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.

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.

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.

Evolution of endemism on a young tropical mountain.

Tropical mountains are hot spots of biodiversity and endemism, but the evolutionary origins of their unique biotas are poorly understood. In varying degrees, local and regional extinction, long-distance colonization, and local recruitment may all contribute to the exceptional character of these communities. Also, it is debated whether mountain endemics mostly originate from local lowland taxa, or from lineages that reach the mountain by long-range dispersal from cool localities elsewhere. Here we investigate the evolutionary routes to endemism by sampling an entire tropical mountain biota on the 4,095-metre-high Mount Kinabalu in Sabah, East Malaysia. We discover that most of its unique biodiversity is younger than the mountain itself (6 million years), and comprises a mix of immigrant pre-adapted lineages and descendants from local lowland ancestors, although substantial shifts from lower to higher vegetation zones in this latter group were rare. These insights could improve forecasts of the likelihood of extinction and 'evolutionary rescue' in montane biodiversity hot spots under climate change scenarios.

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.

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.

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.

A few-gene plastid phylogenetic framework for mycoheterotrophic monocots.

PREMISE OF THE STUDY: Few-gene studies with broad taxon sampling have provided major insights into phylogeny and underpin plant classification. However, they have typically excluded heterotrophic plants because of loss, pseudogenization, or rapid evolution of plastid genes. Here we performed a phylogenetic survey of three commonly retained plastid genes to assess their utility in placing mycoheterotrophs. METHODS: We surveyed accD, clpP, and matK for 34 taxa from seven monocot families that include full mycoheterotrophs and a broad sampling of photosynthetic taxa. After screening for weak contaminants, we conducted phylogenetic analyses and characterized among-lineage rate variation. KEY RESULTS: Likelihood analyses strongly supported local placements of fully mycoheterotrophic taxa for Corsiaceae, Iridaceae, Orchidaceae, and Petrosaviaceae, in positions consistent with other studies. Depression of likelihood bootstrap support values near mycoheterotrophic clades was alleviated when each mycoheterotrophic family was considered separately. Triuridaceae (Sciaphila) monophyly was recovered in a partitioned likelihood analysis, and the family then placed as sister to Cyclanthaceae-Pandanaceae. Burmanniaceae placed in Dioscoreales with weak to strong support depending on analysis details, and we inferred a plastid-based phylogeny for the family. Thismiaceae species may retain a plastid genome, based on accD retention. The inferred position of Thismiaceae is unstable, but was close to Taccaceae (Dioscoreales) in some analyses. CONCLUSIONS: Long branches/elevated substitution rates, missing genes, and occasional contaminants are challenges for plastid-based phylogenetic inference with full mycoheterotrophs. However, most mycoheterotrophs can be readily integrated into the broad picture of plant phylogeny using several plastid genes and broad taxonomic sampling.

Mycorrhizal diversity, seed germination and long-term changes in population size across nine populations of the terrestrial orchid Neottia ovata.

In plant species that rely on mycorrhizal symbioses for germination and seedling establishment, seedling recruitment and temporal changes in abundance can be expected to depend on fungal community composition and local environmental conditions. However, disentangling the precise factors that determine recruitment success in species that critically rely on mycorrhizal fungi represents a major challenge. In this study, we used seed germination experiments, 454 amplicon pyrosequencing and assessment of soil conditions to investigate the factors driving changes in local abundance in 28 populations of the orchid Neottia ovata. Comparison of population sizes measured in 2003 and 2013 showed that nearly 60% of the studied populations had declined in size (average growth rate across all populations: -0.01). Investigation of the mycorrhizal fungi in both the roots and soil revealed a total of 68 species of putatively mycorrhizal fungi, 21 of which occurred exclusively in roots, 25 that occurred solely in soil and 22 that were observed in both the soil and roots. Seed germination was limited and significantly and positively related to soil moisture content and soil pH, but not to fungal community composition. Large populations or populations with high population growth rates showed significantly higher germination than small populations or populations declining in size, but no significant relationships were found between population size or growth and mycorrhizal diversity. Overall, these results indicate that temporal changes in abundance were related to the ability of seeds to germinate, but at the same time they provided limited evidence that variation in fungal communities played an important role in determining population dynamics.

Evolution of mycoheterotrophy in Polygalaceae: The case of Epirixanthes.

PREMISE OF THE STUDY: The mycoheterotrophic lifestyle has enabled some plant lineages to obtain carbon from their mycorrhizal symbionts. The mycoheterotrophic genus Epirixanthes (Polygalaceae) consists of six species from tropical Asia. Although it is probably closely related to the chlorophyllous genus Salomonia and linked to arbuscular mycorrhizal fungi, lack of DNA sequence data has thus far prevented these hypotheses from being tested. Therefore, the evolutionary history of Epirixanthes remains largely unknown. METHODS: We reconstructed the phylogenetic relationships of Epirixanthes based on nuclear ITS and plastid matK data. Divergence times were inferred using a Bayesian relaxed clock approach, and we phylogenetically analyzed its mycorrhizal symbionts. We furthermore assigned these symbionts to operational taxonomic units, compared them with symbionts of other Polygalaceae, and measured their phylogenetic diversity. KEY RESULTS: We found that Epirixanthes is placed in tribe Polygaleae as sister to Salomonia. Epirixanthes has a Miocene-Oligocene stem age and grows exclusively in symbiosis with fungi of Glomeraceae. Salomonia and some Polygala species are linked to both Glomeraceae and Acaulosporaceae, resulting in higher phylogenetic diversity values. The majority of the symbionts of Epirixanthes are not found in Salomonia or Polygala, although a few shared fungal taxa are found. CONCLUSIONS: Epirixanthes forms a relatively young mycoheterotrophic lineage. The Oligocene-Miocene origin suggests its evolution was influenced by the environmental dynamics in Southeast Asia during this time. Although comparison of fungi from Epirixanthes with those from Salomonia and Polygala suggests some specialization, many other mycoheterotrophic plants are linked to a more narrow set of Glomeraceae.

Coexisting orchid species have distinct mycorrhizal communities and display strong spatial segregation.

Because orchids are dependent on mycorrhizal fungi for germination and establishment of seedlings, differences in the mycorrhizal communities associating with orchids can be expected to mediate the abundance, spatial distribution and coexistence of terrestrial orchids in natural communities. We assessed the small-scale spatial distribution of seven orchid species co-occurring in 25 × 25 m plots in two Mediterranean grasslands. In order to characterize the mycorrhizal community associating with each orchid species, 454 pyrosequencing was used. The extent of spatial clustering was assessed using techniques of spatial point pattern analysis. The community of mycorrhizal fungi consisted mainly of members of the Tulasnellaceae, Thelephoraceae and Ceratobasidiaceae, although sporadically members of the Sebacinaceae, Russulaceae and Cortinariaceae were observed. Pronounced differences in mycorrhizal communities were observed between species, whereas strong clustering and significant segregation characterized the spatial distribution of orchid species. However, spatial segregation was not significantly related to phylogenetic dissimilarity of fungal communities. Our results indicate that co-occurring orchid species have distinctive mycorrhizal communities and show strong spatial segregation, suggesting that mycorrhizal fungi are important factors driving niche partitioning in terrestrial orchids and may therefore contribute to orchid coexistence.

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.

New insights in the long-debated evolutionary history of Triuridaceae (Pandanales).

The mycoheterotrophic plant family Triuridaceae (Pandanales) is hypothesized to be an old family, mainly based on its pantropical distribution. The existence of fossils from the Upper Cretaceous, assigned to Triuridaceae may form additional support for a great age of the family, although the affinity of these fossils to Triuridaceae is questioned. Although the circumscription of Triuridaceae has never been problematic, probably due to its distinct morphological characters, its systematic relationship has been under debate since the family was described around 1840. The lack of synapomorphies suitable for resolving higher taxonomic relationships is a function of the family's reduced vegetative growth and the highly modified floral structures. Molecular studies have assigned Triuridaceae to Pandanales, but its exact phylogenetic position remains unknown. In the present study the phylogeny of the Pandanales was reconstructed using four molecular markers and the divergence age estimates were obtained with a relaxed molecular clock method. We found that Triuridaceae are monophyletic and most likely descent form the second major split in Pandanales. The relationships between the other Pandanales families (Cyclanthaceae, Pandanaceae, Stemonaceae and Velloziaceae) are otherwise in accordance with earlier studies. Velloziaceae are sister to the rest of the Pandanales, Stemonaceae are most likely sister to a clade consisting of Pandanaceae and Cyclanthaceae, and the latter two families are sister to each other. All currently recognized tribes within Triuridaceae are also monophyletic at current taxon sampling. We estimate that the family has a Cretaceous (or Lower Paleocene) stem age, which is in accordance with earlier predictions. This old age, along with elevated mutation rates indicated by long branch lengths and the family's mycoheterotrophic lifestyle, might account for the substantial morphological differences between Triuridaceae and its closest relatives.

Phylogenetic relationships of the mycoheterotrophic genus Voyria and the implications for the biogeographic history of Gentianaceae.

PREMISE OF THE STUDY: The angiosperm family Gentianaceae comprises over 1700 species in 91 genera. Gentianaceae are distributed worldwide, but most species occur in temperate zones. Phylogenetic studies demonstrate that the family consists of six monophyletic tribes. However, the phylogenetic position of the mycoheterotrophic genus Voyria, with a remarkable trans-Atlantic distribution, remained unknown. METHODS: We used nuclear ITS and 18S rDNA and mitochondrial apt1 and matR data to infer the phylogenetic position of Voyria in Gentianaceae. In addition, with Bayesian relaxed molecular clock analyses we obtained age estimates for the diversification of Voyria and Gentianaceae in general and used these results to reconstruct the ancestral areas associated with the early diversification events in Gentianaceae. KEY RESULTS: Our results demonstrate that Voyria is an early diverging lineage within Gentianaceae with no close relationships to other mycoheterotrophic Gentianaceae lineages. Voyria originated in the neotropics during the Early Eocene but only reached its current transoceanic distribution around the end of the Oligocene. The neotropics were an important area for the early diversification events in Gentianaceae, most of which occurred during the Eocene. CONCLUSIONS: Voyria is an old, phylogenetically isolated lineage within Gentianaceae, and the current distribution of the genus is indicative of the ancestral area in which the early diversification events of Gentianaceae occurred. In parallel with many other pantropical families, our results suggest that migration of tropical taxa through Laurasia during the Early Eocene has played an important role in shaping the current global distribution of Gentianaceae.

Mycoheterotrophy.

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

Mycoheterotrophic interactions are not limited to a narrow phylogenetic range of arbuscular mycorrhizal fungi.

The majority of achlorophyllous mycoheterotrophic plant species associate with arbuscular mycorrhizal fungi (AMF). Previous studies have shown that some species are highly specialized towards narrow lineages of AMF and have suggested that only particular lineages of these fungi are targeted by mycoheterotrophic plants. To test this hypothesis, we analyzed all available partial SSU sequences of AMF associated with mycoheterotrophic plants including data from 13 additional specimens from French Guiana, Gabon and Australia. Sequences were assigned to 'virtual taxa' (VT) according to the MaarjAM database. We found that 20% of all known Glomeromycota VT are involved in mycoheterotrophic interactions and the majority of associations involve Glomeraceae (Glomus Group A) fungi. While some mycoheterotrophic plant species have been found growing with only a single VT, many species are able to associate with a wide range of AMF. We calculated significant phylogenetic clustering of Glomeromycota VT involved in mycoheterotrophic interactions, suggesting that associations between mycoheterotrophic plants and AMF are influenced by the phylogenetic relationships of the fungi. Our results demonstrate that many lineages of AMF are prone to exploitation by mycoheterotrophic plants. However, mycoheterotrophs from different plant lineages and different geographical regions tend to be dependent on lineages of AMF that are phylogenetically related.

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.