaction with barcoded NS31/AML2 primers. Amplicons were sequenced on an Illumina MiSeq sequencer using version 2, 2 × 250-bp paired-end chemistry, and analyzed using QIIME and RDP Classifier.

Results: Sequencing captured 196 to 6416 operational taxonomic units (OTUs; depending on clustering parameters) representing nine AMF genera. Regardless of clustering parameters, ∼20 OTUs dominated AMF communities (78-87% reads) with the remaining reads distributed among other OTUs. Analyses also showed significant biogeographic differences in AMF communities and that community composition could be linked to specific edaphic factors.

Discussion: Barcoded NS31/AML2 primers and Illumina MiSeq sequencing provide a powerful approach to address AMF diversity and variations in fungal assemblages across host plants, ecosystems, and responses to environmental drivers including global change.

Keywords: Glomeromycota; arbuscular mycorrhizal fungi; barcoding; community composition; diversity; tropical dry forest.

Fig. 1.

Alpha rarefaction curves for 90% similar (A), 95% similar (B), and 97% similar (C) operational taxonomic units (OTUs) in samples from El Eden (red) and La Higuera (blue); Venn diagrams illustrating the number of 90% similar (D), 95% similar (E), and 97% similar (F) OTUs unique to El Eden (EE) or La Higuera (LH), or present at both sites. Data are shown for the most inclusive, all OTUs data sets.

Fig. 2.

Averaged alpha rarefaction curves of observed operational taxonomic unit (OTU) richness at 90%, 95%, and 97% similarity clustering thresholds in El Eden (EE) and La Higuera (LH). Vertical bars represent the standard deviation of the mean.

Fig. 3.

The percentage of operational taxonomic units (OTUs) (A, B) and percentage of sequence reads (C, D) assigned to arbuscular mycorrhizal fungal genera at El Eden (EE) and La Higuera (LH) study sites. Data are shown for 97% similar OTUs at two inclusivity levels: all clustered OTUs (A, C) and ≥10-ton OTUs (B, D). Colors indicate genus assigned by RDP Classifier: Glomus (green); Ambispora, Archaeospora, and Gigaspora (white); Scutellospora (orange); Acaulospora (black); Paraglomus (aqua); Claroideoglomus (yellow); Diversispora (blue); and no genus-level assignment (red).

Fig. 4.

Principal coordinates analysis (PCoA) ordination plots of arbuscular mycorrhizal fungal (AMF) communities sampled at El Eden (EE, red circles) and La Higuera (LH, blue triangles) using Bray–Curtis dissimilarities based on all operational taxonomic units (OTUs) clustered at 90% (A), 95% (B), and 97% (C) similarity thresholds. Percentage values on the axes represent the variation in AMF community dissimilarity explained by each axis. Ellipses represent the central tendency of communities at each site. Vectors denote the magnitude and direction of statistically significant effects of soil properties on AMF community dissimilarity.

Fig. 5.

Relationship between operational taxonomic unit (OTU) richness and soil NH₄ (A) and pH (B) in the 97% ≥10-ton data set, which is representative of patterns observed across all data sets. Red dashed lines show the best fit of linear regression models with P < 0.003.

Fig. 6.

Principal coordinates analysis (PCoA) ordination plot of soil properties (A) and arbuscular mycorrhizal fungal (AMF) communities (B) in El Eden (EE) and La Higuera (LH). Percentage values on the axes represent the variation in soil properties (A) and AMF operational taxonomic unit (OTU) read abundance (B) explained by each axis. Ellipses represent the central tendency of communities, and vectors denote the magnitude and direction of the effects of significant soil nutrients on AMF communities.