Contrasting genetic structuring in the closely related basidiomycetes Trichaptum abietinum and Trichaptum fuscoviolaceum (Hymenochaetales).

Trichaptum abietinum and Trichaptum fuscoviolaceum (Hymenochaetales, Basidiomycota) are closely related saprotrophic fungi, widely distributed on coniferous wood in temperate regions worldwide. Three intersterility groups have previously been detected in T. abietinum, while no prezygotic barriers have been proven within T. fuscoviolaceum. The aim of this study was to reveal the phylogeography and genetic relationship between these two closely related species and to explore whether the previously observed intersterility groups in T. abietinum are reflected in the genetic data. We assembled worldwide fruit body collections of both species (N = 314) and generated DNA sequences from three nuclear (ITS2, LSU, IGS) and one mitochondrial rDNA region (mtLSU). The two species are genetically well separated in all analyses. In correspondence with observations from earlier mating studies, our results revealed that T. fuscoviolaceum is genetically more uniform than T. abietinum. Multiple genetic sub-groups exist in T. abietinum that may correspond to the previously observed intersterility groups. However, there is low consistency across the investigated loci in delimiting the different sub-groups, except for a consistent North American group. As for many other widespread fungi, a complex phylogeographic pattern is found in T. abietinum which may have been formed by geographic, as well as multiple genetic intersterility barriers.

A single ectomycorrhizal plant root system includes a diverse and spatially structured fungal community.

Although only a relatively small proportion of plant species form ectomycorrhizae with fungi, it is crucial for growth and survival for a number of widespread woody plant species. Few studies have attempted to investigate the fine scale spatial structure of entire root systems of adult ectomycorrhizal (EcM) plants. Here, we use the herbaceous perennial Bistorta vivipara to map the entire root system of an adult EcM plant and investigate the spatial structure of its root-associated fungi. All EcM root tips were sampled, mapped and identified using a direct PCR approach and Sanger sequencing of the internal transcribed spacer region. A total of 32.1% of all sampled root tips (739 of 2302) were successfully sequenced and clustered into 41 operational taxonomic units (OTUs). We observed a clear spatial structuring of the root-associated fungi within the root system. Clusters of individual OTUs were observed in the younger parts of the root system, consistent with observations of priority effects in previous studies, but were absent from the older parts of the root system. This may suggest a succession and fragmentation of the root-associated fungi even at a very fine scale, where competition likely comes into play at different successional stages within the root system.

Fungal diversity and seasonal succession in ash leaves infected by the invasive ascomycete Hymenoscyphus fraxineus.

High biodiversity is regarded as a barrier against biological invasions. We hypothesized that the invasion success of the pathogenic ascomycete Hymenoscyphus fraxineus threatening common ash in Europe relates to differences in dispersal and colonization success between the invader and the diverse native competitors. Ash leaf mycobiome was monitored by high-throughput sequencing of the fungal internal transcribed spacer region (ITS) and quantitative PCR profiling of H. fraxineus DNA. Initiation of ascospore production by H. fraxineus after overwintering was followed by pathogen accumulation in asymptomatic leaves. The induction of necrotic leaf lesions coincided with escalation of H. fraxineus DNA levels and changes in proportion of biotrophs, followed by an increase of ubiquitous endophytes with pathogenic potential. H. fraxineus uses high propagule pressure to establish in leaves as quiescent thalli that switch to pathogenic mode once these thalli reach a certain threshold - the massive feedback from the saprophytic phase enables this fungus to challenge host defenses and the resident competitors in mid-season when their density in host tissues is still low. Despite the general correspondence between the ITS-1 and ITS-2 datasets, marker biases were observed, which suggests that multiple barcodes provide better overall representation of mycobiomes.

Primary succession of Bistorta vivipara (L.) Delabre (Polygonaceae) root-associated fungi mirrors plant succession in two glacial chronosequences.

Glacier chronosequences are important sites for primary succession studies and have yielded well-defined primary succession models for plants that identify environmental resistance as an important determinant of the successional trajectory. Whether plant-associated fungal communities follow those same successional trajectories and also respond to environmental resistance is an open question. In this study, 454 amplicon pyrosequencing was used to compare the root-associated fungal communities of the ectomycorrhizal (ECM) herb Bistorta vivipara along two primary succession gradients with different environmental resistance (alpine versus arctic) and different successional trajectories in the vascular plant communities (directional replacement versus directional non-replacement). At both sites, the root-associated fungal communities were dominated by ECM basidiomycetes and community composition shifted with increasing time since deglaciation. However, the fungal community's successional trajectory mirrored the pattern observed in the surrounding plant community at both sites: the alpine site displayed a directional-replacement successional trajectory, and the arctic site displayed a directional-non-replacement successional trajectory. This suggests that, like in plant communities, environmental resistance is key in determining succession patterns in root-associated fungi. The need for further replicated study, including in other host species, is emphasized.

Mycorrhizal fungal communities in coastal sand dunes and heaths investigated by pyrosequencing analyses.

Maritime sand dunes and coastal ericaceous heaths are unstable and dynamic habitats for mycorrhizal fungi. Creeping willow (Salix repens) is an important host plant in these habitats in parts of Europe. In this study, we wanted to assess which mycorrhizal fungi are associated with S. repens in four different coastal vegetation types in Southern Norway, three types from sand dunes and one from heaths. Moreover, we investigated which ecological factors are important for the fungal community structure in these vegetation types. Mycorrhizal fungi on S. repens root samples were identified by 454 pyrosequencing of tag-encoded internal transcribed spacer 1 (ITS1) amplicons. Significantly higher fungal richness was observed in hummock dunes and dune slacks compared to eroded dune vegetation. The compositional variation was mainly accounted for by location (plot) and vegetation type and was significantly correlated to content of carbon, nitrogen and phosphorus in soil. The investigated maritime sand dunes and coastal ericaceous heaths hosted mycorrhizal taxa mainly associated with Helotiales, Sebacinales, Thelephorales and Agaricales.

Low host specificity of root-associated fungi at an Arctic site.

In High Arctic ecosystems, plant growth and reproduction are limited by low soil moisture and nutrient availability, low soil and air temperatures, and a short growing season. Mycorrhizal associations facilitate plant nutrient acquisition and water uptake and may therefore be particularly ecologically important in nutrition-poor and dry environments, such as parts of the Arctic. Similarly, endophytic root associates are thought to play a protective role, increasing plants' stress tolerance, and likely have an important ecosystem function. Despite the importance of these root-associated fungi, little is known about their host specificity in the Arctic. We investigated the host specificity of root-associated fungi in the common, widely distributed arctic plant species Bistorta vivipara, Salix polaris and Dryas octopetala in the High Arctic archipelago Svalbard. High-throughput sequencing of the internal transcribed spacer 1 (ITS1) amplified from whole root systems generated no evidence of host specificity and no spatial autocorrelation within two 3 m × 3 m sample plots. The lack of spatial structure at small spatial scales indicates that Common Mycelial Networks (CMNs) are rare in marginal arctic environments. Moreover, no significant differences in fungal OTU richness were observed across the three plant species, although their root system characteristics (size, biomass) differed considerably. Reasons for lack of host specificity could be that association with generalist fungi may allow arctic plants to more rapidly and easily colonize newly available habitats, and it may be favourable to establish symbiotic relationships with fungi possessing different physiological attributes.

Arctic root-associated fungal community composition reflects environmental filtering.

There is growing evidence that root-associated fungi have important roles in Arctic ecosystems. Here, we assess the diversity of fungal communities associated with roots of the ectomycorrhizal perennial herb Bistorta vivipara on the Arctic archipelago of Svalbard and investigate whether spatial separation and bioclimatic variation are important structuring factors of fungal community composition. We sampled 160 plants of B. vivipara from 32 localities across Svalbard. DNA was extracted from entire root systems, and 454 pyrosequencing of ITS1 amplicons was used to profile the fungal communities. The fungal communities were predominantly composed of Basidiomycota (55% of reads) and Ascomycota (35%), with the orders Thelephorales (24%), Agaricales (13.8%), Pezizales (12.6%) and Sebacinales (11.3%) accounting for most of the reads. Plants from the same site or region had more similar fungal communities to one another than plants from other sites or regions, and sites clustered together along a weak latitudinal gradient. Furthermore, a decrease in per-plant OTU richness with increasing latitude was observed. However, no statistically significant spatial autocorrelation between sites was detected, suggesting that environmental filtering, not dispersal limitation, causes the observed patterns. Our analyses suggest that while latitudinal patterns in community composition and richness might reflect bioclimatic influences at global spatial scales, at the smaller spatial scale of the Svalbard archipelago, these changes more likely reflect varied bedrock composition and associated edaphic factors. The need for further studies focusing on identifying those specific bioclimatic and edaphic factors structuring root-associated fungal community composition at both global and local scales is emphasized.

Different bacterial communities in ectomycorrhizae and surrounding soil.

Several eukaryotic symbioses have shown to host a rich diversity of prokaryotes that interact with their hosts. Here, we study bacterial communities associated with ectomycorrhizal root systems of Bistorta vivipara compared to bacterial communities in bulk soil using pyrosequencing of 16S rRNA amplicons. A high richness of Operational Taxonomic Units (OTUs) was found in plant roots (3,571 OTUs) and surrounding soil (3,476 OTUs). The community composition differed markedly between these two environments. Actinobacteria, Armatimonadetes, Chloroflexi and OTUs unclassified at phylum level were significantly more abundant in plant roots than in soil. A large proportion of the OTUs, especially those in plant roots, presented low similarity to Sanger 16S rRNA reference sequences, suggesting novel bacterial diversity in ectomycorrhizae. Furthermore, the bacterial communities of the plant roots were spatially structured up to a distance of 60 cm, which may be explained by bacteria using fungal hyphae as a transport vector. The analyzed ectomycorrhizae presents a distinct microbiome, which likely influence the functioning of the plant-fungus symbiosis.

Substantial compositional turnover of fungal communities in an alpine ridge-to-snowbed gradient.

The main gradient in vascular plant, bryophyte and lichen species composition in alpine areas, structured by the topographic gradient from wind-exposed ridges to snowbeds, has been extensively studied. Tolerance to environmental stress, resulting from wind abrasion and desiccation towards windswept ridges or reduced growing season due to prolonged snow cover towards snowbeds, is an important ecological mechanism in this gradient. The extent to which belowground fungal communities are structured by the same topographic gradient and the eventual mechanisms involved are less well known. In this study, we analysed variation in fungal diversity and community composition associated with roots of the ectomycorrhizal plant Bistorta vivipara along the ridge-to-snowbed gradient. We collected root samples from fifty B. vivipara plants in ten plots in an alpine area in central Norway. The fungal communities were analysed using 454 pyrosequencing analyses of tag-encoded ITS1 amplicons. A distinct gradient in the fungal community composition was found that coincided with variation from ridge to snowbeds. This gradient was paralleled by change in soil content of carbon, nitrogen and phosphorus. A large proportion (66%) of the detected 801 nonsingleton operational taxonomic units (OTUs) were ascomycetes, while basidiomycetes dominated quantitatively (i.e. with respect to number of reads). Numerous fungal OTUs, many with taxonomic affinity to Sebacinales, Cortinarius and Meliniomyces, showed distinct affinities either to ridge or to snowbed plots, indicating habitat specialization. The compositional turnover of fungal communities along the gradient was not paralleled by a gradient in species richness.

Employing 454 amplicon pyrosequencing to reveal intragenomic divergence in the internal transcribed spacer rDNA region in fungi.

The rDNA internal transcribed spacer (ITS) region has been accepted as a DNA barcoding marker for fungi and is widely used in phylogenetic studies; however, intragenomic ITS variability has been observed in a broad range of taxa, including prokaryotes, plants, animals, and fungi, and this variability has the potential to inflate species richness estimates in molecular investigations of environmental samples. In this study 454 amplicon pyrosequencing of the ITS1 region was applied to 99 phylogenetically diverse axenic single-spore cultures of fungi (Dikarya: Ascomycota and Basidiomycota) to investigate levels of intragenomic variation. Three species (one Basidiomycota and two Ascomycota), in addition to a positive control species known to contain ITS paralogs, displayed levels of molecular variation indicative of intragenomic variation; taxon inflation due to presumed intragenomic variation was ≈9%. Intragenomic variability in the ITS region appears to be widespread but relatively rare in fungi (≈3-5% of species investigated in this study), suggesting this problem may have minor impacts on species richness estimates relative to PCR and/or pyrosequencing errors. Our results indicate that 454 amplicon pyrosequencing represents a powerful tool for investigating levels of ITS intragenomic variability across taxa, which may be valuable for better understanding the fundamental mechanisms underlying concerted evolution of repetitive DNA regions.

Fungal community analysis by high-throughput sequencing of amplified markers--a user's guide.

Novel high-throughput sequencing methods outperform earlier approaches in terms of resolution and magnitude. They enable identification and relative quantification of community members and offer new insights into fungal community ecology. These methods are currently taking over as the primary tool to assess fungal communities of plant-associated endophytes, pathogens, and mycorrhizal symbionts, as well as free-living saprotrophs. Taking advantage of the collective experience of six research groups, we here review the different stages involved in fungal community analysis, from field sampling via laboratory procedures to bioinformatics and data interpretation. We discuss potential pitfalls, alternatives, and solutions. Highlighted topics are challenges involved in: obtaining representative DNA/RNA samples and replicates that encompass the targeted variation in community composition, selection of marker regions and primers, options for amplification and multiplexing, handling of sequencing errors, and taxonomic identification. Without awareness of methodological biases, limitations of markers, and bioinformatics challenges, large-scale sequencing projects risk yielding artificial results and misleading conclusions.

Microsatellite markers for Hylocomium splendens (Hylocomiaceae).

PREMISE OF THE STUDY: The perennial feather moss Hylocomium splendens is one of the most widely distributed and common bryophytes in the Northern Hemisphere and has, because of its capacity to grow under a wide range of environmental conditions, been used as a biomonitor for atmospheric metal deposition in Europe. METHODS AND RESULTS: We present a multiplex approach for the analysis of 14 microsatellite markers tested on 194 H. splendens gametophytes. Ten of the markers are developed recently, and are presented for the first time in this paper, whereas four were previously developed but have not been used for population genetic investigations. CONCLUSIONS: The microsatellite markers reported here will provide a powerful tool for further research on population genetic structure in H. splendens.

Genome wide AFLP markers support cryptic species in Coniophora (Boletales).

Numerous fungal morphospecies include cryptic species that routinely are detected by sequencing a few unlinked DNA loci. However, whether the patterns observed by multi-locus sequencing are compatible with genome wide data, such as amplified fragment length polymorphisms (AFLPs), is not well known for fungi. In this study we compared the ability of three DNA loci and AFLP data to discern between cryptic fungal lineages in the three morphospecies Coniophora olivacea, Coniophora arida, and Coniophora puteana. The sequences and the AFLP data were highly congruent in delimiting the morphotaxa into multiple cryptic species. However, while the DNA sequences indicated introgression or hybridization between some of the cryptic lineages the AFLP data did not. We conclude that as few as three polymorphic DNA loci was sufficient to recognize cryptic lineages within the studied Coniophora taxa. However, based on analyses of a few (three) sequenced loci the hybridization could not easily be distinguished from incomplete lineage sorting. Hence, great caution should be taken when concluding about hybridization based on data from just a few loci.

Changes in the root-associated fungal communities along a primary succession gradient analysed by 454 pyrosequencing.

We investigated changes in the root-associated fungal communities associated with the ectomycorrhizal herb Bistorta vivipara along a primary succession gradient using 454 amplicon sequencing. Our main objective was to assess the degree of variation in fungal richness and community composition as vegetation cover increases along the chronosequence. Sixty root systems of B. vivipara were sampled in vegetation zones delimited by dated moraines in front of a retreating glacier in Norway. We extracted DNA from rinsed root systems, amplified the ITS1 region using fungal-specific primers and analysed the amplicons using 454 sequencing. Between 437 and 5063 sequences were obtained from each root system. Clustering analyses using a 98.5% sequence similarity cut-off yielded a total of 470 operational taxonomic units (OTUs), excluding singletons. Between eight and 41 fungal OTUs were detected within each root system. Already in the first stage of succession, a high fungal diversity was present in the B. vivipara root systems. Total number of OTUs increased significantly along the gradient towards climax vegetation, but the average number of OTUs per root system stayed unchanged. There was a high patchiness in distribution of fungal OTUs across root systems, indicating that stochastic processes to a large extent structure the fungal communities. However, time since deglaciation had impact on the fungal community structure, as a systematic shift in the community composition was observed along the chronosequence. Ectomycorrhizal basidiomycetes were the dominant fungi in the roots of B. vivipara, when it comes to both number of OTUs and number of sequences.

Microsatellite markers for Bistorta vivipara (Polygonaceae).

PREMISE OF THE STUDY: Using genomic shotgun 454 sequencing, 50 candidate microsatellite markers were targeted for the arctic-alpine polyploid perennial herb Bistorta vivipara to distinguish between individual genets and ramets within a population. METHODS AND RESULTS: Out of the 50 markers, 31 were polymorphic for seven test samples. We have developed a multiplex protocol for 16 of these microsatellite markers. CONCLUSIONS: Our results show that the microsatellite markers provide a powerful tool for the research on genetic variation of B. vivipara.

Pleistocene climatic oscillations and the speciation history of an alpine endemic and a widespread arctic-alpine plant.

Even in cases in which geographic isolation appears to have driven the speciation of regional endemics, range shifts during the Pleistocene climatic oscillations may also have influenced their evolutionary history. Elucidating speciation history can provide novel insights into evolutionary dynamics following climatic oscillations. We demonstrated a sister relationship between the Japanese alpine endemic Cardamine nipponica and the currently allopatric, widespread arctic-alpine Cardamine bellidifolia (Brassicaceae) based on internal transcribed spacer (ITS) sequences and 10 other nuclear genes. Speciation history was inferred using demographic parameters under the isolation with migration model. The estimated demographic parameters showed that the population size of C. nipponica was similar to that of C. bellidifolia and that gene flow occurred exclusively from C. nipponica to C. bellidifolia after speciation. The inferred speciation history, which included gene flow, suggests that geographic barriers between the peripheral C. nipponica and the widespread C. bellidifolia were reduced during the Pleistocene. The asymmetric introgression implies that genetic isolation may have been involved in the speciation of C. nipponica. Our results suggest that even currently allopatric species may not have diverged solely under geographic isolation, and that their evolutionary history may have been influenced by Pleistocene range dynamics.

High consistency between replicate 454 pyrosequencing analyses of ectomycorrhizal plant root samples.

In this methodological study, we compare 454 sequencing and a conventional cloning and Sanger sequencing approach in their ability to characterize fungal communities PCR amplified from four root systems of the ectomycorrhizal plant Bistorta vivipara. To examine variation introduced by stochastic processes during the laboratory work, we replicated all analyses using two independently obtained DNA extractions from the same root systems. The ITS1 region was used as DNA barcode and the sequences were clustered into OTUs as proxies for species using single linkage clustering (BLASTC: lust) and 97% sequence similarity cut-off. A relatively low overlap in fungal OTUs was observed between the 454 and the clone library datasets - even among the most abundant OTUs. In a non-metric multidimensional scaling analysis, the samples grouped more according to methodology compared to plant. Some OTUs frequently detected by 454, most notably those OTUs with taxonomic affinity to Glomales, were not detected in the Sanger dataset. Likewise, a few OTUs, including Cenococcum sp., only appeared in the clone libraries. Surprisingly, we observed a significant relationship between GC/AT content of the OTUs and their proportional abundances in the 454 versus the clone library datasets. Reassuringly, a very good consistency in OTU recovery was observed between replicate runs of both sequencing methods. This indicates that stochastic processes had little impact when applying the same sequencing technique on replicate samples.

Evolutionary history of Serpulaceae (Basidiomycota): molecular phylogeny, historical biogeography and evidence for a single transition of nutritional mode.

BACKGROUND: The fungal genus Serpula (Serpulaceae, Boletales) comprises several saprotrophic (brown rot) taxa, including the aggressive house-infecting dry rot fungus Serpula lacrymans. Recent phylogenetic analyses have indicated that the ectomycorrhiza forming genera Austropaxillus and Gymnopaxillus cluster within Serpula. In this study we use DNA sequence data to investigate phylogenetic relationships, historical biogeography of, and nutritional mode transitions in Serpulaceae. RESULTS: Our results corroborate that the two ectomycorrhiza-forming genera, Austropaxillus and Gymnopaxillus, form a monophyletic group nested within the saprotrophic genus Serpula, and that the Serpula species S. lacrymans and S. himantioides constitute the sister group to the Austropaxillus-Gymnopaxillus clade. We found that both vicariance (Beringian) and long distance dispersal events are needed to explain the phylogeny and current distributions of taxa within Serpulaceae. Our results also show that the transition from brown rot to mycorrhiza has happened only once in a monophyletic Serpulaceae, probably between 50 and 22 million years before present. CONCLUSIONS: This study supports the growing understanding that the same geographical barriers that limit plant- and animal dispersal also limit the spread of fungi, as a combination of vicariance and long distance dispersal events are needed to explain the present patterns of distribution in Serpulaceae. Our results verify the transition from brown rot to ECM within Serpulaceae between 50 and 22 MyBP.

CLOTU: an online pipeline for processing and clustering of 454 amplicon reads into OTUs followed by taxonomic annotation.

BACKGROUND: The implementation of high throughput sequencing for exploring biodiversity poses high demands on bioinformatics applications for automated data processing. Here we introduce CLOTU, an online and open access pipeline for processing 454 amplicon reads. CLOTU has been constructed to be highly user-friendly and flexible, since different types of analyses are needed for different datasets. RESULTS: In CLOTU, the user can filter out low quality sequences, trim tags, primers, adaptors, perform clustering of sequence reads, and run BLAST against NCBInr or a customized database in a high performance computing environment. The resulting data may be browsed in a user-friendly manner and easily forwarded to downstream analyses. Although CLOTU is specifically designed for analyzing 454 amplicon reads, other types of DNA sequence data can also be processed. A fungal ITS sequence dataset generated by 454 sequencing of environmental samples is used to demonstrate the utility of CLOTU. CONCLUSIONS: CLOTU is a flexible and easy to use bioinformatics pipeline that includes different options for filtering, trimming, clustering and taxonomic annotation of high throughput sequence reads. Some of these options are not included in comparable pipelines. CLOTU is implemented in a Linux computer cluster and is freely accessible to academic users through the Bioportal web-based bioinformatics service (http://www.bioportal.uio.no).