Mycoheterotrophy in the wood-wide web.

The prevalence and potential functions of common mycorrhizal networks, or the 'wood-wide web', resulting from the simultaneous interaction of mycorrhizal fungi and roots of different neighbouring plants have been increasingly capturing the interest of science and society, sometimes leading to hyperbole and misinterpretation. Several recent reviews conclude that popular claims regarding the widespread nature of these networks in forests and their role in the transfer of resources and information between plants lack evidence. Here we argue that mycoheterotrophic plants associated with ectomycorrhizal or arbuscular mycorrhizal fungi require resource transfer through common mycorrhizal networks and thus are natural evidence for the occurrence and function of these networks, offering a largely overlooked window into this methodologically challenging underground phenomenon. The wide evolutionary and geographic distribution of mycoheterotrophs and their interactions with a broad phylogenetic range of mycorrhizal fungi indicate that common mycorrhizal networks are prevalent, particularly in forests, and result in net carbon transfer among diverse plants through shared mycorrhizal fungi. On the basis of the available scientific evidence, we propose a continuum of carbon transfer options within common mycorrhizal networks, and we discuss how knowledge on the biology of mycoheterotrophic plants can be instrumental for the study of mycorrhizal-mediated transfers between plants.

Metabarcoding read abundances of orchid mycorrhizal fungi are correlated to copy numbers estimated using ddPCR.

Quantifying the abundances of fungi is key to understanding natural variation in mycorrhizal communities in relation to plant ecophysiology and environmental heterogeneity. High-throughput metabarcoding approaches have transformed our ability to characterize and compare complex mycorrhizal communities. However, it remains unclear how well metabarcoding read counts correlate with actual read abundances in the sample, potentially limiting their use as a proxy for species abundances. Here, we use droplet digital PCR (ddPCR) to evaluate the reliability of ITS2 metabarcoding data for quantitative assessments of mycorrhizal communities in the orchid species Neottia ovata sampled at multiple sites. We performed specific ddPCR assays for eight families of orchid mycorrhizal fungi and compared the results with read counts obtained from metabarcoding. Our results demonstrate a significant correlation between DNA copy numbers measured by ddPCR assays and metabarcoding read counts of major mycorrhizal partners of N. ovata, highlighting the usefulness of metabarcoding for quantifying the abundance of orchid mycorrhizal fungi. Yet, the levels of correlation between the two methods and the numbers of false zero values varied across fungal families, which warrants cautious evaluation of the reliability of low-abundance families. This study underscores the potential of metabarcoding data for more quantitative analyses of mycorrhizal communities and presents practical workflows for metabarcoding and ddPCR to achieve a more comprehensive understanding of orchid mycorrhizal communities.

Mycoheterotrophy.

Merckx and Gomes introduce mycoheterotrophy, the ability of a plant to take up its carbon from a close fungal partner.

Plastid phylogenomics and molecular evolution of Thismiaceae (Dioscoreales).

PREMISE: Species in Thismiaceae can no longer photosynthesize and instead obtain carbon from soil fungi. Here we infer Thismiaceae phylogeny using plastid genome data and characterize the molecular evolution of this genome. METHODS: We assembled five Thismiaceae plastid genomes from genome skimming data, adding to previously published data for phylogenomic inference. We investigated plastid-genome structural changes, considering locally colinear blocks (LCBs). We also characterized possible shifts in selection pressure in retained genes by considering changes in the ratio of nonsynonymous to synonymous changes (ω). RESULTS: Thismiaceae experienced two major pulses of gene loss around the early diversification of the family, with subsequent scattered gene losses across descendent lineages. In addition to massive size reduction, Thismiaceae plastid genomes experienced occasional inversions, and there were likely two independent losses of the plastid inverted repeat (IR) region. Retained plastid genes remain under generally strong purifying selection (ω << 1), with significant and sporadic weakening or strengthening in several instances. The bifunctional trnE-UUC gene of Thismia huangii may retain a secondary role in heme biosynthesis, despite a probable loss of functionality in protein translation. Several cis-spliced group IIA introns have been retained, despite the loss of the plastid intron maturase, matK. CONCLUSIONS: We infer that most gene losses in Thismiaceae occurred early and rapidly, following the initial loss of photosynthesis in its stem lineage. As a species-rich, fully mycoheterotrophic lineage, Thismiaceae provide a model system for uncovering the unique and divergent ways in which plastid genomes evolve in heterotrophic plants.

Mycoheterotrophic plants preferentially target arbuscular mycorrhizal fungi that are highly connected to autotrophic plants.

How mycoheterotrophic plants that obtain carbon and soil nutrients from fungi are integrated in the usually mutualistic arbuscular mycorrhizal networks is unknown. Here, we compare autotrophic and mycoheterotrophic plant associations with arbuscular mycorrhizal fungi and use network analysis to investigate interaction preferences in the tripartite network. We sequenced root tips from autotrophic and mycoheterotrophic plants to assemble the combined tripartite network between autotrophic plants, mycorrhizal fungi and mycoheterotrophic plants. We compared plant-fungi interactions between mutualistic and antagonist networks, and searched for a diamond-like module defined by a mycoheterotrophic and an autotrophic plant interacting with the same pair of fungi to investigate whether pairs of fungi simultaneously linked to plant species from each interaction type were overrepresented throughout the network. Mycoheterotrophic plants as a group interacted with a subset of the fungi detected in autotrophs but are indirectly linked to all autotrophic plants, and fungi with a high overlap in autotrophic partners tended to interact with a similar set of mycoheterotrophs. Moreover, pairs of fungi sharing the same mycoheterotrophic and autotrophic plant species are overrepresented in the network. We hypothesise that the maintenance of antagonistic interactions is maximised by targeting well linked mutualistic fungi, thereby minimising the risk of carbon supply shortages.

Mitochondrial genomic data are effective at placing mycoheterotrophic lineages in plant phylogeny.

Fully mycoheterotrophic plants can be difficult to place in plant phylogeny due to elevated substitution rates associated with photosynthesis loss. This potentially limits the effectiveness of downstream analyses of mycoheterotrophy that depend on accurate phylogenetic inference. Although mitochondrial genomic data sets are rarely used in plant phylogenetics, theory predicts that they should be resilient to long-branch artefacts, thanks to their generally slow evolution, coupled with limited rate elevation in heterotrophs. We examined the utility of mitochondrial genomes for resolving contentious higher-order placements of mycoheterotrophic lineages in two test cases: monocots (focusing on Dioscoreales) and Ericaceae. We find Thismiaceae to be distantly related to Burmanniaceae in the monocot order Dioscoreales, conflicting with current classification schemes based on few gene data sets. We confirm that the unusual Afrothismia is related to Taccaceae-Thismiaceae, with a corresponding independent loss of photosynthesis. In Ericaceae we recovered the first well supported relationships among its five major lineages: mycoheterotrophic Ericaceae are not monophyletic, as pyroloids are inferred to be sister to core Ericaceae, and monotropoids to arbutoids. Genes recovered from mitochondrial genomes collectively resolved previously ambiguous mycoheterotroph higher-order relationships. We propose that mitochondrial genomic data should be considered in standardised gene panels for inferring overall plant phylogeny.

Diversity of Fusarium associated banana wilt in northern Viet Nam.

Fusarium is one of the most important fungal genera of plant pathogens that affect the cultivation of a wide range of crops. Agricultural losses caused by Fusariumoxysporumf.sp.cubense (Foc) directly affect the income, subsistence, and nourishment of thousands of farmers worldwide. For Viet Nam, predictions on the impact of Foc for the future are dramatic, with an estimated loss in the banana production area of 8% within the next five years and up to 71% within the next 25 years. In the current study, we applied a combined morphological-molecular approach to assess the taxonomic identity and phylogenetic position of the different Foc isolates collected in northern Viet Nam. In addition, we aimed to estimate the proportion of the different Fusarium races infecting bananas in northern Viet Nam. The morphology of the isolates was investigated by growing the collected Fusarium isolates on four distinct nutritious media (PDA, SNA, CLA, and OMA). Molecular phylogenetic relationships were inferred by sequencing partial rpb1, rpb2, and tef1a genes and adding the obtained sequences into a phylogenetic framework. Molecular characterization shows that c. 74% of the Fusarium isolates obtained from infected banana pseudostem tissue belong to F.tardichlamydosporum. Compared to F.tardichlamydosporum, F.odoratissimum accounts for c.10% of the Fusarium wilt in northern Viet Nam, demonstrating that Foc TR4 is not yet a dominant strain in the region. Fusariumcugenangense - considered to cause Race 2 infections among bananas - is only found in c. 10% of the tissue material that was obtained from infected Vietnamese bananas. Additionally, one of the isolates cultured from diseased bananas was phylogenetically not positioned within the F.oxysporum species complex (FOSC), but in contrast, fell within the Fusariumfujikuroi species complex (FFSC). As a result, a possible new pathogen for bananas may have been found. Besides being present on several ABB 'Tay banana', F.tardichlamydosporum was also derived from infected tissue of a wild Musalutea, showing the importance of wild bananas as a possible sink for Foc.

Fungal diversity driven by bark features affects phorophyte preference in epiphytic orchids from southern China.

Epiphytic orchids exhibit varying degrees of phorophyte tree specificity. We performed a pilot study to investigate why epiphytic orchids prefer or avoid certain trees. We selected two orchid species, Panisea uniflora and Bulbophyllum odoratissimum co-occurring in a forest habitat in southern China, where they showed a specific association with Quercus yiwuensis and Pistacia weinmannifolia trees, respectively. We analysed a number of environmental factors potentially influencing the relationship between orchids and trees. Difference in bark features, such as water holding capacity and pH were recorded between Q. yiwuensis and P. weinmannifolia, which could influence both orchid seed germination and fungal diversity on the two phorophytes. Morphological and molecular culture-based methods, combined with metabarcoding analyses, were used to assess fungal communities associated with studied orchids and trees. A total of 162 fungal species in 74 genera were isolated from bark samples. Only two genera, Acremonium and Verticillium, were shared by the two phorophyte species. Metabarcoding analysis confirmed the presence of significantly different fungal communities on the investigated tree and orchid species, with considerable similarity between each orchid species and its host tree, suggesting that the orchid-host tree association is influenced by the fungal communities of the host tree bark.

Symbiont switching and trophic mode shifts in Orchidaceae.

Mycorrhizal fungi are central to the biology of land plants. However, to what extent mycorrhizal shifts - broad evolutionary transitions in root-associated fungal symbionts - are related to changes in plant trophic modes remains poorly understood. We built a comprehensive DNA dataset of Orchidaceae fungal symbionts and a dated plant molecular phylogeny to test the hypothesis that shifts in orchid trophic modes follow a stepwise pattern, from autotrophy over partial mycoheterotrophy (mixotrophy) to full mycoheterotrophy, and that these shifts are accompanied by switches in fungal symbionts. We estimate that at least 17 independent shifts from autotrophy towards full mycoheterotrophy occurred in orchids, mostly through an intermediate state of partial mycoheterotrophy. A wide range of fungal partners was inferred to occur in the roots of the common ancestor of this family, including 'rhizoctonias', ectomycorrhizal, and wood- or litter-decaying saprotrophic fungi. Phylogenetic hypothesis tests further show that associations with ectomycorrhizal or saprotrophic fungi were most likely a prerequisite for evolutionary shifts towards full mycoheterotrophy. We show that shifts in trophic mode often coincided with switches in fungal symbionts, suggesting that the loss of photosynthesis selects for different fungal communities in orchids. We conclude that changes in symbiotic associations and ecophysiological traits are tightly correlated throughout the diversification of orchids.

Specificity of assemblage, not fungal partner species, explains mycorrhizal partnerships of mycoheterotrophic Burmannia plants.

Mycoheterotrophic plants (MHPs) growing on arbuscular mycorrhizal fungi (AMF) usually maintain specialized mycorrhizal associations. The level of specificity varies between MHPs, although it remains largely unknown whether interactions with mycorrhizal fungi differ by plant lineage, species, and/or by population. Here, we investigate the mycorrhizal interactions among Burmannia species (Burmanniaceae) with different trophic modes using high-throughput DNA sequencing. We characterized the inter- and intraspecific dynamics of the fungal communities by assessing the composition and diversity of fungi among sites. We found that fully mycoheterotrophic species are more specialized in their fungal associations than chlorophyllous species, and that this specialization possibly results from the gradual loss of some fungal groups. In particular, although many fungal species were shared by different Burmannia species, fully MHP species typically host species-specific fungal assemblages, suggesting that they have a preference for the selected fungi. Although no apparent cophylogenetic relationship was detected between fungi and plants, we observe that evolutionarily closely related plants tend to have a greater proportion of shared or closely related fungal partners. Our findings suggest a host preference and specialization toward fungal assemblages in Burmannia, improving understanding of interactions between MHPs and fungi.

Symbiosis: Herbivory Alters Mycorrhizal Nutrient Exchange.

A new study shows that a plant gives less carbon to its root-associated mycorrhizal fungus when targeted by herbivores, but the fungus doesn't retaliate.

Fungal metabarcoding data integration framework for the MycoDiversity DataBase (MDDB).

Fungi have crucial roles in ecosystems, and are important associates for many organisms. They are adapted to a wide variety of habitats, however their global distribution and diversity remains poorly documented. The exponential growth of DNA barcode information retrieved from the environment is assisting considerably the traditional ways for unraveling fungal diversity and detection. The raw DNA data in association to environmental descriptors of metabarcoding studies are made available in public sequence read archives. While this is potentially a valuable source of information for the investigation of Fungi across diverse environmental conditions, the annotation used to describe environment is heterogenous. Moreover, a uniform processing pipeline still needs to be applied to the available raw DNA data. Hence, a comprehensive framework to analyses these data in a large context is still lacking. We introduce the MycoDiversity DataBase, a database which includes public fungal metabarcoding data of environmental samples for the study of biodiversity patterns of Fungi. The framework we propose will contribute to our understanding of fungal biodiversity and aims to become a valuable source for large-scale analyses of patterns in space and time, in addition to assisting evolutionary and ecological research on Fungi.

Environmental drivers for cheaters of arbuscular mycorrhizal symbiosis in tropical rainforests.

Hundreds of nonphotosynthetic mycoheterotrophic plant species cheat the arbuscular mycorrhizal symbiosis. Their patchy local occurrence suggests constraints by biotic and abiotic factors, among which the role of soil chemistry and nutrient status has not been investigated. Here, we examine the edaphic drivers predicting the local-scale distribution of mycoheterotrophic plants in two lowland rainforests in South America. We compared soil chemistry and nutrient status in plots where mycoheterotrophic plants were present with those without these plants. Soil pH, soil nitrate, and the interaction between soil potassium and nitrate concentrations were the best predictors for the occurrence of mycoheterotrophic plants in these tropical rainforests. Mycoheterotrophic plant occurrences decreased with a rise in each of these predictors. This indicates that these plants are associated with low-fertility patches. Such low-fertility conditions coincide with conditions that potentially favour a weak mutualism between plants and arbuscular mycorrhizal fungi according to the trade balance model. Our study points out which soil properties favour the cheating of arbuscular mycorrhizal networks in tropical forests. The patchy occurrence of mycoheterotrophic plants suggests that local soil heterogeneity causes the stability of arbuscular mycorrhizal networks to vary at a very small scale.

Evolutionary dynamics of mycorrhizal symbiosis in land plant diversification.

Mycorrhizal symbiosis between soil fungi and land plants is one of the most widespread and ecologically important mutualisms on earth. It has long been hypothesized that the Glomeromycotina, the mycorrhizal symbionts of the majority of plants, facilitated colonization of land by plants in the Ordovician. This view was recently challenged by the discovery of mycorrhiza-like associations with Mucoromycotina in several early diverging lineages of land plants. Utilizing a large, species-level database of plants' mycorrhiza-like associations and a Bayesian approach to state transition dynamics we here show that the recruitment of Mucoromycotina is the best supported transition from a non-mycorrhizal state. We further found that transitions between different combinations of either or both of Mucoromycotina and Glomeromycotina occur at high rates, and found similar promiscuity among combinations that include either or both of Glomeromycotina and Ascomycota with a nearly fixed association with Basidiomycota. Our results portray an evolutionary scenario of evolution of mycorrhizal symbiosis with a prominent role for Mucoromycotina in the early stages of land plant diversification.

Phylogenomic inference in extremis: A case study with mycoheterotroph plastomes.

PREMISE OF THE STUDY: Phylogenomic studies employing large numbers of genes, including those based on plastid genomes (plastomes), are becoming common. Nonphotosynthetic plants such as mycoheterotrophs (which rely on root-associated fungi for essential nutrients, including carbon) tend to have highly elevated rates of plastome evolution, substantial genome reduction, or both. Mycoheterotroph plastomes therefore provide excellent test cases for investigating how extreme conditions impact phylogenomic inference. METHODS: We used parsimony and likelihood analysis of protein-coding gene sets from published and newly completed plastomes to infer the phylogenetic placement of taxa from the 10 angiosperm families in which mycoheterotrophy evolved. KEY RESULTS: Despite multiple very long branches that reflect elevated substitution rates, and frequently patchy gene recovery due to genome reduction, inferred phylogenetic placements of most mycoheterotrophic lineages in DNA-based likelihood analyses are both well supported and congruent with other studies. Amino-acid-based likelihood placements are broadly consistent with DNA-based inferences, but extremely rate-elevated taxa can have unexpected placements-albeit with weak support. In contrast, parsimony analysis is strongly misled by long-branch attraction among many distantly related mycoheterotrophic monocots. CONCLUSIONS: Mycoheterotrophic plastomes provide challenging cases for phylogenomic inference, as substitutional rates can be elevated and genome reduction can lead to sparse gene recovery. Nonetheless, diverse likelihood frameworks provide generally well-supported and mutually concordant phylogenetic placements of mycoheterotrophs, consistent with recent phylogenetic studies and angiosperm-wide classifications. Previous predictions of parallel photosynthesis loss within families are supported for Burmanniaceae, Ericaceae, Gentianaceae, and Orchidaceae. Burmanniaceae and Thismiaceae should not be combined as a single family in Dioscoreales.

Biological invasions increase the richness of arbuscular mycorrhizal fungi from a Hawaiian subtropical ecosystem.

Biological invasions can have various impacts on the diversity of important microbial mutualists such as mycorrhizal fungi, but few studies have tested whether the effects of invasions on mycorrhizal diversity are consistent across spatial gradients. Furthermore, few of these studies have taken place in tropical ecosystems that experience an inordinate rate of invasions into native habitats. Here, we examined the effects of plant invasions dominated by non-native tree species on the diversity of arbuscular mycorrhizal (AM) fungi in Hawaii. To test the hypothesis that invasions result in consistent changes in AM fungal diversity across spatial gradients relative to native forest habitats, we sampled soil in paired native and invaded sites from three watersheds and used amplicon sequencing to characterize AM fungal communities. Whether our analyses considered phylogenetic relatedness or not, we found that invasions consistently increased the richness of AM fungi. However, AM fungal species composition was not related to invasion status of the vegetation nor local environment, but stratified by watershed. Our results suggest that while invasions can lead to an overall increase in the diversity of microbial mutualists, the effects of plant host identity or geographic structuring potentially outweigh those of invasive species in determining the community membership of AM fungi. Thus, host specificity and spatial factors such as dispersal need to be taken into consideration when examining the effects of biological invasions on symbiotic microbes.

Fungal-host diversity among mycoheterotrophic plants increases proportionally to their fungal-host overlap.

The vast majority of plants obtain an important proportion of vital resources from soil through mycorrhizal fungi. Generally, this happens in exchange of photosynthetically fixed carbon, but occasionally the interaction is mycoheterotrophic, and plants obtain carbon from mycorrhizal fungi. This process results in an antagonistic interaction between mycoheterotrophic plants and their fungal hosts. Importantly, the fungal-host diversity available for plants is restricted as mycoheterotrophic interactions often involve narrow lineages of fungal hosts. Unfortunately, little is known whether fungal-host diversity may be additionally modulated by plant-plant interactions through shared hosts. Yet, this may have important implications for plant competition and coexistence. Here, we use DNA sequencing data to investigate the interaction patterns between mycoheterotrophic plants and arbuscular mycorrhizal fungi. We find no phylogenetic signal on the number of fungal hosts nor on the fungal hosts shared among mycoheterotrophic plants. However, we observe a potential trend toward increased phylogenetic diversity of fungal hosts among mycoheterotrophic plants with increasing overlap in their fungal hosts. While these patterns remain for groups of plants regardless of location, we do find higher levels of overlap and diversity among plants from the same location. These findings suggest that species coexistence cannot be fully understood without attention to the two sides of ecological interactions.

Plastomes on the edge: the evolutionary breakdown of mycoheterotroph plastid genomes.

Contents 48 I. 48 II. 50 III. 53 54 References 54 SUMMARY: We examine recent evidence for ratchet-like genome degradation in mycoheterotrophs, plants that obtain nutrition from fungi. Initial loss of the NADH dehydrogenase-like (NDH) complex may often set off an irreversible evolutionary cascade of photosynthetic gene losses. Genes for plastid-encoded subunits of RNA polymerase and photosynthetic enzymes with secondary functions (Rubisco and ATP synthase) can persist initially, with nonsynchronous and quite broad windows in the relative timing of their loss. Delayed losses of five core nonbioenergetic genes (especially trnE and accD, which respectively code for glutamyl tRNA and a subunit of acetyl-CoA carboxylase) probably explain long-term persistence of heterotrophic plastomes. The observed range of changes of mycoheterotroph plastomes is similar to that of holoparasites, although greater diversity of both probably remains to be discovered. These patterns of gene loss/retention can inform research programs on plastome function.

Arbuscular mycorrhizal interactions of mycoheterotrophic Thismia are more specialized than in autotrophic plants.

In general, plants and arbuscular mycorrhizal (AM) fungi exchange photosynthetically fixed carbon for soil nutrients, but occasionally nonphotosynthetic plants obtain carbon from AM fungi. The interactions of these mycoheterotrophic plants with AM fungi are suggested to be more specialized than those of green plants, although direct comparisons are lacking. We investigated the mycorrhizal interactions of both green and mycoheterotrophic plants. We used next-generation DNA sequencing to compare the AM communities from roots of five closely related mycoheterotrophic species of Thismia (Thismiaceae), roots of surrounding green plants, and soil, sampled over the entire temperate distribution of Thismia in Australia and New Zealand. We observed that the fungal communities of mycoheterotrophic and green plants are phylogenetically more similar within than between these groups of plants, suggesting a specific association pattern according to plant trophic mode. Moreover, mycoheterotrophic plants follow a more restricted association with their fungal partners in terms of phylogenetic diversity when compared with green plants, targeting more clustered lineages of fungi, independent of geographic origin. Our findings demonstrate that these mycoheterotrophic plants target more narrow lineages of fungi than green plants, despite the larger fungal pool available in the soil, and thus they are more specialized towards mycorrhizal fungi than autotrophic plants.

Habitat-driven variation in mycorrhizal communities in the terrestrial orchid genus Dactylorhiza.

Orchid species are critically dependent on mycorrhizal fungi for completion of their life cycle, particularly during the early stages of their development when nutritional resources are scarce. As such, orchid mycorrhizal fungi play an important role in the population dynamics, abundance, and spatial distribution of orchid species. However, less is known about the ecology and distribution of orchid mycorrhizal fungi. In this study, we used 454 amplicon pyrosequencing to investigate ecological and geographic variation in mycorrhizal associations in fourteen species of the orchid genus Dactylorhiza. More specifically, we tested the hypothesis that variation in orchid mycorrhizal communities resulted primarily from differences in habitat conditions where the species were growing. The results showed that all investigated Dactylorhiza species associated with a large number of fungal OTUs, the majority belonging to the Tulasnellaceae, Ceratobasidiaceae and Sebacinales. Mycorrhizal specificity was low, but significant variation in mycorrhizal community composition was observed between species inhabiting different ecological habitats. Although several fungi had a broad geographic distribution, Species Indicator Analysis revealed some fungi that were characteristic for specific habitats. Overall, these results indicate that orchid mycorrhizal fungi may have a broad geographic distribution, but that their occurrence is bounded by specific habitat conditions.