Summary Although the molecular phylogeny, evolution and biodiversity of arbuscular mycorrhizal fungi (AMF) are becoming clearer, phylotaxonomically reliable sequence data are still limited. To fill this gap, a data set allowing resolution and environmental tracing across all taxonomic levels is provided. Two overlapping nuclear DNA regions, totalling c. 3 kb, were analysed: the small subunit (SSU) rRNA gene (up to 1800 bp) and a fragment spanning c. 250 bp of the SSU rDNA, the internal transcribed spacer (ITS) region (c. 475–520 bp) and c. 800 bp of the large subunit (LSU) rRNA gene. Both DNA regions together could be analysed for 35 described species, the SSU rDNA for c. 76 named and 18 as yet undefined species, and the ITS region or LSU rDNA, or a combination of both, for c. 91 named and 16 as yet undefined species. Present phylogenetic analyses, based on the three rDNA markers, provide reliable and robust resolution from phylum to species level. Altogether, 109 named species and 27 cultures representing as yet undefined species were analysed. This study provides a reference data set for molecular systematics and environmental community analyses of AMF, including analyses based on deep sequencing.

Summary At present, molecular ecological studies of arbuscular mycorrhizal fungi (AMF) are only possible above species level when targeting entire communities. To improve molecular species characterization and to allow species level community analyses in the field, a set of newly designed AMF specific PCR primers was successfully tested. Nuclear rDNA fragments from diverse phylogenetic AMF lineages were sequenced and analysed to design four primer mixtures, each targeting one binding site in the small subunit (SSU) or large subunit (LSU) rDNA. To allow species resolution, they span a fragment covering the partial SSU, whole internal transcribed spacer (ITS) rDNA region and partial LSU. The new primers are suitable for specifically amplifying AMF rDNA from material that may be contaminated by other organisms (e.g., samples from pot cultures or the field), characterizing the diversity of AMF species from field samples, and amplifying a SSU‐ITS‐LSU fragment that allows phylogenetic analyses with species level resolution. The PCR primers can be used to monitor entire AMF field communities, based on a single rDNA marker region. Their application will improve the base for deep sequencing approaches; moreover, they can be efficiently used as DNA barcoding primers.

Sexual reproduction is ubiquitous among eukaryotes, and fully asexual lineages are extremely rare. Prominent among ancient asexual lineages are the arbuscular mycorrhizal fungi (AMF), a group of plant symbionts with a multinucleate cytoplasm. Genomic divergence among co-existing nuclei was proposed to drive the evolutionary success of AMF in the absence of sex(1), but this hypothesis has been contradicted by recent genome analyses that failed to find significant genetic diversity within an AMF isolate(2,3). Here, we set out to resolve issues surrounding the genome organization and sexual potential of AMF by exploring the genomes of five isolates of Rhizophagus irregularis, a model AMF. We find that genetic diversity in this species varies among isolates and is structured in a homo-dikaryon-like manner usually linked with the existence of a sexual life cycle. We also identify a putative AMF mating-type locus, containing two genes with structural and evolutionary similarities with the mating-type locus of some Dikarya. Our analyses suggest that this locus may be multi-allelic and that AMF could be heterothallic and bipolar. These findings reconcile opposing views on the genome organization of these ubiquitous plant symbionts and open avenues for strain improvement and environmental application of these organisms.

Arbuscular mycorrhizal fungi (AMF) are known to improve plant fitness through the establishment of mycorrhizal symbioses. Genetic and phenotypic variations among closely related AMF isolates can significantly affect plant growth, but the genomic changes underlying this variability are unclear. To address this issue, we improved the genome assembly and gene annotation of the model strain Rhizophagus irregularis DAOM197198, and compared its gene content with five isolates of R. irregularis sampled in the same field. All isolates harbor striking genome variations, with large numbers of isolate-specific genes, gene family expansions, and evidence of interisolate genetic exchange. The observed variability affects all gene ontology terms and PFAM protein domains, as well as putative mycorrhiza-induced small secreted effector-like proteins and other symbiosis differentially expressed genes. High variability is also found in active transposable elements. Overall, these findings indicate a substantial divergence in the functioning capacity of isolates harvested from the same field, and thus their genetic potential for adaptation to biotic and abiotic changes. Our data also provide a first glimpse into the genome diversity that resides within natural populations of these symbionts, and open avenues for future analyses of plant-AMF interactions that link AMF genome variation with plant phenotype and fitness.

Summary Currently, no official DNA barcode region is defined for the Fungi. The COX1 gene DNA barcode is difficult to apply. The internal transcribed spacer (ITS) region has been suggested as a primary barcode candidate, but for arbuscular mycorrhizal fungi (AMF; Glomeromycota) the region is exceptionably variable and does not resolve closely related species. DNA barcoding analyses were performed with datasets from several phylogenetic lineages of the Glomeromycota. We tested a c. 1500 bp fragment spanning small subunit (SSU), ITS region, and large subunit (LSU) nuclear ribosomal DNA for species resolving power. Subfragments covering the complete ITS region, c. 800 bp of the LSU rDNA, and three c. 400 bp fragments spanning the ITS2, the LSU‐D1 or LSU‐D2 domains were also analysed. Barcode gap analyses did not resolve all species, but neighbour joining analyses, using Kimura two‐parameter (K2P) distances, resolved all species when based on the 1500 bp fragment. The shorter fragments failed to separate closely related species. We recommend the complete 1500 bp fragment as a basis for AMF DNA barcoding. This will also allow future identification of AMF at species level based on 400 or 1000 bp amplicons in deep sequencing approaches.

Ecosystem retrogression following long-term pedogenesis is attributed to phosphorus (P) limitation of primary productivity. Arbuscular mycorrhizal fungi (AMF) enhance P acquisition for most terrestrial plants, but it has been suggested that this strategy becomes less effective in strongly weathered soils with extremely low P availability. Using next generation sequencing of the large subunit ribosomal RNA gene in roots and soil, we compared the composition and diversity of AMF communities in three contrasting stages of a retrogressive >2-million-year dune chronosequence in a global biodiversity hotspot. This chronosequence shows a ~60-fold decline in total soil P concentration, with the oldest stage representing some of the most severely P-impoverished soils found in any terrestrial ecosystem. The richness of AMF operational taxonomic units was low on young (1000's of years), moderately P-rich soils, greatest on relatively old (~120 000 years) low-P soils, and low again on the oldest (>2 000 000 years) soils that were lowest in P availability. A similar decline in AMF phylogenetic diversity on the oldest soils occurred, despite invariant host plant diversity and only small declines in host cover along the chronosequence. Differences in AMF community composition were greatest between the youngest and the two oldest soils, and this was best explained by differences in soil P concentrations. Our results point to a threshold in soil P availability during ecosystem regression below which AMF diversity declines, suggesting environmental filtering of AMF insufficiently adapted to extremely low P availability.

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BackgroundUnderstanding the mechanisms underlying biological phenomena, such as evolutionarily conservative trait inheritance, is predicated on knowledge of the natural relationships among organisms. However, despite their enormous ecological significance, many of the ubiquitous soil inhabiting and plant symbiotic arbuscular mycorrhizal fungi (AMF, phylum Glomeromycota) are incorrectly classified.Methodology/Principal FindingsHere, we focused on a frequently used model AMF registered as culture BEG47. This fungus is a descendent of the ex-type culture-lineage of Glomus epigaeum, which in 1983 was synonymised with Glomus versiforme. It has since then been used as ‘G. versiforme BEG47’. We show by morphological comparisons, based on type material, collected 1860–61, of G. versiforme and on type material and living ex-type cultures of G. epigaeum, that these two AMF species cannot be conspecific, and by molecular phylogenetics that BEG47 is a member of the genus Diversispora.ConclusionsThis study highlights that experimental works published during the last >25 years on an AMF named ‘G. versiforme’ or ‘BEG47’ refer to D. epigaea, a species that is actually evolutionarily separated by hundreds of millions of years from all members of the genera in the Glomerales and thus from most other commonly used AMF ‘laboratory strains’. Detailed redescriptions substantiate the renaming of G. epigaeum (BEG47) as D. epigaea, positioning it systematically in the order Diversisporales, thus enabling an evolutionary understanding of genetical, physiological, and ecological traits, relative to those of other AMF. Diversispora epigaea is widely cultured as a laboratory strain of AMF, whereas G. versiforme appears not to have been cultured nor found in the field since its original description.