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Epulopiscium sp. type B (Lachnospiraceae) is an exceptionally large, highly polyploid, intestinal symbiont of the coral reef dwelling surgeonfish Naso tonganus. These obligate anaerobes do not form mature endospores and reproduce solely through the production of multiple intracellular offspring. This likely makes them dependent on immediate transfer to a receptive host for dispersal. During reproduction, only a small proportion of Epulopiscium mother-cell DNA is inherited. To explore the impact of this unusual viviparous lifestyle on symbiont population dynamics, we investigated Epulopiscium sp. type B and their fish hosts collected over the course of two decades, at island and reef habitats near Lizard Island, Australia. Using multi-locus sequence analysis, we found that recombination plays an important role in maintaining diversity of these symbionts and yet populations exhibit linkage disequilibrium (LD). Symbiont populations showed spatial but not temporal partitioning. Surgeonfish are long-lived and capable of traveling long distances, yet the population structures of Epulopiscium suggest that adult fish tend to not roam beyond a limited locale. Codiversification analyses and traits of this partnership suggest that while symbionts are obligately dependent on their host, the host has a facultative association with Epulopiscium. We suggest that congression of unlinked markers contributes to LD estimates in this and other recombinant populations of bacteria. The findings here inform our understanding of evolutionary processes within intestinal Lachnospiraceae populations.

Arbuscular mycorrhizal (AM) fungi form an intimate symbiosis with roots of more than 80% of land plants without eliciting a significant defense response, and how they do so is yet to be determined. Typically, plants mount a defense response upon sensing chitin in fungal walls, and to counteract this response, plant-pathogenic fungi secrete small effector proteins with chitin-binding LysM domains. In the AM fungus, Rhizophagus irregularis, a small, putatively-secreted LysM protein, which we refer to as RiSLM, is among the most highly expressed effector-like proteins during symbiosis. Here, we show that RiSLM expression is reduced during non-functional symbiosis with Medicago mutants, mtpt4-2 and vapyrin. We demonstrate that RiSLM can bind to both chitin and chitosan, and we model the protein-ligand interaction to identify possible binding sites. Finally, we have identified RiSLM homologs in five published R. irregularis isolate genomes and demonstrate that the gene is subject to a high rate of evolution and is experiencing positive selection, while still conserving putative function. Our results present important clues for elucidating a role for a LysM effector, RiSLM, in AM symbiosis.

Animals and plants interact with microbes by engaging specific surveillance systems, regulatory networks, and response modules that allow for accommodation of mutualists and defense against antagonists. Antimicrobial defense responses are mediated in both animals and plants by innate immunity systems that owe their functional similarities to convergent evolution. Like animals and plants, fungi interact with bacteria. However, the principles governing these relations are only now being discovered. In a study system of host and nonhost fungi interacting with a bacterium isolated from the host, we found that bacteria used a common gene repertoire to engage both partners. In contrast, fungal responses to bacteria differed dramatically between the host and nonhost. These findings suggest that as in animals and plants, the genetic makeup of the fungus determines whether bacterial partners are perceived as mutualists or antagonists and what specific regulatory networks and response modules are initiated during each encounter., Fungal-bacterial symbioses range from antagonisms to mutualisms and remain one of the least understood interdomain interactions despite their ubiquity as well as ecological and medical importance. To build a predictive conceptual framework for understanding interactions between fungi and bacteria in different types of symbioses, we surveyed fungal and bacterial transcriptional responses in the mutualism between Rhizopus microsporus (Rm) (ATCC 52813, host) and its Mycetohabitans (formerly Burkholderia) endobacteria versus the antagonism between a nonhost Rm (ATCC 11559) and Mycetohabitans isolated from the host, at two time points, before and after partner physical contact. We found that bacteria and fungi sensed each other before contact and altered gene expression patterns accordingly. Mycetohabitans did not discriminate between the host and nonhost and engaged a common set of genes encoding known as well as novel symbiosis factors. In contrast, responses of the host versus nonhost to endobacteria were dramatically different, converging on the altered expression of genes involved in cell wall biosynthesis and reactive oxygen species (ROS) metabolism. On the basis of the observed patterns, we formulated a set of hypotheses describing fungal-bacterial interactions and tested some of them. By conducting ROS measurements, we confirmed that nonhost fungi increased production of ROS in response to endobacteria, whereas host fungi quenched their ROS output, suggesting that ROS metabolism contributes to the nonhost resistance to bacterial infection and the host ability to form a mutualism. Overall, our study offers a testable framework of predictions describing interactions of early divergent Mucoromycotina fungi with bacteria.

Significance Endosymbiotic bacteria are found in diverse fungi, but little is known about how they communicate with their hosts. Some plant pathogenic bacteria use type III-translocated TAL effectors to control host transcription, and TAL-like proteins are encoded in genomes of the fungal endosymbiotic bacterium Mycetohabitans rhizoxinica. In this paper, we present evidence that these proteins are, like TAL effectors, type III-secreted, nuclear-localizing effectors that perturb host transcription and show that one enhances tolerance of the fungal host to cell membrane stress. Our characterization of an effector in a bacterial–fungal symbiosis opens a new door to molecular understanding of these interkingdom partnerships. Our findings also provide insight into the functional diversity and evolution of the TAL effector protein family., Symbioses of bacteria with fungi have only recently been described and are poorly understood. In the symbiosis of Mycetohabitans (formerly Burkholderia) rhizoxinica with the fungus Rhizopus microsporus, bacterial type III (T3) secretion is known to be essential. Proteins resembling T3-secreted transcription activator-like (TAL) effectors of plant pathogenic bacteria are encoded in the three sequenced Mycetohabitans spp. genomes. TAL effectors nuclear-localize in plants, where they bind and activate genes important in disease. The Burkholderia TAL-like (Btl) proteins bind DNA but lack the N- and C-terminal regions, in which TAL effectors harbor their T3 and nuclear localization signals, and activation domain. We characterized a Btl protein, Btl19-13, and found that, despite the structural differences, it can be T3-secreted and can nuclear-localize. A btl19-13 gene knockout did not prevent the bacterium from infecting the fungus, but the fungus became less tolerant to cell membrane stress. Btl19-13 did not alter transcription in a plant-based reporter assay, but 15 R. microsporus genes were differentially expressed in comparisons both of the fungus infected with the wild-type bacterium vs. the mutant and with the mutant vs. a complemented strain. Southern blotting revealed btl genes in 14 diverse Mycetohabitans isolates. However, banding patterns and available sequences suggest variation, and the btl19-13 phenotype could not be rescued by a btl gene from a different strain. Our findings support the conclusion that Btl proteins are effectors that act on host DNA and play important but varied or possibly host genotype-specific roles in the M. rhizoxinica–R. microsporus symbiosis.

Symbioses of bacteria with fungi have only recently been described and are poorly understood. In the symbiosis of Mycetohabitans (formerly Burkholderia) rhizoxinica with the fungus Rhizopus microsporus, bacterial type III (T3) secretion is known to be essential. Proteins resembling T3-secreted transcription activator-like (TAL) effectors of plant pathogenic bacteria are encoded in the three sequenced Mycetohabitans spp. genomes. TAL effectors nuclear localize in plants, where they bind and activate genes important in disease. The Burkholderia TAL-like (Btl) proteins bind DNA but lack the N- and C-terminal regions in which TAL effectors harbor their T3 and nuclear localization signals, and activation domain. We characterized a Btl protein, Btl19-13, and found that, despite the structural differences, it can be T3-secreted and can nuclear localize. A btl19-13 gene knockout did not prevent the bacterium from infecting the fungus, but the fungus became less tolerant to cell membrane stress. Btl19-13 did not alter transcription in a plant-based reporter assay, but 15 R. microsporus genes were differentially expressed in comparisons both of the fungus infected with the wildtype bacterium vs the mutant and with the mutant vs. a complemented strain. Southern blotting revealed btl genes in 14 diverse Mycetohabitans isolates. However, banding patterns and available sequences suggest variation, and the btl19-13 phenotype could not be rescued by a btl gene from a different strain. Our findings support the conclusion that Btl proteins are effectors that act on host DNA and play important but varied or possibly host-genotype-specific roles in the M. rhizoxinica-R. microsporus symbiosis.

The recent accumulation of newly discovered fungal-bacterial mutualisms challenges the paradigm that fungi and bacteria are natural antagonists. To understand the mechanisms that govern the establishment and maintenance over evolutionary time of mutualisms between fungi and bacteria, we studied a symbiosis of the fungus Rhizopus microsporus (Mucoromycotina) and its Burkholderia endobacteria. We found that nonhost R. microsporus, as well as other mucoralean fungi, interact antagonistically with endobacteria derived from the host and are not invaded by them. Comparison of gene expression profiles of host and nonhost fungi during interaction with endobacteria revealed dramatic changes in expression of lipid metabolic genes in the host. Analysis of the host lipidome confirmed that symbiosis establishment was accompanied by specific changes in the fungal lipid profile. Diacylglycerol kinase (DGK) activity was important for these lipid metabolic changes, as its inhibition altered the fungal lipid profile and caused a shift in the host-bacterial interaction into an antagonism. We conclude that adjustments in host lipid metabolism during symbiosis establishment, mediated by DGKs, are required for the mutualistic outcome of the Rhizopus-Burkholderia symbiosis. In addition, the neutral and phospholipid profiles of R. microsporus provide important insights into lipid metabolism in an understudied group of oleaginous Mucoromycotina. Lastly, our study revealed that the DGKs involved in the symbiosis form a previously uncharacterized clade of DGK domain proteins.

Arbuscular mycorrhizal fungi (AMF, Glomeromycotina), in addition to forming symbioses with the majority of land plants, harbor vertically transmitted endosymbiotic bacteria 'Candidatus Glomeribacter gigasporarum' (CaGg) and 'Candidatus Moeniiplasma glomeromycotorum' (CaMg). CaGg is a nonessential mutualist of AMF, whereas the lifestyle of CaMg is unknown. To start unraveling the interactions between AMF and their endosymbionts in nature, we examined diversity and distribution of AMF-associated endobacteria in North Atlantic dunes at Cape Cod. Of nearly 500 foredune AMF isolates successfully genotyped during a systematic study, 94% were classified as Gigasporaceae. Two percent of all AMF spores harbored CaGg, and 88% contained CaMg. CaGg was found only in the Gigasporaceae, whereas CaMg was present in Gigasporaceae, Acaulosporaceae, and Diversisporaceae. Incidence of CaGg across AMF was not affected by any of the environmental parameters measured, whereas distribution of CaMg in one of the fungal hosts was impacted by plant density. CaMg populations associated with AMF individuals displayed high levels of genetic diversity but no evidence of gene flow, suggesting that host physical proximity is not sufficient to facilitate horizontal transmission of CaMg. Finally, in addition to a novel lineage of CaGg, we discovered that AMF likely harbor Burkholderia-related bacteria with close phylogenetic affinity to free-living Burkholderia and endobacteria of other Mucoromycota fungi.

The mycoplasma-related endobacteria (MRE), representing a recently discovered lineage of Mollicutes, are widely distributed across arbuscular mycorrhizal fungi (AMF, Glomeromycota). AMF colonize roots of most terrestrial plants and improve plant mineral nutrient uptake in return for plant-assimilated carbon. The role of MRE in the biology of their fungal hosts is unknown. To start characterizing this association, we assessed partitioning of MRE genetic diversity within AMF individuals and across the AMF phylogeographic range. We further used molecular evolution patterns to make inferences about MRE codivergence with AMF, their lifestyle and antiquity of the Glomeromycota-MRE association. While we did not detect differentiation between MRE derived from different continents, high levels of diversity were apparent in MRE populations within AMF host individuals. MRE exhibited significant codiversification with AMF over ecological time and the absence of codivergence over evolutionary time. Moreover, genetic recombination was evident in MRE. These patterns indicate that, while MRE transmission is predominantly vertical, their complex intrahost populations are likely generated by horizontal transmission and recombination. Based on predictions of evolutionary theory, we interpreted these observations as a suggestion that MRE may be antagonists of AMF. Finally, we detected a marginally significant signature of codivergence of MRE with Glomeromycota and the Endogone lineage of Mucoromycotina, implying that the symbiosis between MRE and fungi may predate the divergence between these two groups of fungi.

Despite its importance to energy flow and nutrient cycling the process of fine root decomposition has received comparatively little detailed research. Disruption of the fine root-soil interface during preparation of root litterbags for decomposition studies could affect decay rates and nutrient mobilization in part by altering the community of decay organisms. We compared rates of decomposition and nutrient release from fine roots of pine between litterbags and intact cores and characterized the fungal community in the decomposing roots. Fine root decomposition was about twice as fast overall for intact cores than litterbags, and rapid mobilization of N and P was observed for roots in cores whereas nutrients were immobilized in litterbags. Fungal communities characterized using 454 pyrosequencing were considerably different between decaying roots in intact cores and litterbags. Most interesting, taxa from ectomycorrhizal fungal orders such as Boletales, Thelephorales and Cantharellales appeared to be more common in decaying roots from cores than litterbags. Moreover, the rate of N and P mobilization from decaying fine roots was highly correlated with taxa from two orders of ectomycorrhizal fungi (Thelephorales, Cantharellales). Although we caution that DNA identified from the decaying roots cannot be conclusively ascribed to active fungi, the results provide tentative support for a significant role of ectomycorrhizal fungi in decomposition and nutrient mobilization from fine roots of pine.

Arbuscular mycorrhizal fungi (AMF, subphylum Glomeromycotina) are symbionts of most terrestrial plants. They commonly harbour endobacteria of a largely unknown biology, referred to as MRE ( M ollicutes /mycoplasma-related endobacteria). Here, we propose to accommodate MRE in the novel genus ‘Candidatus Moeniiplasma.’ Phylogeny reconstructions based on the 16S rRNA gene sequences cluster ‘Ca. Moeniiplasma ’ with representatives of the class Mollicutes , whereas phylogenies derived from amino acid sequences of 19 genes indicate that it is a discrete lineage sharing ancestry with the members of the family Mycoplasmataceae . Cells of ‘Ca. Moeniiplasma ’ reside directly in the host cytoplasm and have not yet been cultivated. They are coccoid, ~500 nm in diameter, with an electron-dense layer outside the plasma membrane. However, the draft genomes of ‘Ca. Moeniiplasma ’ suggest that this structure is not a Gram-positive cell wall. The evolution of ‘Ca. Moeniiplasma ’ appears to be driven by an ultrarapid rate of mutation accumulation related to the loss of DNA repair mechanisms. Moreover, molecular evolution patterns suggest that, in addition to vertical transmission, ‘Ca. Moeniiplasma ’ is able to transmit horizontally among distinct Glomeromycotina host lineages and exchange genes. On the basis of these unique lifestyle features, the new species ‘Candidatus Moeniiplasma glomeromycotorum’ is proposed.

Arbuscular mycorrhizal (AM) fungi (phylum Glomeromycota) are important components of natural and managed ecosystems. We explored the AM fungal assemblage in a selection of maize fields across a landscape in eastern New York State and characterized their diversity, dominance, and species abundance distribution. In this managed agroecosystem, we could investigate environment-influenced composition and diversity patterns unencumbered by immediate host species effects. We found that AM fungal taxon abundances were distributed lognormally, which suggests that the fungal community structure is shaped in a complex manner by many interacting niche-related factors rather than by only a single factor of disturbance associated with agricultural management. In addition to species abundance distribution, the focal assemblage shared with natural AM fungal communities a pattern of very strong dominance of certain taxa. To quantify this pattern, we developed two new indices “overdominance” and “inequitability”. Contrary to expectations based on observations of natural AM fungal communities, we found that most of the individual field communities were dominated by taxa from within a narrow phylogenetic range. At the landscape scale, we did not find an inverse relationship between the levels of taxonomic richness and phylogenetic relatedness expected in complex communities shaped by competitive exclusion.

The arbuscular mycorrhizal (AM) fungi (phylum Glomeromycota) are important for the functioning of terrestrial ecosystems because of their influence on plant nutrient relations and plant responses to stress. To assess the impact of dispersal limitation and identify niche-related environmental gradients affecting AM fungal community composition and structure, we studied AM fungal communities in an assemblage of maize fields across an eastern New York State landscape. We expressed AM fungal community differences in terms of abundance structure (Bray–Curtis dissimilarities), the presence of unique phylogenetic lineages (UniFrac), and mean phylogenetic relatedness between samples (mean patristic distance, MPD). We did not find strong evidence of dispersal limitation or isolation by distance within or between field sites. To identify environmental factors that may be related to community differentiation, we projected vectors of edaphic variables onto nonmetric multidimensional scaling (NMDS) ordinations of community dissimilarity measures. Of these factors, soil textural components appeared most strongly related to AM fungal community differences. We speculate that this pattern may be explained by the relationship between texture and soil moisture availability. In addition to soil textural components, phylogenetic measures of community differentiation suggested that AM fungal community structure was affected by nutrient concentrations, particularly Mg. Of the two phylogenetic indices of community differentiation, MPD was more consistent and stable with our data, whereas the UniFrac metric failed to be interpretable in several cases. Overall, our data suggest that, rather than phosphorus or pH, soil texture may have an influence on AM fungal community structure over large agroecological scales.

Heritable endobacteria, which are transmitted from one host generation to the next, are subjected to evolutionary forces that are different from those experienced by free-living bacteria. In particular, they suffer consequences of Muller’s ratchet, a mechanism that leads to extinction of small asexual populations due to fixation of slightly deleterious mutations combined with the random loss of the most-fit genotypes, which cannot be recreated without recombination. Mycoplasma-related endobacteria (MRE) are heritable symbionts of fungi from two ancient lineages, Glomeromycota (arbuscular mycorrhizal fungi) and Mucoromycotina . Previous studies revealed that MRE maintain unusually diverse populations inside their hosts and may have been associated with fungi already in the early Paleozoic. Here we show that MRE are vulnerable to genomic degeneration and propose that they defy Muller’s ratchet thanks to retention of recombination and genome plasticity. We suggest that other endobacteria may be capable of raising similar defenses against Muller’s ratchet.

Bacterial endosymbionts are critical to the existence of many eukaryotes. Among them, vertically transmitted endobacteria are uniquely typified by reduced genomes and molecular evolution rate acceleration relative to free-living taxa. These patterns are attributable to genetic drift-dominated degenerative processes associated with reproductive dependence on the host. The degenerative evolution scenario is well supported in endobacteria with strict vertical transmission, such as essential mutualists of insects. In contrast, heritable endosymbionts that are nonessential to their hosts and engage occasionally in horizontal transmission are expected to display deviations from the degenerative evolution model. To explore evolution patterns in such nonessential endobacteria, we focused on Candidatus Glomeribacter gigasporarum ancient heritable mutualists of fungi. Using a collection of genomes, we estimated in Glomeribacter mutation rate at 2.03 × 10(-9) substitutions per site per year and effective population size at 1.44 × 10(8) Both fall within the range of values observed in free-living bacteria. To assess the ability of Glomeribacter to purge slightly deleterious mutations, we examined genome-wide dN/dS values and distribution patterns. We found that these dN/dS profiles cluster Glomeribacter with free-living bacteria as well as with other nonessential endosymbionts, while distinguishing it from essential heritable mutualists of insects. Finally, our evolutionary simulations revealed that the molecular evolution rate acceleration in Glomeribacter is caused by limited recombination in a largely clonal population rather than by increased fixation of slightly deleterious mutations. Based on these patterns, we propose that genome evolution in Glomeribacter is nondegenerative and exemplifies a departure from the model of degenerative evolution in heritable endosymbionts.

Recent accumulation of data from newly discovered bacterial–fungal symbioses revealed that many of these associations are suitable as models to understand evolution of symbioses in general. Thus far, alliances formed by early divergent lineages of Mucoromycotina and Glomeromycota with heritable beta-proteobacteria and Mollicutes yielded novel insights into the forms of evolutionary trajectories in mutualisms and into mechanisms of symbiont genome evolution. Future work is expected to reveal molecular underpinnings of symbiont vertical transmission and forces that drive coevolution between the partners.

Arbuscular mycorrhizal fungi (phylum Glomeromycota) are among the oldest and most successful symbionts of land plants. With no evidence of sexual reproduction, their evolutionary success is inconsistent with the prediction that asexual taxa are vulnerable to extinction due to accumulation of deleterious mutations. To explore why Glomeromycota defy this prediction, we studied ribosomal RNA (rRNA) gene evolution in the Claroideoglomus lineage and estimated effective population size, N(e) , in C. etunicatum. We found that rRNA genes of these fungi exhibit unusual and complex patterns of molecular evolution. In C. etunicatum, these patterns can be collectively explained by an unexpectedly large N(e) combined with imperfect genome-wide and population-level rRNA gene repeat homogenization. The mutations accumulated in rRNA gene sequences indicate that natural selection is effective at purging deleterious mutations in the Claroideoglomus lineage, which is also consistent with the large N(e) of C. etunicatum. We propose that in the near absence of recombination, asexual reproduction involving massively multinucleate spores typical for Glomeromycota is responsible for the improved efficacy of selection relative to drift. We postulate that large effective population sizes contribute to the evolutionary longevity of Glomeromycota.

Many eukaryotes interact with heritable endobacteria to satisfy diverse metabolic needs. Some of these interactions are facultative symbioses, in which one partner is not essential to the other. Facultative symbioses are expected to be transitional stages along an evolutionary trajectory toward obligate relationships. We tested this evolutionary theory prediction in Ca. Glomeribacter gigasporarum, nonessential endosymbionts of arbuscular mycorrhizal fungi (Glomeromycota). We found that heritable facultative mutualisms can be both ancient and evolutionarily stable. We detected significant patterns of codivergence between the partners that we would only expect in obligate associations. Using codiverging partner pairs and the fungal fossil record, we established that the Glomeromycota‐Glomeribacter symbiosis is at least 400 million years old. Despite clear signs of codivergence, we determined that the Glomeribacter endobacteria engage in recombination and host switching, which display patterns indicating that the association is not evolving toward reciprocal dependence. We postulate that low frequency of recombination in heritable endosymbionts together with host switching stabilize facultative mutualisms over extended evolutionary times.

Arbuscular mycorrhizal (AM) fungi in the phylum Glomeromycota colonize roots of the majority of land plants and assist them in the uptake of mineral nutrients in exchange for plant-assimilated carbon. In the absence of sexual reproductive structures and with asexual spore morphology conserved since the Ordovician, Glomeromycota may be one of the oldest eukaryotic lineages that rely predominantly on asexual reproduction for gene transmission. Clonal population structure detected in the majority of AM fungi examined to date supports this hypothesis. However, evidence of recombination found in few local populations suggests that genetic exchanges may be more common in these organisms than is currently recognized. To explore the significance of clonal expansion versus genetic recombination in the life history of modern Glomeromycota, we examined the global population of a cosmopolitan fungus Glomus etunicatum and made inferences about the population structure and the occurrence of recombination in the history of this species. We sampled eight loci from 84 isolates. We found that although the global population of G. etunicatum showed a pattern of significant differentiation, several haplotypes had a broad geographic distribution spanning multiple continents. Molecular variation among the sampled isolates indicated an overwhelmingly clonal population structure and suggested that clonal expansion plays an important role in the ecological success of modern Glomeromycota. In contrast, a pattern of homoplasy consistent with a history of recombination suggested that gene exchanges are not completely absent from the life history of these organisms, although they are likely to be very rare.

Arbuscular mycorrhizal fungi (AMF, Glomeromycota) colonize roots of the majority of terrestrial plants. They provide essential minerals to their plant hosts and receive photosynthates in return. All major lineages of AMF harbor endobacteria classified as Mollicutes, and known as mycoplasma-related endobacteria (MRE). Except for their substantial intrahost genetic diversity and ability to transmit vertically, virtually nothing is known about the life history of these endobacteria. To understand MRE biology, we sequenced metagenomes of three MRE populations, each associated with divergent AMF hosts. We found that each AMF species harbored a genetically distinct group of MRE. Despite vertical transmission, all MRE populations showed extensive chromosomal rearrangements, which we attributed to genetic recombination, activity of mobile elements, and a history of plectroviral invasion. The MRE genomes are characterized by a highly reduced gene content, indicating metabolic dependence on the fungal host, with the mechanism of energy production remaining unclear. Several MRE genes encode proteins with domains involved in protein–protein interactions with eukaryotic hosts. In addition, the MRE genomes harbor genes horizontally acquired from AMF. Some of these genes encode small ubiquitin-like modifier (SUMO) proteases specific to the SUMOylation systems of eukaryotes, which MRE likely use to manipulate their fungal host. The extent of MRE genome plasticity and reduction, along with the large number of horizontally acquired host genes, suggests a high degree of adaptation to the fungal host. These features, together with the ubiquity of the MRE–Glomeromycota associations, emphasize the significance of MRE in the biology of Glomeromycota.

Arbuscular mycorrhizal (AM) fungi (Glomeromycota) colonize roots of the majority of land plants and facilitate their mineral nutrient uptake. Consequently, AM fungi play an important role in terrestrial ecosystems and are becoming a component of sustainable land management practices. The absence of sexual reproductive structures in modern Glomeromycota combined with their long evolutionary history suggest that these fungi may represent an ancient asexual lineage of great potential interest to evolutionary biology. However, many aspects of basic AM fungal biology, including genome structure, within-individual genetic variation, and reproductive mode are poorly understood. These knowledge gaps hinder research on the mechanisms of AM fungal interactions with individual plants and plant communities, and utilization of AM fungi in agricultural practices. I present here the current state of research on the reproduction in AM fungi and indicate what new findings can be expected in the future.

The rate of global deposition of Cd, Pb, and Zn has decreased over the past few decades, but heavy metals already in the soil may be mobilized by local and global changes in soil conditions and exert toxic effects on soil microorganisms. We examined in vitro effects of Cd, Pb, and Zn on critical life stages in metal-sensitive ecotypes of arbuscular mycorrhizal (AM) fungi, including spore germination, presymbiotic hyphal extension, presymbiotic sporulation, symbiotic extraradical mycelium expansion, and symbiotic sporulation. Despite long-term culturing under the same low-metal conditions, two species, Glomus etunicatum and Glomus intraradices , had different levels of sensitivity to metal stress. G. etunicatum was more sensitive to all three metals than was G. intraradices . A unique response of increased presymbiotic hyphal extension occurred in G. intraradices exposed to Cd and Pb. Presymbiotic hyphae of G. intraradices formed presymbiotic spores, whose initiation was more affected by heavy metals than was presymbiotic hyphal extension. In G. intraradices grown in compartmentalized habitats with only a portion of the extraradical mycelium exposed to metal stress, inhibitory effects of elevated metal concentrations on symbiotic mycelial expansion and symbiotic sporulation were limited to the metal-enriched compartment. Symbiotic sporulation was more sensitive to metal exposure than symbiotic mycelium expansion. Patterns exhibited by G. intraradices spore germination, presymbiotic hyphal extension, symbiotic extraradical mycelium expansion, and sporulation under elevated metal concentrations suggest that AM fungi may be able to survive in heavy metal-contaminated environments by using a metal avoidance strategy.

Arbuscular mycorrhizal (AM) fungi (Glomeromycota) are thought to be the oldest group of asexual multicellular organisms. They colonize the roots of most land plants, where they facilitate mineral uptake from the soil in exchange for plant-assimilated carbon1. Cells of AM fungi contain hundreds of nuclei. Unusual polymorphism of ribosomal DNA observed in individual spores of AM fungi inspired a hypothesis that heterokaryosis—that is, the coexistence of many dissimilar nuclei in cells—occurs throughout the AM fungal life history2,3. Here we report a genetic approach to test the hypothesis of heterokaryosis in AM fungi. Our study of the transmission of polymorphic genetic markers in natural isolates of Glomus etunicatum, coupled with direct amplification of rDNA from microdissected nuclei by polymerase chain reaction, supports the alternative hypothesis of homokaryosis, in which nuclei populating AM fungal individuals are genetically uniform. Intrasporal rDNA polymorphism contained in each nucleus signals a relaxation of concerted evolution4, a recombination-driven process that is responsible for homogenizing rDNA repeats5. Polyploid organization of glomeromycotan genomes could accommodate intranuclear rDNA polymorphism and buffer these apparently asexual organisms against the effects of accumulating mutations.

Arbuscular mycorrhizal (AM) fungi (phylum Glomeromycota) are one of the most important yet least understood groups of soil microorganisms. This chapter discusses recent findings concerning the genome structure in Glomeromycota in the context of the evolutionary theory predictions. The extent of the intraindividual polymorphism of the PLS1 and rRNA genes combined with the uncommonly large genome size estimates available at the time led Pawlowska and Taylor to speculate that genomes of Glomeromycota may be polyploid. A solution to the dilemma of how multiple gene copies are distributed among the nuclei of Glomeromycota is expected to emerge from the complete genome sequence of Glomus intraradices, which is anticipated to be available in 2007. This genome sequence and future projects to sequence genomes of other AM fungi are also likely to shed light on the source of the huge diversity of genome sizes in various species of Glomeromycota that was uncovered recently. To better understand the patterns of the rDNA variation in Glomeromycota, the chapter first explores general mechanisms behind the process of concerted evolution and possible causes of deviations from the molecular pattern of concerted evolution. The conflicting inferences about the genetic makeup and genome structure of Glomeromycota are clearly a reflection of the scarcity of experimental data. Some of the outstanding questions will be resolved by the examination of genome sequence data of representative AM fungi when these become available in the near future.

Arbuscular mycorrhizal fungi (AMF) are important members of the plant microbiome. They are obligate biotrophs that colonize the roots of most land plants and enhance host nutrient acquisition. Many AMF themselves harbor endobacteria in their hyphae and spores. Two types of endobacteria are known in Glomeromycota: rod-shaped Gram-negative Candidatus Glomeribacter gigasporarum, CaGg, limited in distribution to members of the Gigasporaceae family, and coccoid Mollicutes-related endobacteria, Mre, widely distributed across different lineages of AMF. The goal of the present study is to investigate the patterns of distribution and coexistence of the two endosymbionts, CaGg and Mre, in spore samples of several strains of Gigaspora margarita. Based on previous observations, we hypothesized that some AMF could host populations of both endobacteria. To test this hypothesis, we performed an extensive investigation of both endosymbionts in G. margarita spores sampled from Cameroonian soils as well as in the Japanese G. margarita MAFF520054 isolate using different approaches (molecular phylotyping, electron microscopy, fluorescence in situ hybridization and quantitative real-time PCR). We found that a single AMF host can harbour both types of endobacteria, with Mre population being more abundant, variable and prone to recombination than the CaGg one. Both endosymbionts seem to retain their genetic and lifestyle peculiarities regardless of whether they colonize the host alone or together. These findings show for the first time that fungi support an intracellular bacterial microbiome, in which distinct types of endobacteria coexist in a single cell.

Progress in understanding the biology of arbuscular mycorrhizal fungi is hampered by the limited number of species that can be successfully propagated and studied in vitro . We report the establishment of monoxenic cultures of Glomus etunicatum in association with excised Ri T-DNA transformed carrot roots. The fungus can be propagated in vitro using monoxenically formed resting spores and/or colonized root fragments. Modified White's medium buffered with 10 mM MES (pH 6) or MOPSO (pH 6.5) was most optimal for the host root growth as well as for G. etunicatum spore germination and mycorrhiza formation. The number of resting spores formed in vitro correlated positively with the length of roots occupied by arbuscular mycorrhizal structures, including arbuscules and vesicles. Spores first appeared in dual cultures within two weeks of root inoculation. Sporulation was asynchronous and continued until root senescence. Under applied culture conditions, spores achieved mature appearance within 5–7 d after their initiation. Approximately 6% of monoxenic spores were aborted at different stages of their development. Although G. etunicatum spores formed in vitro exhibited general morphological and anatomical similarity to soil-borne inoculum, they were significantly smaller and had thicker spore walls than their soil-borne counterparts. Caution should, therefore, be exercised in utilizing the in vitro system as a model of growth and development of glomalean fungi in soil.

The arbuscular mycorrhizal (AM) status of two plant communities on a calamine spoil mound (rich in cadmium, lead and zinc) in southern Poland was surveyed: an undisturbed grassland community and an early succession community that developed after complete removal of the surface layer of the calamine substrate about 10 years earlier. The undisturbed site harbored 40 herbaceous species making up 87% of the absolute cover. AM colonization was recorded in 25 species accounting for 77% of the relative cover. Species with 51–75% AM root colonization such as Festuca ovina and Leontodon hispidus dominated the undisturbed turf, contributing 45% to the relative cover. Carex ssp. were the most abundant nonmycorrhizal plants and accounted for 9% of the relative cover. Spores of Glomus aggregatum, G. constrictum, G. fasciculatum, G. pansihalos, Glomus sp. and Entrophospora sp. averaged 25 per 100 g dry substrate at the undisturbed site. The disturbed site was colonized by 25 species accounting for 17% of the absolute cover. Among the AM plants, most abundant were the species with up to 20% AM root colonization, such as Agrostis stolonifera and Thymus pulegioides, wich accounted for 24% of the relative cover. Nonmycorrhizal species, such as Biscutella laevigata,Cardaminopsis arenosa, Gypsophila fastigiata and Silene vulgaris, dominated the early succession community and contributed 64% to the relative cover. Spores of G. fasciculatum and Entrophospora sp. averaged 20 per 100 g dry substrate at the disturbed site.

Arbuscular mycorrhizal fungi (phylum Glomeromycota) are among the oldest and most successful symbionts of land plants. With no evidence of sexual reproduction, their evolutionary success is inconsistent with the prediction that asexual taxa are vulnerable to extinction due to accumulation of deleterious mutations. To explore why Glomeromycota defy this prediction, we studied ribosomal RNA (rRNA) gene evolution in the Claroideoglomus lineage and estimated effective population size, N(e) , in C. etunicatum. We found that rRNA genes of these fungi exhibit unusual and complex patterns of molecular evolution. In C. etunicatum, these patterns can be collectively explained by an unexpectedly large N(e) combined with imperfect genome-wide and population-level rRNA gene repeat homogenization. The mutations accumulated in rRNA gene sequences indicate that natural selection is effective at purging deleterious mutations in the Claroideoglomus lineage, which is also consistent with the large N(e) of C. etunicatum. We propose that in the near absence of recombination, asexual reproduction involving massively multinucleate spores typical for Glomeromycota is responsible for the improved efficacy of selection relative to drift. We postulate that large effective population sizes contribute to the evolutionary longevity of Glomeromycota.

Many eukaryotes interact with heritable endobacteria to satisfy diverse metabolic needs. Some of these interactions are facultative symbioses, in which one partner is not essential to the other. Facultative symbioses are expected to be transitional stages along an evolutionary trajectory toward obligate relationships. We tested this evolutionary theory prediction in Ca. Glomeribacter gigasporarum, nonessential endosymbionts of arbuscular mycorrhizal fungi (Glomeromycota). We found that heritable facultative mutualisms can be both ancient and evolutionarily stable. We detected significant patterns of codivergence between the partners that we would only expect in obligate associations. Using codiverging partner pairs and the fungal fossil record, we established that the Glomeromycota-Glomeribacter symbiosis is at least 400 million years old. Despite clear signs of codivergence, we determined that the Glomeribacter endobacteria engage in recombination and host switching, which display patterns indicating that the association is not evolving toward reciprocal dependence. We postulate that low frequency of recombination in heritable endosymbionts together with host switching stabilize facultative mutualisms over extended evolutionary times.

Interactions with soil microbiota determine the success of restoring plants to their native habitats. The goal of our study was to understand the effects of restoration practices on interactions of giant sequoia Sequoiadendron giganteum with arbuscular mycorrhizal (AM) fungi (Glomeromycota). Natural regeneration of Sequoiadendron is threatened by the absence of severe fires that create forest canopy gaps. Generating artificial canopy gaps offers an alternative tool for giant sequoia restoration. We investigated the effect of regeneration practices, including (i) sapling location within gaps, (ii) gap size and (iii) soil substrate, on AM fungal colonization of giant sequoia sapling roots in a native giant sequoia grove of the Sierra Nevada, California. We found that the extent of AM fungal root colonization was positively correlated with sapling height and light availability, which were related to the location of the sapling within the gap and the gap size. While colonization frequency by arbuscules in saplings on ash substrate was higher relative to saplings in mineral soil, the total AM fungal root colonization was similar between the substrates. A negative correlation between root colonization by Glomeromycota and non-AM fungal species indicated antagonistic interactions between different classes of root-associated fungi. Using DNA genotyping, we identified six AM fungal taxa representing genera Glomus and Ambispora present in Sequoiadendron roots. Overall, we found that AM fungal colonization of giant sequoia roots was associated with availability of plant-assimilated carbon to the fungus rather than with the AM fungal supply of mineral nutrients to the roots. We conclude that restoration practices affecting light availability and carbon assimilation alter feedbacks between sapling growth and activity of AM fungi in the roots.

The movement of mobile genetic elements (MGEs), including bacteriophages, insertion sequence (IS) elements, and integrative and conjugative elements (ICEs) can have profound effects on bacterial evolution by introducing novel genes, or disrupting the existing ones. Obligate endobacteria are a distinctive group of bacteria that reside within the intracellular compartments of their eukaryotic hosts. Many obligate endobacteria are reproductively dependent on their hosts. Vertical transmission, in addition to degenerative genome contraction and loss of MGEs, makes heritable endobacteria vulnerable to Muller's ratchet, a process that jeopardizes evolutionary longevity of small populations. Mycoplasma-related endobacteria (MRE) are ancient heritable endosymbionts of arbuscular mycorrhizal fungi. Their genomes harbour numerous MGEs. To explore the significance of MGEs in the evolution of MRE and other obligate endobacteria, we analyze the impact of transmission mode, recombination, and evolutionary age on the maintenance of MGEs. Furthermore, we discuss the ability of MGEs to act as sites of gene conversion and recombination in endobacterial genomes. We propose that MGEs are important instruments of genome shuffling, contributing to population heterogeneity and evolutionary longevity in heritable obligate endobacteria.

Arbuscular mycorrhizal fungi (Glomeromycota) are the dominant symbionts of land plants and one of the oldest multicellular lineages that exist without evidence of sexual reproduction. The mechanisms that protect these organisms from extinction due to accumulation of deleterious mutations in the absence of sexual recombination are unclear. Glomeromycota reproduce by spores containing hundreds of nuclei, which represents a departure from the typical eukaryotic developmental pattern, where a multicellular organism is re-created from a uninucleate propagule. To understand whether the multinucleate spore makeup may have contributed to the evolutionary success of Glomeromycota, we examined the dynamics of spore nuclei in Glomus etunicatum using live three-dimensional imaging and mathematical models. We show that the spores are populated by an influx of a stream of nuclei from the surrounding mycelium rather than by divisions of a single founder nucleus. We present evidence that mechanisms of selection are likely to operate at the level of individual nuclei. On the basis of mathematical analyses of the effects that these nuclear dynamics have on the population mutation load, we postulate that the developmental patterns of sporogenesis have adaptive significance for moderating the accumulation of deleterious mutations and may have contributed to the evolutionary longevity of Glomeromycota.

The prediction that progressive coupling of host and symbiont metabolic and reproductive interests leads to reduced mixing of symbiont lineages has been verified extensively in maternally transmitted bacterial endosymbionts of insects. To test whether this prediction is also applicable to associations of bacteria with fungi, we explored patterns of molecular evolution in two lineages of mutualistic endosymbionts of fungi: the Burkholderia endosymbionts of Rhizopus microsporus (Mucormycotina) and Candidatus Glomeribacter gigasporarum endosymbionts of arbuscular mycorrhizal fungi (Glomeromycota). We compared these two lineages with the closely related Candidatus Tremblaya princeps endosymbionts of mealybugs (Hemiptera, Coccoidea, Pseudococcidae) and to free-living Burkholderia species. To make inferences about the life histories of the endosymbionts, we relied on the empirically validated predictions of the nearly neutral theory of molecular evolution that a reduction of the effective population size increases the rate of fixation of slightly deleterious mutations. Our analyses showed that the slightly deleterious mutation accumulation patterns in the Burkholderia endosymbionts of Rhizopus were nearly indistinguishable from those in their free-living relatives. In contrast, Ca. Glomeribacter showed unique patterns of molecular evolution that differentiated them from both the Burkholderia endosymbionts of Rhizopus and from the Ca. Tremblaya endosymbionts of insects. These findings imply that reduced mixing of symbiont lineages is not a universal feature of symbioses between fungi and endocellular bacteria.

The shoot apical meristem (SAM) maintains a pool of indeterminate cells within the SAM proper, while lateral organs are initiated from the SAM periphery. Laser microdissection–microarray technology was used to compare transcriptional profiles within these SAM domains to identify novel maize genes that function during leaf development. Nine hundred and sixty-two differentially expressed maize genes were detected; control genes known to be upregulated in the initiating leaf (P0/P1) or in the SAM proper verified the precision of the microdissections. Genes involved in cell division/growth, cell wall biosynthesis, chromatin remodeling, RNA binding, and translation are especially upregulated in initiating leaves, whereas genes functioning during protein fate and DNA repair are more abundant in the SAM proper. In situ hybridization analyses confirmed the expression patterns of six previously uncharacterized maize genes upregulated in the P0/P1. P0/P1-upregulated genes that were also shown to be downregulated in leaf-arrested shoots treated with an auxin transport inhibitor are especially implicated to function during early events in maize leaf initiation. Reverse genetic analyses of asceapen1 (asc1), a maize D4-cyclin gene upregulated in the P0/P1, revealed novel leaf phenotypes, less genetic redundancy, and expanded D4-CYCLIN function during maize shoot development as compared to Arabidopsis. These analyses generated a unique SAM domain-specific database that provides new insight into SAM function and a useful platform for reverse genetic analyses of shoot development in maize., Author Summary All the organs of plant shoots are derived from the shoot apical meristem (SAM), a pool of plant stem cells that are both organogenic and self-sustaining. These dual SAM functions take place in distinct yet adjacent meristematic domains; leaves are derived from the peripheral zone (PZ) of the SAM whereas cells lost during organogenesis are replenished from the central zone (CZ). Deciphering the global patterns of differential gene expression within these discrete SAM functional domains is integral toward understanding the molecular-signaling networks that regulate plant development. We utilized laser-microdissection technology to isolate tissues from the SAM crown and center (SAM-proper) and from initiating leaves (P0/P1) at the SAM periphery for use in microarray comparisons of gene expression within these SAM functional domains. Nine hundred and sixty-two maize genes were differentially expressed, confirming that the distinct functions of these meristematic domains involve widespread differences in gene expression. Genes involved in cell division, cell wall biosynthesis, chromatin structure, and RNA binding are especially upregulated in initiating leaves, whereas genes regulating protein stability and DNA repair are upregulated in the SAM proper. Mutations in a D-cyclin gene that was upregulated in the P0/P1 render narrow-leafed mutant plants with defective stomatal patterning, providing functional genetic data for a previously uncharacterized maize gene.

Phytoextraction involves use of plants to remove toxic metals from soil. We examined the effects of phytoextraction practices with three plant species ( Silene vulgaris , Thlaspi caerulescens , and Zea mays ) and a factorial variation of soil amendments (either an ammonium or nitrate source of nitrogen and the presence or absence of an elemental sulfur supplement) on arbuscular mycorrhizal (AM) fungi (Glomales, Zygomycetes) at a moderately metal-contaminated landfill located in St. Paul, Minn. Specifically, we tested whether the applied treatments affected the density of glomalean spores and AM root colonization in maize. Glomalean fungi from the landfill were grouped into two morphotypes characterized by either light-colored spores (LCS) or dark-colored spores (DCS). Dominant species of the LCS morphotype were Glomus mosseae and an unidentified Glomus sp., whereas the DCS morphotype was dominated by Glomus constrictum . The density of spores of the LCS morphotype from the phytoremediated area was lower than the density of these spores in the untreated landfill soil. Within the experimental area, spore density of the LCS morphotype in the rhizosphere of mycorrhizal maize was significantly higher than in rhizospheres of nonmycorrhizal S. vulgaris or T. caerulescens . Sulfur supplement increased vesicular root colonization in maize and exerted a negative effect on spore density in maize rhizosphere. We conclude that phytoextraction practices, e.g., the choice of plant species and soil amendments, may have a great impact on the quantity and species composition of glomalean propagules as well as on mycorrhiza functioning during long-term metal-remediation treatments.

Pawlowska and Taylor reply - To challenge the hypothesis of multigenomic structure of arbuscular mycorrhizal (AM) fungi1,2, we presented three lines of evidence consistent with the homokaryotic organization of within-individual genetic variation, including distribution of polymorphic genetic markers among and within field isolates of an AM fungus, and distribution of ribosomal DNA variants among individually microdissected nuclei3. Bever and Wang suggest4 that our data can be explained equally well by heterokaryosis, proposing a model that relies on the assumption that fusions of hyphae of genetically non-identical individuals contribute to the creation and maintenance of a multigenomic status of AM fungal cells. However, we do not believe that this assumption is supported by existing biological evidence.