Nhu H. Nguyen

Towards a unified paradigm for sequence‐based identification of fungi

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

Measuring ectomycorrhizal fungal dispersal: macroecological patterns driven by microscopic propagules.

Dispersal plays a prominent role in most conceptual models of community assembly. However, direct measurement of dispersal across a whole community is difficult at ecologically relevant spatial scales. For cryptic organisms, such as fungi and bacteria, the scale and importance of dispersal limitation has become a major point of debate. We use an experimental island biogeographic approach to measure the effects of dispersal limitation on the ecological dynamics of an important group of plant symbionts, ectomycorrhizal fungi. We manipulated the isolation of uncolonized host seedlings across a natural landscape and used a range of molecular techniques to measure the dispersal rates of ectomycorrhizal propagules and host colonization. Some species were prolific dispersers, producing annual spore loads on the order of trillions of spores per km2. However, fungal propagules reaching host seedlings decreased rapidly with increasing distance from potential spore sources, causing a concomitant reduction in ectomycorrhizal species richness, host colonization and host biomass. There were also strong differences in dispersal ability across species, which correlated well with the predictable composition of ectomycorrhizal communities associated with establishing pine forest. The use of molecular tools to measure whole community dispersal provides a direct confirmation for a key mechanism underlying island biogeography theory and has the potential to make microbial systems a model for understanding the role of dispersal in ecological theory.

Parsing ecological signal from noise in next generation amplicon sequencing

It is clear that the use of next generation sequencing (NGS) applied to environmental DNA is changing the way researchers conduct experiments and significantly deepening our understanding of microbial communities around the globe (Amend et al., 2010; Caporaso et al., 2011; Bik et al., 2012; Bates et al., 2013). The lower per unit cost and sheer number of sequences relative to traditional methods provide tremendous advantages in characterizing the richness and composition of highly diverse microbial systems (Bokulich et al., 2013). In a recent volume of New Phytologist, Lindahl et al. (2013) presented an excellent introduction into high-throughput sequencing of amplified gene markers for fungi, and broadly discussed field sampling and handling,DNA extraction, markers, primers, amplicon library construction, sequencing platform, bioinformatic analyses and data interpretation.We applaud their overview as an important general guide, but we have found that there are significant additional issues regarding NGS that have not been well articulated in the literature, especially when applied to fungi. Below we highlight a series of platformindependent recommendations based on our recent experiences with NGS, which we think are critical for maximizing the signal: noise ratio in molecular ecological analyses.

Inoculum potential of Rhizopogon spores increases with time over the first 4 yr of a 99-yr spore burial experiment

In disturbed or pioneer settings, spores and sclerotia of ectomycorrhizal fungi serve as the necessary inoculum for establishment of ectomycorrhizal-dependent trees. Yet, little is known about the persistence of these propagules through time. Here, live field soil was inoculated with known quantities of basidiospores from four pine-associated species of Rhizopogon; these samples were then buried in retrievable containers, and pine seedling bioassays of serially diluted spore samples were used to measure spore viability. In the first 4 yr, no evidence of loss of spore viability was found in the four Rhizopogon species tested, but all four species exhibited dormancy in which a maximum of 1-8% of their spores were initially receptive to pine roots. There were some differences between species in overall inoculum potential of their spores, but all species broke dormancy at a statistically similar rate. This result provides evidence for spore dormancy in a common ectomycorrhizal genus, but it also precludes our ability to estimate the longevity of the spores accurately. Nevertheless these results, coupled with the observed patterns of Rhizopogon spore banks, suggest that at least decade-long durations are likely. As this experiment progresses, the true longevity of the spores will eventually be revealed.

Nine new Candida species near C. membranifaciens isolated from insects

14 different yeasts were isolated from the gut of a variety of insects, including beetles, lacewings, fishflies, craneflies, and a cockroach. One of the yeasts was found both in the gut and on the body surface of a beetle larva. Based on ribosomal DNA sequence comparisons and phenotypic characters, the yeasts were identified as Candida membranifaciens, C. tenuis, Pichia nakazawae, and nine undescribed taxa in Saccharomycotina. All the undescribed taxa reproduced only asexually, and they fit within the limits of the polyphyletic genus Candida. The new species and their type strains are Candida blattariae NRRL Y-27703T, C. amphixiae NRRL Y-27704T, C. michaelii NRRL Y-27705T, C. cerambycidarum NRRL Y-27706T, C. gorgasii NRRL Y-27707T, C. endomychidarum NRRL Y-27708T, C. temnochilae NRRL Y-27763T, C. sinolaborantium NRRL Y-27765T, and C. lessepsii NRRL Y-27766T spp. nov. Phylogenetic analysis of combined small and large subunit ribosomal DNA sequences placed C. amphixiae, C. michaelii, C. cerambycidarum, C. gorgasii, C. endomychidarum, and C. lessepsii in a statistically well supported clade with C. blattariae, C. membranifaciens, C. friedrichii, and C. buinensis as sisters to the clade. The other two new taxa, C. temnochilae and C. sinolaborantium, formed an independent clade basal to the major clade containing C. membranifaciens and closely related taxa. C. sinolaborantium occurred in both Panama and the USA, but there were genetic differences between the isolates from the two places.

Compartmentalized microbial composition, oxygen gradients and nitrogen fixation in the gut of Odontotaenius disjunctus

Coarse woody debris is an important biomass pool in forest ecosystems that numerous groups of insects have evolved to take advantage of. These insects are ecologically important and represent useful natural analogs for biomass to biofuel conversion. Using a range of molecular approaches combined with microelectrode measurements of oxygen, we have characterized the gut microbiome and physiology of Odontotaenius disjunctus, a wood-feeding beetle native to the eastern United States. We hypothesized that morphological and physiological differences among gut regions would correspond to distinct microbial populations and activities. In fact, significantly different communities were found in the foregut (FG), midgut (MG)/posterior hindgut (PHG) and anterior hindgut (AHG), with Actinobacteria and Rhizobiales being more abundant toward the FG and PHG. Conversely, fermentative bacteria such as Bacteroidetes and Clostridia were more abundant in the AHG, and also the sole region where methanogenic Archaea were detected. Although each gut region possessed an anaerobic core, micron-scale profiling identified radial gradients in oxygen concentration in all regions. Nitrogen fixation was confirmed by 15N2 incorporation, and nitrogenase gene (nifH) expression was greatest in the AHG. Phylogenetic analysis of nifH identified the most abundant transcript as related to Ni–Fe nitrogenase of a Bacteroidetes species, Paludibacter propionicigenes. Overall, we demonstrate not only a compartmentalized microbiome in this beetle digestive tract but also sharp oxygen gradients that may permit aerobic and anaerobic metabolism to occur within the same regions in close proximity. We provide evidence for the microbial fixation of N2 that is important for this beetle to subsist on woody biomass.

The status and characterization of Enteroramus dimorphus: a xylose-fermenting yeast attached to the gut of beetles

Enteroramus dimorphus from the gut of the passalid beetle Odontotaenius disjunctus was described originally as a yeast-like fungus of unknown taxonomic affiliation. The fungus can be observed in situ, attached by a specialized cell to the beetle hindgut wall. In a recent study of yeast endosymbionts from a variety of beetles, we discovered that E. dimorphus is a member of the Pichia stipitis (Saccharomycetes) clade, known for xylose fermentation and assimilation. The closest relative of E. dimorphus is the PASS1 isolate, repeatedly acquired from passalid beetles in eastern North America from Pennsylvania to Louisiana. In addition to xylose fermentation and assimilation, these yeasts are characterized by the production of hat-shaped ascospores in culture, assimilation of a wide range of sugars, and synthesis of several vitamins. Enteroramus dimorphus, however, can be distinguished from close relatives by several physiological characteristics and rDNA sequences, which vary slightly from the more widespread PASS1 genotype. We present an amended description of E. dimorphus and discuss its symbiotic phase in association with O. disjunctus, including a holdfast that parallels those of unrelated symbiotic yeasts associated with nematodes.

The Microbiome of Pinus muricata Ectomycorrhizae: Community Assemblages, Fungal Species Effects, and Burkholderia as Important Bacteria in Multipartnered Symbioses.

Bacteria have been observed to grow with fungi, and those that associate with ectomycorrhizal fungi have often been thought of as symbionts that may either increase or decrease ectomycorrhizal formation rate or provide other unaccounted benefits. To explore this symbiosis from a community ecology perspective, we sampled ectomycorrhizal root tips over a 3-year period and used 454 pyrosequencing to identify the bacteria that live inside the ectomycorrhizal root tips. The results showed that fungal community composition within the same soil core and fungal taxonomic identity had a stronger effect on bacterial community composition than sample year or site. Members of the Burkholderiales and Rhizobiales were most highly represented, reflecting many previous reports of these bacteria in association with fungi. The repeated occurrences of these two bacterial orders suggest that they may be symbiotic with their fungal hosts, although the nature of such mechanisms, be it symbiotic diazotrophy or otherwise, remains to be thoroughly tested.

Ectomycorrhizal fungal diversity and saprotrophic fungal diversity are linked to different tree community attributes in a field-based tree experiment.

Exploring the link between above‐ and belowground biodiversity has been a major theme of recent ecological research, due in large part to the increasingly well‐recognized role that soil microorganisms play in driving plant community processes. In this study, we utilized a field‐based tree experiment in Minnesota, USA, to assess the effect of changes in plant species richness and phylogenetic diversity on the richness and composition of both ectomycorrhizal and saprotrophic fungal communities. We found that ectomycorrhizal fungal species richness was significantly positively influenced by increasing plant phylogenetic diversity, while saprotrophic fungal species richness was significantly affected by plant leaf nitrogen content, specific root length and standing biomass. The increasing ectomycorrhizal fungal richness associated with increasing plant phylogenetic diversity was driven by the combined presence of ectomycorrhizal fungal specialists in plots with both gymnosperm and angiosperm hosts. Although the species composition of both the ectomycorrhizal and saprotrophic fungal communities changed significantly in response to changes in plant species composition, the effect was much greater for ectomycorrhizal fungi. In addition, ectomycorrhizal but not saprotrophic fungal species composition was significantly influenced by both plant phylum (angiosperm, gymnosperm, both) and origin (Europe, America, both). The phylum effect was caused by differences in ectomycorrhizal fungal community composition, while the origin effect was attributable to differences in community heterogeneity. Taken together, this study emphasizes that plant‐associated effects on soil fungal communities are largely guild‐specific and provides a mechanistic basis for the positive link between plant phylogenetic diversity and ectomycorrhizal fungal richness.

Missing checkerboards? An absence of competitive signal in Alnus-associated ectomycorrhizal fungal communities

A number of recent studies suggest that interspecific competition plays a key role in determining the structure of ectomycorrhizal (ECM) fungal communities. Despite this growing consensus, there has been limited study of ECM fungal community dynamics in abiotically stressful environments, which are often dominated by positive rather than antagonistic interactions. In this study, we examined the ECM fungal communities associated with the host genus Alnus, which live in soils high in both nitrate and acidity. The nature of ECM fungal species interactions (i.e., antagonistic, neutral, or positive) was assessed using taxon co-occurrence and DNA sequence abundance correlational analyses. ECM fungal communities were sampled from root tips or mesh in-growth bags in three monodominant A. rubra plots at a site in Oregon, USA and identified using Illumina-based amplification of the ITS1 gene region. We found a total of 175 ECM fungal taxa; 16 of which were closely related to known Alnus-associated ECM fungi. Contrary to previous studies of ECM fungal communities, taxon co-occurrence analyses on both the total and Alnus-associated ECM datasets indicated that the ECM fungal communities in this system were not structured by interspecific competition. Instead, the co-occurrence patterns were consistent with either random assembly or significant positive interactions. Pair-wise correlational analyses were also more consistent with neutral or positive interactions. Taken together, our results suggest that interspecific competition does not appear to determine the structure of all ECM fungal communities and that abiotic conditions may be important in determining the specific type of interaction occurring among ECM fungi.