Arthur Schüßler

Towards a unified paradigm for sequence‐based identification of fungi

The nuclear ribosomal internal transcribed spacer (ITS) region is the formal fungal barcode and in most cases the marker of choice for the exploration of fungal diversity in environmental samples. Two problems are particularly acute in the pursuit of satisfactory taxonomic assignment of newly generated ITS sequences: (i) the lack of an inclusive, reliable public reference data set and (ii) the lack of means to refer to fungal species, for which no Latin name is available in a standardized stable way. Here, we report on progress in these regards through further development of the UNITE database (http://unite.ut.ee) for molecular identification of fungi. All fungal species represented by at least two ITS sequences in the international nucleotide sequence databases are now given a unique, stable name of the accession number type (e.g. Hymenoscyphus pseudoalbidus|GU586904|SH133781.05FU), and their taxonomic and ecological annotations were corrected as far as possible through a distributed, third‐party annotation effort. We introduce the term ‘species hypothesis’ (SH) for the taxa discovered in clustering on different similarity thresholds (97–99%). An automatically or manually designated sequence is chosen to represent each such SH. These reference sequences are released (http://unite.ut.ee/repository.php) for use by the scientific community in, for example, local sequence similarity searches and in the QIIME pipeline. The system and the data will be updated automatically as the number of public fungal ITS sequences grows. We invite everybody in the position to improve the annotation or metadata associated with their particular fungal lineages of expertise to do so through the new Web‐based sequence management system in UNITE.

Phylogenetic reference data for systematics and phylotaxonomy of arbuscular mycorrhizal fungi from phylum to species level

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.

An evidence-based consensus for the classification of arbuscular mycorrhizal fungi (Glomeromycota)

The publication of a large number of taxon names at all levels within the arbuscular mycorrhizal fungi (Glomeromycota) has resulted in conflicting systematic schemes and generated considerable confusion among biologists working with these important plant symbionts. A group of biologists with more than a century of collective experience in the systematics of Glomeromycota examined all available molecular–phylogenetic evidence within the framework of phylogenetic hypotheses, incorporating morphological characters when they were congruent. This study is the outcome, wherein the classification of Glomeromycota is revised by rejecting some new names on the grounds that they are founded in error and by synonymizing others that, while validly published, are not evidence-based. The proposed “consensus” will provide a framework for additional original research aimed at clarifying the evolutionary history of this important group of symbiotic fungi.

DNA-based species level detection of Glomeromycota: one PCR primer set for all arbuscular mycorrhizal fungi

* 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.

‘Glomus intraradices DAOM197198’, a model fungus in arbuscular mycorrhiza research, is not Glomus intraradices

Glomus intraradices-like fungi are the most intensely studied arbuscular mycorrhizal (AM) fungi. However, there are several AM fungi named as G. intraradices that may not be conspecific. Therefore, the hypothesis was tested that DAOM197198 and similar AM fungi, such as BEG195, correspond to the type of G. intraradices. The G. intraradices isotype material, a descendant (INVAM FL208) of the type culture, and a morphologically corresponding AM fungus (MUCL49410) isolated from the type locality were studied and compared with several cultures of DAOM197198 and BEG195. Phylogenetic analyses of the partial small subunit (SSU), complete internal transcribed spacer (ITS) and partial large subunit (LSU) nuclear rDNA regions revealed two clades, one including G. intraradices FL208 and MUCL49410, the other containing DAOM197198 and BEG195. The two clades were clearly separated by sequence analyses, despite the high intraspecific and intrasporal ITS region sequence divergence of up to > 23%. We conclude that the AM fungi with the identifiers DAOM197198 and BEG195 are not G. intraradices, but fall in a clade that contains the recently described species G. irregulare.

DNA barcoding of arbuscular mycorrhizal fungi

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.

Diversispora celata sp. nov: molecular ecology and phylotaxonomy of an inconspicuous arbuscular mycorrhizal fungus

The increasing numbers of taxonomically unassigned phylotypes reported in molecular ecological studies contrast with the few formally described arbuscular mycorrhizal fungi (AMF; Glomeromycota). Here, a species new to science with Glomus-like spores is phylogenetically, morphologically and ecologically characterized. From single spore isolates of a previously recognized member of the Diversisporaceae from Swiss agricultural grassland, 17 new nuclear internal transcribed spacer (ITS), large subunit (LSU) and small subunit (SSU) ribosomal RNA (rRNA) gene sequences were determined and compared with 14 newly generated sequences of two close relatives and public database sequences, including environmental sequences, of known geographic origin. SSU ribosomal DNA (rDNA) sequence signatures and phylogenies based on ITS, LSU and SSU rDNA sequences show that the fungus belongs to the genus Diversispora. It is described as Diversispora celata sp. nov. Comparison with environmental sequences in the public domain confirmed its molecular genetic distinctiveness and revealed a cross-continental distribution of close relatives. The value of combining morphology and phylogeny to characterize AMF was reinforced by the morphological similarity to other species and the inconspicuous nature of D. celata spores and mycorrhizas. Inclusion of all three nuclear rDNA regions in species descriptions will facilitate species determination from environmental phylotypes.

Functional Domain Analysis of the Remorin Protein LjSYMREM1 in Lotus japonicus

In legumes rhizobial infection during root nodule symbiosis (RNS) is controlled by a conserved set of receptor proteins and downstream components. MtSYMREM1, a protein of the Remorin family in Medicago truncatula, was shown to interact with at least three receptor-like kinases (RLKs) that are essential for RNS. Remorins are comprised of a conserved C-terminal domain and a variable N-terminal region that defines the six different Remorin groups. While both N- and C-terminal regions of Remorins belonging to the same phylogenetic group are similar to each other throughout the plant kingdom, the N-terminal domains of legume-specific group 2 Remorins show exceptional high degrees of sequence divergence suggesting evolutionary specialization of this protein within this clade. We therefore identified and characterized the MtSYMREM1 ortholog from Lotus japonicus (LjSYMREM1), a model legume that forms determinate root nodules. Here, we resolved its spatio-temporal regulation and showed that over-expression of LjSYMREM1 increases nodulation on transgenic roots. Using a structure-function approach we show that protein interactions including Remorin oligomerization are mainly mediated and stabilized by the Remorin C-terminal region with its coiled-coil domain while the RLK kinase domains transiently interact in vivo and phosphorylate a residue in the N-terminal region of the LjSYMREM1 protein in vitro. These data provide novel insights into the mechanism of this putative molecular scaffold protein and underline its importance during rhizobial infection.

Mosaic genome of endobacteria in arbuscular mycorrhizal fungi: Transkingdom gene transfer in an ancient mycoplasma-fungus association.

Significance Obligate plant-symbiotic, arbuscular mycorrhizal fungi (AMF) are major drivers of terrestrial ecosystems and host enigmatic Mollicutes-related endobacteria (MRE) in their cytoplasm. The genome analysis of a MRE living in the AMF Dentiscutata heterogama revealed it to represent a previously unidentified bacterial lineage of Mycoplasma-related species. DhMRE shows strongly reduced metabolic capacity and underwent trans-kingdom gene transfer: its genome codes for an arsenal of eukaryotic-like putative effector proteins, with nuclear encoded homologues in AMF and Mortierella. The MRE-fungus (-plant) association probably evolved in ancestors of Glomeromycota and Mucoromycotina. This calls for a targeted search for ancient effector proteins that play crucial roles in the MRE interaction with fungal hosts, and putatively also with plants. For more than 450 million years, arbuscular mycorrhizal fungi (AMF) have formed intimate, mutualistic symbioses with the vast majority of land plants and are major drivers in almost all terrestrial ecosystems. The obligate plant-symbiotic AMF host additional symbionts, so-called Mollicutes-related endobacteria (MRE). To uncover putative functional roles of these widespread but yet enigmatic MRE, we sequenced the genome of DhMRE living in the AMF Dentiscutata heterogama. Multilocus phylogenetic analyses showed that MRE form a previously unidentified lineage sister to the hominis group of Mycoplasma species. DhMRE possesses a strongly reduced metabolic capacity with 55% of the proteins having unknown function, which reflects unique adaptations to an intracellular lifestyle. We found evidence for transkingdom gene transfer between MRE and their AMF host. At least 27 annotated DhMRE proteins show similarities to nuclear-encoded proteins of the AMF Rhizophagus irregularis, which itself lacks MRE. Nuclear-encoded homologs could moreover be identified for another AMF, Gigaspora margarita, and surprisingly, also the non-AMF Mortierella verticillata. Our data indicate a possible origin of the MRE-fungus association in ancestors of the Glomeromycota and Mucoromycotina. The DhMRE genome encodes an arsenal of putative regulatory proteins with eukaryotic-like domains, some of them encoded in putative genomic islands. MRE are highly interesting candidates to study the evolution and interactions between an ancient, obligate endosymbiotic prokaryote with its obligate plant-symbiotic fungal host. Our data moreover may be used for further targeted searches for ancient effector-like proteins that may be key components in the regulation of the arbuscular mycorrhiza symbiosis.

7 Evolution of the ‘Plant-Symbiotic’ Fungal Phylum, Glomeromycota

The most widespread and prominent symbiosis between land plants and fungi is the arbuscular mycorrhiza (AM). This type of mycorrhiza symbiosis is formed between approximately 80% of land plants and a monophyletic group of obligate symbiotic, multikaryotic and asexual fungi, the Glomeromycota. Despite the enormous ecological and economical importance of AM fungi, their biology is poorly understood. The focus here is, after reporting some historical aspects, on the recently advanced understanding of molecular phylogenetic relationships, the evolution and biogeography, and the obligate symbiotic endobacteria of AM fungi. Fossils and molecular clock estimates date the origin of AM symbioses to at least 460 MY ago, and AM fungi and land plants coevolved since then. Possibly, the fungi in the Glomeromycota were already ‘on the symbiotic track’ long before land plants originated. Aspects regarding the asexual evolution and heterokaryotic nature of glomeromycotan fungi and a feasible species concept are discussed.