Metabarcoding of soil environmental DNA to estimate plant diversity globally.

Introduction: Traditional approaches to collecting large-scale biodiversity data pose huge logistical and technical challenges. We aimed to assess how a comparatively simple method based on sequencing environmental DNA (eDNA) characterises global variation in plant diversity and community composition compared with data derived from traditional plant inventory methods. Methods: We sequenced a short fragment (P6 loop) of the chloroplast trnL intron from from 325 globally distributed soil samples and compared estimates of diversity and composition with those derived from traditional sources based on empirical (GBIF) or extrapolated plant distribution and diversity data. Results: Large-scale plant diversity and community composition patterns revealed by sequencing eDNA were broadly in accordance with those derived from traditional sources. The success of the eDNA taxonomy assignment, and the overlap of taxon lists between eDNA and GBIF, was greatest at moderate to high latitudes of the northern hemisphere. On average, around half (mean: 51.5% SD 17.6) of local GBIF records were represented in eDNA databases at the species level, depending on the geographic region. Discussion: eDNA trnL gene sequencing data accurately represent global patterns in plant diversity and composition and thus can provide a basis for large-scale vegetation studies. Important experimental considerations for plant eDNA studies include using a sampling volume and design to maximise the number of taxa detected and optimising the sequencing depth. However, increasing the coverage of reference sequence databases would yield the most significant improvements in the accuracy of taxonomic assignments made using the P6 loop of the trnL region.

Global diversity and distribution of nitrogen-fixing bacteria in the soil.

Our knowledge of microbial biogeography has advanced in recent years, yet we lack knowledge of the global diversity of some important functional groups. Here, we used environmental DNA from 327 globally collected soil samples to investigate the biodiversity patterns of nitrogen-fixing bacteria by focusing on the nifH gene but also amplifying the general prokaryotic 16S SSU region. Globally, N-fixing prokaryotic communities are driven mainly by climatic conditions, with most groups being positively correlated with stable hot or seasonally humid climates. Among soil parameters, pH, but also soil N content were most often shown to correlate with the diversity of N-fixer groups. However, specific groups of N-fixing prokaryotes show contrasting responses to the same variables, notably in Cyanobacteria that were negatively correlated with stable hot climates, and showed a U-shaped correlation with soil pH, contrary to other N-fixers. Also, the non-N-fixing prokaryotic community composition was differentially correlated with the diversity and abundance of N-fixer groups, showing the often-neglected impact of biotic interactions among bacteria.

Elevation and plant species identity jointly shape a diverse arbuscular mycorrhizal fungal community in the High Arctic.

Knowledge about the distribution and local diversity patterns of arbuscular mycorrhizal (AM) fungi are limited for extreme environments such as the Arctic, where most studies have focused on spore morphology or root colonization. We here studied the joint effects of plant species identity and elevation on AM fungal distribution and diversity. We sampled roots of 19 plant species in 18 locations in Northeast Greenland, using next generation sequencing to identify AM fungi. We studied the joint effect of plant species, elevation and selected abiotic conditions on AM fungal presence, richness and composition. We identified 29 AM fungal virtual taxa (VT), of which six represent putatively new VT. Arbuscular mycorrhizal fungal presence increased with elevation, and as vegetation cover and the active soil layer decreased. Arbuscular mycorrhizal fungal composition was shaped jointly by elevation and plant species identity. We demonstrate that the Arctic harbours a relatively species-rich and nonrandomly distributed diversity of AM fungi. Given the high diversity and general lack of knowledge exposed herein, we encourage further research into the diversity, drivers and functional role of AM fungi in the Arctic. Such insight is urgently needed for an area with some of the globally highest rates of climate change.

Dominance, diversity, and niche breadth in arbuscular mycorrhizal fungal communities.

Classical theory identifies resource competition as the major structuring force of biotic communities and predicts that (i) levels of dominance and richness in communities are inversely related, (ii) narrow niches allow dense "packing" in niche space and thus promote diversity, and (iii) dominants are generalists with wide niches, such that locally abundant taxa also exhibit wide distributions. Current empirical support, however, is mixed. We tested these expectations using published data on arbuscular mycorrhizal (AM) fungal community composition worldwide. We recorded the expected negative relationship between dominance and richness and, to a degree, the positive association between local and global dominance. However, contrary to expectations, dominance was pronounced in communities where more specialists were present and, conversely, richness was higher in communities with more generalists. Thus, resource competition and niche packing appear to be of limited importance in AM fungal community assembly; rather, patterns of dominance and diversity seem more consistent with habitat filtering and stochastic processes.

Global taxonomic and phylogenetic assembly of AM fungi.

Arbuscular mycorrhizal (AM) fungi are a ubiquitous group of plant symbionts, yet processes underlying their global assembly - in particular the roles of dispersal limitation and historical drivers - remain poorly understood. Because earlier studies have reported niche conservatism in AM fungi, we hypothesized that variation in taxonomic community composition (i.e., unweighted by taxon relatedness) should resemble variation in phylogenetic community composition (i.e., weighted by taxon relatedness) which reflects ancestral adaptations to historical habitat gradients. Because of the presumed strong dispersal ability of AM fungi, we also anticipated that the large-scale structure of AM fungal communities would track environmental conditions without regional discontinuity. We used recently published AM fungal sequence data (small-subunit ribosomal RNA gene) from soil samples collected worldwide to reconstruct global patterns in taxonomic and phylogenetic community variation. The taxonomic structure of AM fungal communities was primarily driven by habitat conditions, with limited regional differentiation, and there were two well-supported clusters of communities - occurring in cold and warm conditions. Phylogenetic structure was driven by the same factors, though all relationships were markedly weaker. This suggests that niche conservatism with respect to habitat associations is weakly expressed in AM fungal communities. We conclude that the composition of AM fungal communities tracks major climatic and edaphic gradients, with the effects of dispersal limitation and historic factors considerably less apparent than those of climate and soil.

Legacy effects of temporary grassland in annual crop rotation on soil ecosystem services.

The introduction of temporary grassland into an annual crop rotation is recognized to improve soil ecosystem services, and resulting legacies can be beneficial for the following crops. In this context, the aim of the present study was to evaluate legacy effects of introducing temporary grassland into an annual crop rotation on five ecosystem services (i) soil structure maintenance (aggregate stability), (ii) water regulation (saturated hydraulic conductivity), (iii) biodiversity conservation (microbial biomass and microbial metabolic activity, as well as microorganism, enchytraeid, springtail and earthworm communities), (iv) pathogen regulation (soil suppressiveness to Verticillium dahliae), and (v) forage production and quality. Three crop rotation schemes, maintained for twelve years, were compared in four random blocks, one being an annual crop rotation without grassland (0%), another with a medium percentage of grassland (50%, corresponding to 3 years of continuous grassland in the crop rotation), and a third one with a high percentage of grassland in the crop rotation (75%, corresponding to 6 years of continuous grassland in the crop rotation). The results showed that the grassland introduction into an annual crop rotation improved, whatever the duration of the grassland, soil structure maintenance and biodiversity conservation, while it decreased pathogen regulation and did not modify water regulation. Comparing the two crop rotations that included grassland, indicated a stronger beneficial grassland legacy effect for the higher proportion of grassland concerning soil structure maintenance and biodiversity conservation. By contrast, water regulation, pathogen regulation and forage production were not affected by the legacy of the 75% grassland during the rotation. Overall, our findings demonstrated the extent to which grassland legacies are affecting the current state of soil properties and possible ecosystem services provided. To improve ecosystem services, soil management should take legacy effects into account and consider longer timeframes to apply beneficial practices.

User-friendly bioinformatics pipeline gDAT (graphical downstream analysis tool) for analysing rDNA sequences.

High-throughput sequencing (HTS) of multiple organisms in parallel (metabarcoding) has become a routine and cost-effective method for the analysis of microbial communities in environmental samples. However, careful data treatment is required to identify potential errors in HTS data, and the large volume of data generated by HTS requires in-house experience with command line tools for downstream analysis. This paper introduces a pipeline that incorporates the most common command line tools into an easy-to-use graphical interface-gDAT. By using the Python scripting language, the pipeline is compatible with the latest Windows, macOS and Linux operating systems. The pipeline supports analysis of Sanger, 454, IonTorrent, Illumina and PacBio sequences, allows custom modification of quality filtering steps, and implements both open and closed-reference operational taxonomic unit-picking for sequence identification. Predefined parameters are optimized for analysis of small subunit (SSU) rRNA gene amplicons from arbuscular mycorrhizal fungi, but the pipeline is widely applicable to metabarcoding studies targeting a broad range of organisms. The pipeline was additionally tested with data using general eukaryotic primers from the SSU gene region and fungal primers from the internal transcribed spacer (ITS) marker region. We describe the pipeline design and evaluate its performance and speed by conducting analysis of example data sets using different marker regions sequenced on Illumina platforms. The graphical interface, with the option to use the command line if needed, provides an accessible tool for rapid data analysis with repeatability and logging capabilities. Keeping the software open-source maximizes code accessibility, allowing scrutiny and bug fixes by the community.

Arbuscular Mycorrhizal Fungal Communities in the Soils of Desert Habitats.

Deserts cover a significant proportion of the Earth's surface and continue to expand as a consequence of climate change. Mutualistic arbuscular mycorrhizal (AM) fungi are functionally important plant root symbionts, and may be particularly important in drought stressed systems such as deserts. Here we provide a first molecular characterization of the AM fungi occurring in several desert ecosystems worldwide. We sequenced AM fungal DNA from soil samples collected from deserts in six different regions of the globe using the primer pair WANDA-AML2 with Illumina MiSeq. We recorded altogether 50 AM fungal phylotypes. Glomeraceae was the most common family, while Claroideoglomeraceae, Diversisporaceae and Acaulosporaceae were represented with lower frequency and abundance. The most diverse site, with 35 virtual taxa (VT), was in the Israeli Negev desert. Sites representing harsh conditions yielded relatively few reads and low richness estimates, for example, a Saudi Arabian desert site where only three Diversispora VT were recorded. The AM fungal taxa recorded in the desert soils are mostly geographically and ecologically widespread. However, in four sites out of six, communities comprised more desert-affiliated taxa (according to the MaarjAM database) than expected at random. AM fungal VT present in samples were phylogenetically clustered compared with the global taxon pool, suggesting that nonrandom assembly processes, notably habitat filtering, may have shaped desert fungal assemblages.

Plant functional groups associate with distinct arbuscular mycorrhizal fungal communities.

The benefits of the arbuscular mycorrhizal (AM) symbiosis between plants and fungi are modulated by the functional characteristics of both partners. However, it is unknown to what extent functionally distinct groups of plants naturally associate with different AM fungi. We reanalysed 14 high-throughput sequencing data sets describing AM fungal communities associating with plant individuals (2427) belonging to 297 species. We examined how root-associating AM fungal communities varied between plants with different growth forms, photosynthetic pathways, CSR (competitor, stress-tolerator, ruderal) strategies, mycorrhizal statuses and N-fixing statuses. AM fungal community composition differed in relation to all studied plant functional groups. Grasses, C4 and nonruderal plants were characterised by high AM fungal alpha diversity, while C4 , ruderal and obligately mycorrhizal plants were characterised by high beta diversity. The phylogenetic diversity of AM fungi, a potential surrogate for functional diversity, was higher among forbs than other plant growth forms. Putatively ruderal (previously cultured) AM fungi were disproportionately associated with forbs and ruderal plants. There was phylogenetic correlation among AM fungi in the degree of association with different plant growth forms and photosynthetic pathways. Associated AM fungal communities constitute an important component of plant ecological strategies. Functionally different plants associate with distinct AM fungal communities, linking mycorrhizal associations with functional diversity in ecosystems.

Non-random association patterns in a plant-mycorrhizal fungal network reveal host-symbiont specificity.

Arbuscular mycorrhizal (AM) fungi are obligate plant symbionts that have important functions in most terrestrial ecosystems, but there remains an incomplete understanding of host-fungus specificity and the relationships between species and functional groups of plants and AM fungi. Here, we aimed to provide a comprehensive description of plant-AM fungal interactions in a biodiverse semi-natural grassland. We sampled all plant species in a 1,000-m2 homogeneous plot of dry calcareous grassland in two seasons (summer and autumn) and identified root-colonizing AM fungi by SSU rDNA sequencing. In the network of 33 plant and 100 AM fungal species, we found a significant effect of both host plant species and host plant functional group on AM fungal richness and community composition. Comparison with network null models revealed a larger-than-random degree of partner selectivity among plants. Grasses harboured a larger number of AM fungal partners and were more generalist in partner selection, compared with forbs. More generalist partner association and lower specialization were apparent among obligately, compared with facultatively, mycorrhizal plant species and among locally more abundant plant species. This study provides the most complete data set of co-occurring plant and AM fungal taxa to date, showing that at this particular site, the interaction network is assembled non-randomly, with moderate selectivity in associations between plant species and functional groups and their fungal symbionts.

Anthropogenic disturbance equalizes diversity levels in arbuscular mycorrhizal fungal communities.

The arbuscular mycorrhizal (AM) symbiosis is a key plant-microbe interaction in sustainable functioning ecosystems. Increasing anthropogenic disturbance poses a threat to AM fungal communities worldwide, but there is little empirical evidence about its potential negative consequences. In this global study, we sequenced AM fungal DNA in soil samples collected from pairs of natural (undisturbed) and anthropogenic (disturbed) plots in two ecosystem types (10 naturally wooded and six naturally unwooded ecosystems). We found that ecosystem type had stronger directional effects than anthropogenic disturbance on AM fungal alpha and beta diversity. However, disturbance increased alpha and beta diversity at sites where natural diversity was low and decreased diversity at sites where natural diversity was high. Cultured AM fungal taxa were more prevalent in anthropogenic than natural plots, probably due to their efficient colonization strategies and ability to recover from disturbance. We conclude that anthropogenic disturbance does not have a consistent directional effect on AM fungal diversity; rather, disturbance equalizes levels of diversity at large scales and causes changes in community functional structure.

Effects of land use on arbuscular mycorrhizal fungal communities in Estonia.

Arbuscular mycorrhizal (AM) fungal communities vary across habitat types, as well as across different land use types. Most relevant research, however, has focused on agricultural or other severely human-impacted ecosystems. Here, we compared AM fungal communities across six habitat types: calcareous grassland, overgrown ungrazed calcareous grassland, wooded meadow, farmyard lawn, boreonemoral forest, and boreonemoral forest clear-cut, exhibiting contrasting modes of land use. AM fungi in the roots of a single host plant species, Prunella vulgaris, and in its rhizosphere soil were identified using 454-sequencing from a total of 103 samples from 12 sites in Estonia. Mean AM fungal taxon richness per sample did not differ among habitats. AM fungal community composition, however, was significantly different among habitat types. Both abandonment and land use intensification (clearcutting; trampling combined with frequent mowing) changed AM fungal community composition. The AM fungal communities in different habitat types were most similar in the roots of the single host plant species and most distinct in soil samples, suggesting a non-random pattern in host-fungal taxon interactions. The results show that AM fungal taxon composition is driven by habitat type and land use intensity, while the plant host may act as an additional filter between the available and realized AM fungal species pool.