Marie L. Davey

Seasonal trends in the biomass and structure of bryophyte-associated fungal communities explored by 454 pyrosequencing

Bryophytes are a dominant vegetation component of the boreal forest, but little is known about their associated fungal communities, including seasonal variation within them. Seasonal variation in the fungal biomass and composition of fungal communities associated with three widespread boreal bryophytes was investigated using HPLC assays of ergosterol and amplicon pyrosequencing of the internal transcribed spacer 2 (ITS2) region of rDNA. The bryophyte phyllosphere community was dominated by Ascomycota. Fungal biomass did not decline appreciably in winter (P=0.272). Significant host-specific patterns in seasonal variation of biomass were detected (P=0.003). Although seasonal effects were not the primary factors structuring community composition, collection date significantly explained (P=0.001) variation not attributed to locality, host, and tissue. Community homogenization and a reduction in turnover occurred with the onset of frost events and subzero air and soil temperatures. Fluctuations in the relative abundance of particular fungal groups seem to reflect the nature of their association with mosses, although conclusions are drawn with caution because of potential methodological bias. The moss-associated fungal community is dynamic, exhibiting seasonal turnover in composition and relative abundance of different fungal groups, and significant fungal biomass is present year-round, suggesting a winter-active fungal community.

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 palaeodiversity revealed using high-throughput metabarcoding of ancient DNA from arctic permafrost.

The taxonomic and ecological diversity of ancient fungal communities was assessed by combining next generation sequencing and metabarcoding of DNA preserved in permafrost. Twenty-six sediment samples dated 16 000-32 000 radiocarbon years old from two localities in Siberia were analysed for fungal ITS. We detected 75 fungal OTUs from 21 orders representing three phyla, although rarefaction analyses suggested that the full diversity was not recovered despite generating an average of 6677 ± 3811 (mean ± SD) sequences per sample and that preservation bias likely has considerable effect on the recovered DNA. Most OTUs (75.4%) represented ascomycetes. Due to insufficient sequencing depth, DNA degradation and putative preservation biases in our samples, the recovered taxa probably do not represent the complete historic fungal community, and it is difficult to determine whether the fungal communities varied geographically or experienced a composition shift within the period of 16 000-32 000 bp. However, annotation of OTUs to functional ecological groups provided a wealth of information on the historic communities. About one-third of the OTUs are presumed plant-associates (pathogens, saprotrophs and endophytes) typical of graminoid- and forb-rich habitats. We also detected putative insect pathogens, coprophiles and keratinophiles likely associated with ancient insect and herbivore faunas. The detection of putative insect pathogens, mycoparasites, aquatic fungi and endophytes broadens our previous knowledge of the diversity of fungi present in Beringian palaeoecosystems. A large group of putatively psychrophilic/psychrotolerant fungi was also detected, most likely representing a modern, metabolically active fungal community.

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.

Amplicon-pyrosequencing-based detection of compositional shifts in bryophyte-associated fungal communities along an elevation gradient

Although bryophytes are a dominant vegetation component of boreal and alpine ecosystems, little is known about their associated fungal communities. HPLC assays of ergosterol (fungal biomass) and amplicon pyrosequencing of the ITS2 region of rDNA were used to investigate how the fungal communities associated with four bryophyte species changed across an elevational gradient transitioning from conifer forest to the low‐alpine. Fungal biomass and OTU richness associated with the four moss hosts did not vary significantly across the gradient (P > 0.05), and both were more strongly affected by host and tissue type. Despite largely constant levels of fungal biomass, distinct shifts in community composition of fungi associated with Hylocomium, Pleurozium and Polytrichum occurred between the elevation zones of the gradient. This likely is a result of influence on fungal communities by major environmental factors such as temperature, directly or indirectly mediated by, or interacting with, the response of other components of the vegetation (i.e. the dominant trees). Fungal communities associated with Dicranum were an exception, exhibiting spatial autocorrelation between plots, and no significant structuring by elevation. Nevertheless, the detection of distinct fungal assemblages associated with a single host growing in different elevation zones along an elevational gradient is of particular relevance in the light of the ongoing changes in vegetation patterns in boreal and alpine systems due to global climate warming.

Fungal biomass associated with the phyllosphere of bryophytes and vascular plants

Little is known about the amount of fungal biomass in the phyllosphere of bryophytes compared to higher plants. In this study, fungal biomass associated with the phyllosphere of three bryophytes (Hylocomium splendens, Pleurozium schreberi, Polytrichum commune) and three vascular plants (Avenella flexuosa, Gymnocarpium dryopteris, Vaccinium myrtillus) was investigated using ergosterol content as a proxy for fungal biomass. Phyllosphere fungi accounted for 0.2-4.0 % of the dry mass of moss gametophytes, representing the first estimation of fungal biomass associated with bryophytes. Significantly more fungal biomass was associated with the phyllosphere of bryophytes than co-occurring vascular plants. The ergosterol present in moss gametophytic tissues differed significantly between species, while the ergosterol present in vascular plant leaf tissues did not. The photosynthetic tissues of mosses had less associated fungal biomass than their senescent tissues, and the magnitude of this difference varied in a species-specific manner. The fungal biomass associated with the vascular plants studied varied significantly between localities, while that of mosses did not. The observed differences in phyllosphere community biomass suggest their size could be affected by host anatomical and physiological attributes, including micro-niche availability and chemical host defenses, in addition to abiotic factors like moisture and nutrient availability.

Pathogenesis of bryophyte hosts by the ascomycete Atradidymella muscivora.

Atradidymella muscivora (Pleosporales) is a bryophyte pathogen that infects the mosses Aulacomnium palustre, Hylocomium splendens, and Polytrichum juniperinum. Light and scanning electron microscopy and extracellular enzyme production were used to characterize the interactions between this fungus and its native hosts and the model host Funaria hygrometrica. Penetration was direct via hyphae or appressoria, and hosts responded by forming layered, darkly pigmented deposits at penetration sites, similar to the papillae formed by vascular plants in response to fungal infection. Infected hosts gradually became chlorotic as hyphae grew intracellularly, presumably killing host cells. Pycnidia of the Phoma anamorph (P. muscivora) and uniloculate pseudothecia were initiated as tightly packed masses of stromatic dematiaceous hyphae within a single host cell. Mature pycnidia and pseudothecia were erumpent. A new microniche among bryophilous fungi is described, whereby A. muscivora supplants the gemmae of Aul. palustre and exploits the normal nutrient-flow of the moss gametophyte. Atradidymella muscivora produced both cellulases and soluble polyphenolic oxidases, allowing it to also function as a saprobe and degrade the cell walls of bryophytes. The saprophytic and pathogenic abilities of A. muscivora suggest it may play a role in nutrient cycling, population dynamics, and small-scale disturbances in boreal ecosystems.

Atradidymella muscivora gen. et sp. nov. (Pleosporales) and its anamorph Phoma muscivora sp. nov.: A new pleomorphic pathogen of boreal bryophytes.

During a survey of bryophilous fungi from boreal and montane habitats, 12 isolates of a hitherto unknown plant pathogenic member of the Pleosporales were recovered from Aulacomnium palustre, Hylocomium splendens, and Polytrichum juniperinum, and described as Atradidymella muscivora gen. et sp. nov. Atradidymella is characterized by minute, unilocular, setose pseudothecia having 2-3 wall layers; brown, fusiform, 1-septate ascospores; and a Phoma anamorph. The genus is distinguished from all other pleosporalean genera with brown, fusiform ascospores on the basis of ascospore and pseudothecium morphology and a highly reduced stroma that is localized within a single host cell. Atradidymella muscivora is distinguished by its minute pseudothecia (<115 μm) and ascospores that are slightly allantoid and constricted at the septum with the upper cell often wider than the lower. Its anamorph, Phoma muscivora sp. nov., is morphologically distinguishable from P. herbarum in having smaller conidia. Parsimony analysis of the ITS rDNA region indicates A. muscivora has affinities to the Phoma-Ascochyta-Didymella clade that is sister to the Phaeosphaeriaceae within the Pleosporales.

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 communitys 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.

Forestry impacts on the hidden fungal biodiversity associated with bryophytes

Recent studies have revealed an unexpectedly high, cryptic diversity of fungi associated with boreal forest bryophytes. Forestry practices heavily influence the boreal forest and fundamentally transform the landscape. However, little is known about how bryophyte-associated fungal communities are affected by these large-scale habitat transformations. This study assesses to what degree bryophyte-associated fungal communities are structured across the forest successional stages created by current forestry practices. Shoots of Hylocomium splendens were collected in Picea abies dominated forests of different ages, and their associated fungal communities were surveyed by pyrosequencing of ITS2 amplicons. Although community richness, diversity and evenness were relatively stable across the forest types and all were consistently dominated by ascomycete taxa, there was a marked shift in fungal community composition between young and old forests. Numerous fungal operational taxonomic units (OTUs) showed distinct affinities for different forest ages. Spatial structure was also detected among the sites, suggesting that environmental gradients resulting from the topography of the study area and dispersal limitations may also significantly affect bryophyte-associated fungal community structure. This study confirms that Hylocomium splendens hosts an immense diversity of fungi and demonstrates that this community is structured in part by forest age, and as such is highly influenced by modern forestry practices.