Strain-specific evolution and host-specific regulation of transposable elements in the model plant symbiont Rhizophagus irregularis.

Transposable elements (TEs) are repetitive DNA that can create genome structure and regulation variability. The genome of Rhizophagus irregularis, a widely studied arbuscular mycorrhizal fungus (AMF), comprises ∼50% repetitive sequences that include TEs. Despite their abundance, two-thirds of TEs remain unclassified, and their regulation among AMF life stages remains unknown. Here, we aimed to improve our understanding of TE diversity and regulation in this model species by curating repeat datasets obtained from chromosome-level assemblies and by investigating their expression across multiple conditions. Our analyses uncovered new TE superfamilies and families in this model symbiont and revealed significant differences in how these sequences evolve both within and between R. irregularis strains. With this curated TE annotation, we also found that the number of upregulated TE families in colonized roots is 4 times higher than in the extraradical mycelium, and their overall expression differs depending on the plant host. This work provides a fine-scale view of TE diversity and evolution in model plant symbionts and highlights their transcriptional dynamism and specificity during host-microbe interactions. We also provide Hidden Markov Model profiles of TE domains for future manual curation of uncharacterized sequences (https://github.com/jordana-olive/TE-manual-curation/tree/main).

Association of mycoheterotrophic Gentianaceae with specific Glomus lineages.

Some plant species took an alternative evolutionary pathway in which they lost their photosynthetic capacity to depend exclusively on carbon supplied by arbuscular mycorrhizal fungi (AMF) in an association called mycoheterotrophy. Among them is Voyriella parviflora, a species of the family Gentianaceae, which is found in tropical regions such as the Amazon basin. Here, we assessed the identity of AMF symbionts associated with this species. DNA was isolated from eight Gentianaceae specimens and from litter and surrounding roots of photosynthetic plants. The atp1 gene was amplified by Sanger sequencing to determine the taxonomic affiliation of the mycoheterotrophic plants. A 280 bp region of the 18S rRNA gene of AMF was amplified with primers NS31/AML2 by high-throughput sequencing. The mycoheterotrophic specimens were assigned to V. parviflora with a bootstrap support of 72%. Glomus was the most abundant AMF genus, both in the mycoheterotrophic plants and in the litter and roots of photosynthetic plants. In addition, a few Glomus genotypes were abundantly enriched in the mycoheterotrophic plants, with only a few specimens colonized by Gigaspora, Acaulospora, and Scutellospora in a low proportion. These genotypes formed a cluster within a larger clade, suggesting that V. parviflora shows a preferential association with a narrow Glomus lineage which is not phylogenetically close to a previously identified V. parviflora's associated lineage. Furthermore, detecting fungi from other families suggests that V. parviflora is colonized by other genera, although with low frequency. These findings provide new insights into the association between AMF and mycoheterotrophic species and highlight the importance of considering trap culture-independent approaches in understanding this symbiosis.

Characterization of the arbuscular mycorrhizal fungal community associated with rosewood in threatened Miombo forests.

Understanding the dynamics of arbuscular mycorrhizal fungi (AMF) in response to land use change is important for the restoration of degraded forests. Here, we investigated the AMF community composition in the roots of Pterocarpus tinctorius sampled from agricultural and forest fallow soils rich in aluminum and iron. By sequencing the large subunit region of the rRNA gene, we identified a total of 30 operational taxonomic units (OTUs) in 33 root samples. These OTUs belonged to the genera Rhizophagus, Dominikia, Glomus, Sclerocystis, and Scutellospora. The majority of these OTUs did not closely match any known AMF species. We found that AMF species richness was significantly influenced by soil properties and overall tree density. Acidic soils with high levels of aluminum and iron had a low mean AMF species richness of 3.2. Indicator species analyses revealed several AMF OTUs associated with base saturation (4 OTUs), high aluminum (3 OTUs), and iron (2 OTUs). OTUs positively correlated with acidity (1 OTU), iron, and available phosphorus (2 OTUs) were assigned to the genus Rhizophagus, suggesting their tolerance to aluminum and iron. The results highlight the potential of leguminous trees in tropical dry forests as a reservoir of unknown AMF species. The baseline data obtained in this study opens new avenues for future studies, including the use of indigenous AMF-based biofertilizers to implement ecological revegetation strategies and improve land use.

Structurally distinct mitoviruses: are they an ancestral lineage of the Mitoviridae exclusive to arbuscular mycorrhizal fungi (Glomeromycotina)?

Mitoviruses in the family Mitoviridae are the mitochondria-replicating "naked RNA viruses" with genomes encoding only the replicase RNA-dependent RNA polymerase (RdRp) and prevalent across fungi, plants, and invertebrates. Arbuscular mycorrhizal fungi in the subphylum Glomeromycotina are obligate plant symbionts that deliver water and nutrients to the host. We discovered distinct mitoviruses in glomeromycotinian fungi, namely "large duamitovirus," encoding unusually large RdRp with a unique N-terminal motif that is endogenized in some host genomes. More than 400 viral sequences similar to the large duamitoviruses are present in metatranscriptome databases. They are globally distributed in soil ecosystems, consistent with the cosmopolitan distribution of glomeromycotinian fungi, and formed the most basal clade of the Mitoviridae in phylogenetic analysis. Given that glomeromycotinian fungi are the only confirmed hosts of these viruses, we propose the hypothesis that large duamitoviruses are the most ancestral lineage of the Mitoviridae that have been maintained exclusively in glomeromycotinian fungi.

Paired Root-Soil Samples and Metabarcoding Reveal Taxon-Based Colonization Strategies in Arbuscular Mycorrhizal Fungi Communities in Japanese Cedar and Cypress Stands.

Arbuscular mycorrhizal fungi (AMF) in the roots and soil surrounding their hosts are typically independently investigated and little is known of the relationships between the communities of the two compartments. We simultaneously collected root and surrounding soil samples from Cryptomeria japonica (Cj) and Chamaecyparis obtusa (Co) at three environmentally different sites. Based on molecular and morphological analyses, we characterized their associated AMF communities. Cj was more densely colonized than Co and that root colonization intensity was significantly correlated with soil AMF diversity. The communities comprised 15 AMF genera dominated by Glomus and Paraglomus and 1443 operational taxonomic units (OTUs) of which 1067 and 1170 were in roots and soil, respectively. AMF communities were significantly different among sites, and the root AMF communities were significantly different from those of soil at each site. The root and soil AMF communities responded differently to soil pH. At the genus level, Glomus and Acaulospora were abundant in roots while Paraglomus and Redeckera were abundant in soil. Our findings suggest that AMF colonizing roots are protected from environmental stresses in soil. However, the root-soil-abundant taxa have adapted to both environments and represent a model AMF symbiont. This evidence of strategic exploitation of the rhizosphere by AMF supports prior hypotheses and provides insights into community ecology.

A highly contiguous genome assembly reveals sources of genomic novelty in the symbiotic fungus Rhizophagus irregularis.

The root systems of most plant species are aided by the soil-foraging capacities of symbiotic arbuscular mycorrhizal (AM) fungi of the Glomeromycotina subphylum. Despite recent advances in our knowledge of the ecology and molecular biology of this mutualistic symbiosis, our understanding of the AM fungi genome biology is just emerging. Presented here is a close to T2T genome assembly of the model AM fungus Rhizophagus irregularis DAOM197198, achieved through Nanopore long-read DNA sequencing and Hi-C data. This haploid genome assembly of R. irregularis, alongside short- and long-read RNA-Sequencing data, was used to produce a comprehensive annotation catalog of gene models, repetitive elements, small RNA loci, and DNA cytosine methylome. A phylostratigraphic gene age inference framework revealed that the birth of genes associated with nutrient transporter activity and transmembrane ion transport systems predates the emergence of Glomeromycotina. While nutrient cycling in AM fungi relies on genes that existed in ancestor lineages, a burst of Glomeromycotina-restricted genetic innovation is also detected. Analysis of the chromosomal distribution of genetic and epigenetic features highlights evolutionarily young genomic regions that produce abundant small RNAs, suggesting active RNA-based monitoring of genetic sequences surrounding recently evolved genes. This chromosome-scale view of the genome of an AM fungus genome reveals previously unexplored sources of genomic novelty in an organism evolving under an obligate symbiotic life cycle.

Divergent Evolution of Early Terrestrial Fungi Reveals the Evolution of Mucormycosis Pathogenicity Factors.

Fungi have evolved over millions of years and their species diversity is predicted to be the second largest on the earth. Fungi have cross-kingdom interactions with many organisms that have mutually shaped their evolutionary trajectories. Zygomycete fungi hold a pivotal position in the fungal tree of life and provide important perspectives on the early evolution of fungi from aquatic to terrestrial environments. Phylogenomic analyses have found that zygomycete fungi diversified into two separate clades, the Mucoromycota which are frequently associated with plants and Zoopagomycota that are commonly animal-associated fungi. Genetic elements that contributed to the fitness and divergence of these lineages may have been shaped by the varied interactions these fungi have had with plants, animals, bacteria, and other microbes. To investigate this, we performed comparative genomic analyses of the two clades of zygomycetes in the context of Kingdom Fungi, benefiting from our generation of a new collection of zygomycete genomes, including nine produced for this study. We identified lineage-specific genomic content that may contribute to the disparate biology observed in these zygomycetes. Our findings include the discovery of undescribed diversity in CotH, a Mucormycosis pathogenicity factor, which was found in a broad set of zygomycetes. Reconciliation analysis identified multiple duplication events and an expansion of CotH copies throughout the Mucoromycotina, Mortierellomycotina, Neocallimastigomycota, and Basidiobolus lineages. A kingdom-level phylogenomic analysis also identified new evolutionary relationships within the subphyla of Mucoromycota and Zoopagomycota, including supporting the sister-clade relationship between Glomeromycotina and Mortierellomycotina and the placement of Basidiobolus as sister to other Zoopagomycota lineages.

Generalist arbuscular mycorrhizal fungi dominated heavy metal polluted soils at two artisanal and small - scale gold mining sites in southeastern Ecuador.

BACKGROUND: Artisanal and small-scale gold mining activities are producing contamination with heavy metals and metalloids (HMM) into soils and water worldwide. The HMM are considered as one of the major abiotic stresses due to their long-term persistence in soil. In this context, arbuscular mycorrhizal fungi (AMF) confer resistance to a variety of abiotic plant stressors including HMM. However, little is known regarding the diversity and composition of AMF communities in heavy metal polluted sites in Ecuador. METHODS: In order to investigate the AMF diversity, root samples and associated soil of six plant species were collected from two sites polluted by heavy metals, located in Zamora-Chinchipe province, Ecuador. The AMF 18S nrDNA genetic region was analyzed and sequenced, and fungal OTUs were defined based on 99% sequence similarity. Results were contrasted with AMF communities from a natural forest and from reforestation sites located in the same province and with available sequences in GenBank. RESULTS: The main pollutants in soils were Pb, Zn, Hg, Cd and Cu with concentrations exceeding the soil reference value for agricultural use. Molecular phylogeny and OTU delimitation showed 19 OTUs, the family Glomeraceae was the most OTU-rich followed by Archaeosporaceae, Acaulosporaceae, Ambisporaceae and Paraglomeraceae. Most of the OTUs (11 of 19) have been found at other locations worldwide, 14 OTUs were proven from nearby non-contaminated sites in Zamora-Chinchipe. CONCLUSION: Our study showed that there are no specialized OTUs at the studied HMM polluted sites, but rather generalists adapted to a wide variety of habitats. Their potential role in phytoremediation approaches remains to be investigated.

Whole genome analyses based on single, field collected spores of the arbuscular mycorrhizal fungus Funneliformis geosporum.

Arbuscular mycorrhizal (AM) fungi are ubiquitous mutualistic symbionts of most terrestrial plants and many complete their lifecycles underground. Whole genome analysis of AM fungi has long been restricted to species and strains that can be maintained under controlled conditions that facilitate collection of biological samples. There is some evidence suggesting that AM fungi can adapt to culture resulting in phenotypic and possibly also genotypic changes in the fungi. In this study, we used field isolated spores of AM fungi and identified them as Funneliformis geosporum based on morphology and phylogenetic analyses. We separately assembled the genomes of two representative spores using DNA sequences of 19 and 22 individually amplified nuclei. The genomes were compared with previously published data from other members of Glomeraceae including two strains of F. mosseae. No significant differences were observed among the species in terms of gene content, while the single nucleotide polymorphism density was higher in the strains of F. geosporum than in the strains of F. mosseae. In this study, we demonstrate that it is possible to sequence and assemble genomes from AM fungal spores sampled in the field, which opens up the possibility to include uncultured AM fungi in phylogenomic and comparative genomic analysis and to study genomic variation in natural populations of these important plant symbionts.

Clonal spore populations in sporocarps of arbuscular mycorrhizal fungi.

Some arbuscular mycorrhizal (AM) fungal species known to form sporocarps (i.e., aggregations of spores) are polyphyletic in two orders, Glomerales and Diversisporales. Spore clusters (sporocarp-like structures) often formed in pot cultures or in vitro conditions are supposed to be clonal populations, while sporocarps in natural habitats with a fungal peridium are morphologically similar to those of epigeous sexual (zygosporic) sporocarps of Endogone species. Thus, in this study, we explored the genetics of sporocarpic spores of two AM fungi with a view to possibilities of clonal or sexual reproduction during sporocarps formation. To examine these possibilities, we investigated single-nucleotide polymorphisms (SNPs) in reduced genomic libraries of spores isolated from sporocarps molecularly identified as Rhizophagus irregularis and Diversispora epigaea. In addition, partial sequences of the MAT locus HD2 gene of R. irregularis were phylogenetically analyzed to determine the nuclear status of the spores. We found that most SNPs were shared among the spores isolated from each sporocarp in both species. Furthermore, all HD2 sequences from spores isolated from three R. irregularis sporocarps were identical. These results indicate that those sporocarps comprise clonal spores. Therefore, sporocarps with clonal spores may have different functions than sexual reproduction, such as massive spore production or spore dispersal via mycophagy.

Atmospheric pollution, soil nutrients and climate effects on Mucoromycota arbuscular mycorrhizal fungi.

Fine root endophyte mycorrhizal fungi in the Endogonales (Mucoromycota arbuscular mycorrhizal fungi, M-AMF) are now recognized as at least as important globally as Glomeromycota AMF (G-AMF), yet little is known about the environmental factors which influence M-AMF diversity and colonization, partly because they typically only co-colonize plants with G-AMF. Wild populations of Lycopodiella inundata predominantly form mycorrhizas with M-AMF and therefore allow focussed study of M-AMF environmental drivers. Using microscopic examination and DNA sequencing we measured M-AMF colonization and diversity over three consecutive seasons and modelled interactions between these response variables and environmental data. Significant relationships were found between M-AMF colonization and soil S, P, C:N ratio, electrical conductivity, and the previously overlooked micronutrient Mn. Estimated N deposition was negatively related to M-AMF colonization. Thirty-nine Endogonales Operational Taxonomic Units (OTUs) were identified in L. inundata roots, a greater diversity than previously recognized in this plant. Endogonales OTU richness correlated negatively with soil C:N while community composition was mostly influenced by soil P. This study provides first evidence that M-AMF have distinct ecological preferences in response to edaphic variables also related to air pollution. Future studies require site-level atmospheric pollution monitoring to guide critical load policy for mycorrhizal fungi in heathlands and grasslands.

Analysing diversification dynamics using barcoding data: The case of an obligate mycorrhizal symbiont.

Analysing diversification dynamics is key to understanding the past evolutionary history of clades that led to present-day biodiversity patterns. While such analyses are widespread in well-characterized groups of species, they are much more challenging in groups for which diversity is mostly known through molecular techniques. Here, we use the largest global database on the small subunit (SSU) rRNA gene of Glomeromycotina, a subphylum of microscopic arbuscular mycorrhizal fungi that provide mineral nutrients to most land plants by forming one of the oldest terrestrial symbioses, to analyse the diversification dynamics of this clade in the past 500 million years. We perform a range of sensitivity analyses and simulations to control for potential biases linked to the nature of the data. We find that Glomeromycotina tend to have low speciation rates compared to other eukaryotes. After a peak of speciations between 200 and 100 million years ago, they experienced an important decline in speciation rates toward the present. Such a decline could be at least partially related to a shrinking of their mycorrhizal niches and to their limited ability to colonize new niches. Our analyses identify patterns of diversification in a group of obligate symbionts of major ecological and evolutionary importance and illustrate that short molecular markers combined with intensive sensitivity analyses can be useful for studying diversification dynamics in microbial groups.

Early branching arbuscular mycorrhizal fungus Paraglomus occultum carries a small and repeat-poor genome compared to relatives in the Glomeromycotina.

The arbuscular mycorrhizal fungi (AMFs) are obligate root symbionts in the subphylum Glomeromycotina that can benefit land plants by increasing their soil nutrient uptake in exchange for photosynthetically fixed carbon sources. To date, annotated genome data from representatives of the AMF orders Glomerales, Diversisporales and Archaeosporales have shown that these organisms have large and highly repeated genomes, and no genes to produce sugars and fatty acids. This led to the hypothesis that the most recent common ancestor (MRCA) of Glomeromycotina was fully dependent on plants for nutrition. Here, we aimed to further test this hypothesis by obtaining annotated genome data from a member of the early diverging order Paraglomerales (Paraglomus occultum). Genome analyses showed this species carries a 39.6 Mb genome and considerably fewer genes and repeats compared to most AMF relatives with annotated genomes. Consistent with phylogenies based on ribosomal genes, our phylogenetic analyses suggest P. occultum as the earliest diverged branch within Glomeromycotina. Overall, our analyses support the view that the MRCA of Glomeromycotina carried hallmarks of obligate plant biotrophy. The small genome size and content of P. occultum could either reflect adaptive reductive processes affecting some early AMF lineages, or indicate that the high gene and repeat family diversity thought to drive AMF adaptability to host and environmental change was not an ancestral feature of these prominent plant symbionts.

Two AMP-Binding Domain Proteins from Rhizophagus irregularis Involved in Import of Exogenous Fatty Acids.

Arbuscular mycorrhizal fungi (AMF) colonize roots, where they provide nutrients in exchange for sugars and lipids. Because AMF lack genes for cytosolic fatty acid de novo synthase (FAS), they depend on host-derived fatty acids. AMF colonization is accompanied by expression of specific lipid genes and synthesis of sn-2 monoacylglycerols (MAGs). It is unknown how host-derived fatty acids are taken up by AMF. We describe the characterization of two AMP-binding domain protein genes from Rhizophagus irregularis, RiFAT1 and RiFAT2, with sequence similarity to Saccharomyces cerevisiae fatty acid transporter 1 (FAT1). Uptake of 13C-myristic acid (14:0) and, to a lesser extent, 13C-palmitic acid (16:0) was enhanced after expression of RiFAT1 or RiFAT2 in S. cerevisiae Δfat1 cells. The uptake of 2H-labeled fatty acids from 2H-myristoylglycerol or 2H-palmitoylglycerol was also increased after RiFAT1 and RiFAT2 expression in Δfat, but intact 2H-MAGs were not detected. RiFAT1 and RiFAT2 expression was induced in colonized roots compared with extraradical mycelium. 13C-label in the AMF-specific palmitvaccenic acid (16:1Δ11) and eicosatrienoic acid (20:3) were detected in colonized roots only when 13C2-acetate was supplemented but not 13C-fatty acids, demonstrating that de novo synthesized, host-derived fatty acids are rapidly taken up by R. irregularis from the roots. The results show that RiFAT1 and RiFAT2 are involved in the uptake of myristic acid (14:0) and palmitic acid (16:0), while fatty acids from MAGs are only taken up after hydrolysis. Therefore, the two proteins might be involved in fatty acid import into the fungal arbuscules in colonized roots.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Environmental identification of arbuscular mycorrhizal fungi using the LSU rDNA gene region: an expanded database and improved pipeline.

Arbuscular mycorrhizal fungi (AMF; Glomeromycota) are difficult to culture; therefore, establishing a robust amplicon-based approach to taxa identification is imperative to describe AMF diversity. Further, due to low and biased sampling of AMF taxa, molecular databases do not represent the breadth of AMF diversity, making database matching approaches suboptimal. Therefore, a full description of AMF diversity requires a tool to determine sequence-based placement in the Glomeromycota clade. Nonetheless, commonly used gene regions, including the SSU and ITS, do not enable reliable phylogenetic placement. Here, we present an improved database and pipeline for the phylogenetic determination of AMF using amplicons from the large subunit (LSU) rRNA gene. We improve our database and backbone tree by including additional outgroup sequences. We also improve an existing bioinformatics pipeline by aligning forward and reverse reads separately, using a universal alignment for all tree building, and implementing a BLAST screening prior to tree building to remove non-homologous sequences. Finally, we present a script to extract AMF belonging to 11 major families as well as an amplicon sequencing variant (ASV) version of our pipeline. We test the utility of the pipeline by testing the placement of known AMF, known non-AMF, and Acaulospora sp. spore sequences. This work represents the most comprehensive database and pipeline for phylogenetic placement of AMF LSU amplicon sequences within the Glomeromycota clade.

Long reads and Hi-C sequencing illuminate the two-compartment genome of the model arbuscular mycorrhizal symbiont Rhizophagus irregularis.

Chromosome folding links genome structure with gene function by generating distinct nuclear compartments and topologically associating domains. In mammals, these undergo preferential interactions and regulate gene expression. However, their role in fungal genome biology is unclear. Here, we combine Nanopore (ONT) sequencing with chromatin conformation capture sequencing (Hi-C) to reveal chromosome and epigenetic diversity in a group of obligate plant symbionts: the arbuscular mycorrhizal fungi (AMF). We find that five phylogenetically distinct strains of the model AMF Rhizophagus irregularis carry 33 chromosomes with substantial within-species variability in size, as well as in gene and repeat content. Strain-specific Hi-C contact maps reveal a 'checkerboard' pattern that underline two dominant euchromatin (A) and heterochromatin (B) compartments. Each compartment differs in the level of gene transcription, regulation of candidate effectors and methylation frequencies. The A-compartment is more gene-dense and contains most core genes, while the B-compartment is more repeat-rich and has higher rates of chromosomal rearrangement. While the B-compartment is transcriptionally repressed, it has significantly more secreted proteins and in planta upregulated candidate effectors, suggesting a possible host-induced change in chromosome conformation. Overall, this study provides a fine-scale view into the genome biology and evolution of model plant symbionts, and opens avenues to study the epigenetic mechanisms that modify chromosome folding during host-microbe interactions.

Symbiotic germination and development of fully mycoheterotrophic plants convergently targeting similar Glomeraceae taxa.

Plants producing dust seeds often meet their carbon demands by exploiting fungi at the seedling stage. This germination strategy (i.e. mycoheterotrophic germination) has been investigated among orchidaceous and ericaceous plants exploiting Ascomycota or Basidiomycota. Although several other angiosperm lineages have evolved fully mycoheterotrophic relationships with Glomeromycota, the fungal identities involved in mycoheterotrophic germination remain largely unknown. Here, we conducted in situ seed baiting and high-throughput DNA barcoding to identify mycobionts associated with seedlings of Burmannia championii (Burmanniaceae: Dioscoreales) and Sciaphila megastyla (Triuridaceae: Pandanales), which have independently evolved full mycoheterotrophy. Subsequently, we revealed that both seedlings and adults in B. championii and S. megastyla predominantly associate with Glomeraceae. However, mycorrhizal communities are somewhat distinct between seedling and adult stages, particularly in S. megastyla. Notably, the dissimilarity of mycorrhizal communities between S. megastyla adult samples and S. megastyla seedling samples is significantly higher than that between B. championi adult samples and S. megastyla adult samples, based on some indices. This pattern is possibly due to both mycorrhizal shifts during ontogenetic development and convergent recruitment of cheating-susceptible fungi. The extensive fungal overlap in two unrelated mycoheterotrophic plants indicates that both species convergently exploit specific AM fungal phylotypes.

Diverse and abundant arbuscular mycorrhizal fungi in ecological floating beds used to treat eutrophic water.

An increasing number of investigations have shown the universal existence of arbuscular mycorrhizal fungi (AMF) in aquatic ecosystems. However, little is known about the accurate distribution and function of AMF inhabiting aquatic ecosystems, especially ecological floating beds (EFBs), which are constructed for the remediation of polluted water bodies. In this study, we collected root samples of Canna generalis, Cyperus alternifolius, and Eichhornia crassipes from three EFBs on two eutrophic lakes in Wuhan, China. We aimed to investigate the resources and distribution of AMF in EFBs using Illumina Mi-seq technology. A total of 229 operational taxonomic units (OTUs) and 21 taxa from 348,799 Glomeromycota sequences were detected. Glomus and Acaulospora were the most dominant and second most dominant genera of AMF in the three EFBs, respectively. Different aquatic plant species showed varying degrees of AMF colonization (3.83-71%), diversity (6-103 OTUs, 3-15 virtual taxa), and abundance (14-57,551 sequences). Low AMF abundance, but relatively high AMF diversity, was found in C. alternifolius, which is usually considered non-mycorrhizal. This finding indicated the high accuracy of Illumina sequencing. Our results also revealed a lognormal species abundance distribution that was observed across AMF taxa in the three plant species. The AMF community composition was closely related to nitrogen and phosphorus contents. Overall, our data show that EFBs harbor diverse and abundant AMF communities. Additionally, the AMF community composition is closely related to the water quality of eutrophic lakes treated by the EFBs, indicating the potential application of AMF in plant-based bioremediation of wastewater. KEYPOINTS: • Aquatic plants in EFBs harbor diverse (229 OTUs) and abundant (348,799 sequences) AMF. • Different plant species host different taxa of AMF. Cyperaceae, originally considered non-mycorrhizal, may in fact be a variable mycorrhizal plant family. • The AMF community composition in EFBs is closely related to nutrient concentrations (nitrogen and phosphorus).

Generation of unequal nuclear genotype proportions in Rhizophagus irregularis progeny causes allelic imbalance in gene transcription.

Arbuscular mycorrhizal fungi (AMF) form mutualisms with most plant species. The model AMF Rhizophagus irregularis is common in many ecosystems and naturally forms homokaryons and dikaryons. Quantitative variation in allele frequencies in clonally dikaryon offspring suggests they disproportionately inherit two distinct nuclear genotypes from their parent. This is interesting, because such progeny strongly and differentially affect plant growth. Neither the frequency and magnitude of this occurrence nor its effect on gene transcription are known. Using reduced representation genome sequencing, transcriptomics, and quantitative analysis tools, we show that progeny of homokaryons and dikaryons are qualitatively genetically identical to the parent. However, dikaryon progeny differ quantitatively due to unequal inheritance of nuclear genotypes. Allele frequencies of actively transcribed biallelic genes resembled the frequencies of the two nuclear genotypes. More biallelic genes showed transcription of both alleles than monoallelic transcription, but biallelic transcription was less likely with greater allelic divergence. Monoallelic transcription levels of biallelic genes were reduced compared with biallelic gene transcription, a finding consistent with genomic conflict. Given that genetic variation in R. irregularis is associated with plant growth, our results establish quantitative genetic variation as a future consideration when selecting AMF lines to improve plant production.

Regulation of mating genes during arbuscular mycorrhizal isolate co-existence-where is the evidence?

A recent study published by Mateus et al. [1] claimed that 18 "mating-related" genes are differentially expressed in the model arbuscular mycorrhizal fungus (AMF) Rhizophagus irregularis when genetically distinct fungal strains co-colonize a host plant. To clarify the level of evidence for this interesting conclusion, we first aimed to validate the functional annotation of these 18 R. irregularis genes using orthology predictions. These analyses revealed that, although sequence relationship exists, only 2 of the claimed 18 R. irregularis mating genes are potential orthologues to validated fungal mating genes. We also investigated the RNA-seq data from Mateus et al. [1] using classical RNA-seq methods and statistics. This analysis found that the over-expression during strain co-existence was not significant at the typical cut-off of the R. irregularis strains DAOM197198 and B1 in plants. Overall, we do not find convincing evidence that the genes involved have functions in mating, or that they are reproducibly up or down regulated during co-existence in plants.