Plant species identity and plant-induced changes in soil physicochemistry-but not plant phylogeny or functional traits - shape the assembly of the root-associated soil microbiome.

The root-associated soil microbiome contributes immensely to support plant health and performance against abiotic and biotic stressors. Understanding the processes that shape microbial assembly in root-associated soils is of interest in microbial ecology and plant health research. In this study, 37 plant species were grown in the same soil mixture for 10 months, whereupon the root-associated soil microbiome was assessed using amplicon sequencing. From this, the contribution of direct and indirect plant effects on microbial assembly was assessed. Plant species and plant-induced changes in soil physicochemistry were the most significant factors that accounted for bacterial and fungal community variation. Considering that all plants were grown in the same starting soil mixture, our results suggest that plants, in part, shape the assembly of their root-associated soil microbiome via their effects on soil physicochemistry. With the increase in phylogenetic ranking from plant species to class, we observed declines in the degree of community variation attributed to phylogenetic origin. That is, plant-microbe associations were unique to each plant species, but the phylogenetic associations between plant species were not important. We observed a large degree of residual variation (> 65%) not accounted for by any plant-related factors, which may be attributed to random community assembly.

Puke or poop? Comparison of regurgitate and faecal samples to infer alpine grasshopper (Paprides nitidus Hutton) diet in experimental plant communities.

Characterising plant-herbivore interactions is important to understanding the processes that influence community structure and ecosystem functioning. Traditional methods used to identify plant-herbivore interactions are being superseded by non-destructive molecular approaches that can infer interactions with greater resolution and accuracy from environmental DNA (e.g. faeces and regurgitate). However, few studies have compared the success of using different sample types and whether they provide similar or contrasting information about species' diet. Here we compared the success of DNA amplification and host plant species identification using restriction fragment length polymorphism (RFLP) applied to faecal and regurgitate samples collected from alpine grasshoppers Paprides nitidus Hutton during a grassland community mesocosm experiment. We found that DNA amplification success was 23% and 86% higher for faecal than regurgitate samples from female and male grasshoppers, respectively. In contrast, successful host plant identification using RFLP was 9% higher for regurgitate than faecal samples. The mean number of host plant species identified per sample (1.40) did not differ between sample types or grasshopper sexes. Of the 136 paired faecal-regurgitate samples, just 41% and 74% produced exactly or partially matching host plant identifications, respectively, indicating that different sample types provided complementary information about herbivore diet. Some plant species were more likely to be identified from faecal samples than expected by chance, and we found that this identification bias skewed towards plant species with higher investment in leaf tissue. We conclude that multiple sample types may be required to fully characterise an invertebrate herbivore species' diet.

Root diameter, host specificity and arbuscular mycorrhizal fungal community composition among native and exotic plant species.

Plant root systems rely on a functionally diverse range of arbuscular mycorrhizal fungi to, among other benefits, extend their nutrient foraging. Extended nutrient foraging is likely of greatest importance to coarse-rooted plants, yet few studies have examined the link between root traits and arbuscular mycorrhizal fungal community composition. Here, we examine the relationship between root diameter and the composition of arbuscular mycorrhizal fungal communities in a range of native and exotic plant species. We characterized the arbuscular mycorrhizal fungal communities of 30 co-occurring native and exotic montane grassland/shrubland plant species in New Zealand. We found that plant root diameter and native/exotic status both strongly correlated with arbuscular mycorrhizal fungal community composition. Coarse-rooted plants had a lower diversity of mycorrhizal fungi compared with fine-rooted plants and associated less with generalist fungal partners. Exotic plants had a lower diversity of fungi and fewer associations with nondominant families of arbuscular mycorrhizal fungi compared with native plants. These observational patterns suggest that plants may differentially associate with fungal partners based on their root traits, with coarse-rooted plants being more specific in their associations. Furthermore, exotic plants may associate with dominant arbuscular mycorrhizal fungal taxa as a strategy in invasion.

A network perspective for sustainable agroecosystems.

Nature-based management aims to improve sustainable agroecosystem production, but its efficacy has been variable. We argue that nature-based agroecosystem management could be significantly improved by explicitly considering and manipulating the underlying networks of species interactions. A network perspective can link species interactions to ecosystem functioning and stability, identify influential species and interactions, and suggest optimal management approaches. Recent advances in predicting the network roles of species from their functional traits could allow direct manipulation of network architecture through additions or removals of species with targeted traits. Combined with improved understanding of the structure and dynamics of networks across spatial and temporal scales and interaction types, including social-ecological, applying these tools to nature-based management can contribute to sustainable agroecosystems.

Agricultural land-use favours Mucoromycotinian, but not Glomeromycotinian, arbuscular mycorrhizal fungi across ten biomes.

Globally, agricultural land-use negatively affects soil biota that contribute to ecosystem functions such as nutrient cycling, yet arbuscular mycorrhizal fungi (AMF) are promoted as essential components of agroecosystems. Arbuscular mycorrhizal fungi include Glomeromycotinian AMF (G-AMF) and the arbuscule-producing fine root endophytes, recently re-classified into the Endogonales order within Mucoromycotina. The correct classification of Mucoromycotinian AMF (M-AMF) and the availability of new molecular tools can guide research to better the understanding of their diversity and ecology. To investigate the impact on G-AMF and M-AMF of agricultural land-use at a continental scale, we sampled DNA from paired farm and native sites across 10 Australian biomes. Glomeromycotinian AMF were present in both native and farm sites in all biomes. Putative M-AMF were favoured by farm sites, rare or absent in native sites, and almost entirely absent in tropical biomes. Temperature, rainfall, and soil pH were strong drivers of richness and community composition of both groups, and plant richness was an important mediator. Both fungal groups occupy different, but overlapping, ecological niches, with M-AMF thriving in temperate agricultural landscapes. Our findings invite exploration of the origin and spread of M-AMF and continued efforts to resolve the phylogeny of this newly reclassified group of AMF.

Environmental and plant community drivers of plant pathogen composition and richness.

Interactions between individual plant pathogens and their environment have been described many times. However, the relative contribution of different environmental parameters as controls of pathogen communities remains largely unknown. Here we investigate the importance of environmental factors, including geomorphology, climate, land use, soil and plant community composition, for a broad range of aboveground and belowground fungal, oomycete and bacterial plant pathogens. We found that plant community composition is the main driver of the composition and richness of plant pathogens after taking into account all other tested parameters, especially those related to climate and soil. In the face of future changes in climate and land use, our results suggest that changes in plant pathogen community composition and richness will primarily be mediated through changes in plant communities, rather than the direct effects of climate or soils.

Land use, but not distance, drives fungal beta diversity.

Fungi are one of the most diverse taxonomic groups on the planet, but much of their diversity and community organization remains unknown, especially at local scales. Indeed, a consensus on how fungal communities change across spatial or temporal gradients-beta diversity-remains nascent. Here, we use a data set of plant-associated fungal communities (leaf, root, and soil) across multiple land uses from a New Zealand-wide study to look at fungal community turnover at small spatial scales (<1 km). Using hierarchical Bayesian beta regressions and Hill-number-based diversity profiles, we show that fungal communities are often markedly dissimilar at even small distances, regardless of land use. Moreover, diversity profile plots indicate that leaf, root, and soil-associated communities show different patterns in the dominance or rarity of dissimilar species. Leaf-associated communities differed from site to site in their low-abundance species, whereas root-associated communities differed between sites in the dominant species; soil-associated communities were intermediate. Land-use differences were largely driven by the lower turnover between high-productivity grassland sites. Further, we discuss the implications and benefits of using diversity profile plots of turnover to draw inferences into the mechanisms of how communities are structured across spatial gradients.

Exotic plants accumulate and share herbivores yet dominate communities via rapid growth.

Herbivores may facilitate or impede exotic plant invasion, depending on their direct and indirect interactions with exotic plants relative to co-occurring natives. However, previous studies investigating direct effects have mostly used pairwise native-exotic comparisons with few enemies, reached conflicting conclusions, and largely overlooked indirect interactions such as apparent competition. Here, we ask whether native and exotic plants differ in their interactions with invertebrate herbivores. We manipulate and measure plant-herbivore and plant-soil biota interactions in 160 experimental mesocosm communities to test several invasion hypotheses. We find that compared with natives, exotic plants support higher herbivore diversity and biomass, and experience larger proportional biomass reductions from herbivory, regardless of whether specialist soil biota are present. Yet, exotics consistently dominate community biomass, likely due to their fast growth rates rather than strong potential to exert apparent competition on neighbors. We conclude that polyphagous invertebrate herbivores are unlikely to play significant direct or indirect roles in mediating plant invasions, especially for fast-growing exotic plants.

Soil sample pooling generates no consistent inference bias: a meta-analysis of 71 plant-soil feedback experiments.

There is current debate on how soil sample pooling affects the measurement of plant-soil feedbacks. Several studies have suggested that pooling soil samples among experimental units reduces variance and can bias estimates of plant-soil feedbacks. However, it is unclear whether pooling has resulted in systematic mismeasurement of plant-soil feedbacks in the literature. Using data from 71 experiments, we tested whether pairwise plant-soil feedback direction, magnitude and variance differed among soil pooling treatments. We also tested whether pooling has altered our understanding of abiotic and biotic drivers that influence pairwise plant-soil feedbacks. Pooling of soil samples among experimental units was used in 42% of examined experiments. Contrary to predictions, pooling did not affect mean pairwise plant-soil feedback effect size or within-experiment variance. Accounting for soil sample pooling also did not significantly alter our understanding of the drivers of pairwise plant-soil feedbacks. We conclude that there is no evidence that soil sample pooling systematically biases estimates of plant-soil feedback direction, magnitude, variance or drivers across many studies. Given the debate of whether to pool soil samples, researchers should be aware of potential criticisms and carefully consider how experimental design and soil pooling methods influence interpretation of experiments.

Evidence for Niche Differentiation in the Environmental Responses of Co-occurring Mucoromycotinian Fine Root Endophytes and Glomeromycotinian Arbuscular Mycorrhizal Fungi.

Fine root endophytes (FRE) were traditionally considered a morphotype of arbuscular mycorrhizal fungi (AMF), but recent genetic studies demonstrate that FRE belong within the subphylum Mucoromycotina, rather than in the subphylum Glomeromycotina with the AMF. These findings prompt enquiry into the fundamental ecology of FRE and AMF. We sampled FRE and AMF in roots of Trifolium subterraneum from 58 sites across temperate southern Australia. We investigated the environmental drivers of composition, richness, and root colonization of FRE and AMF by using structural equation modelling and canonical correspondence analyses. Root colonization by FRE increased with increasing temperature and rainfall but decreased with increasing phosphorus (P). Root colonization by AMF increased with increasing soil organic carbon but decreased with increasing P. Richness of FRE decreased with increasing temperature and soil pH. Richness of AMF increased with increasing temperature and rainfall but decreased with increasing soil aluminium (Al) and pH. Aluminium, soil pH, and rainfall were, in decreasing order, the strongest drivers of community composition of FRE; they were also important drivers of community composition of AMF, along with temperature, in decreasing order: rainfall, Al, temperature, and soil pH. Thus, FRE and AMF showed the same responses to some (e.g. soil P, soil pH) and different responses to other (e.g. temperature) key environmental factors. Overall, our data are evidence for niche differentiation among these co-occurring mycorrhizal associates.

Rarity is a more reliable indicator of land-use impacts on soil invertebrate communities than other diversity metrics.

The effects of land use on soil invertebrates - an important ecosystem component - are poorly understood. We investigated land-use impacts on a comprehensive range of soil invertebrates across New Zealand, measured using DNA metabarcoding and six biodiversity metrics. Rarity and phylogenetic rarity - direct measures of the number of species or the portion of a phylogeny unique to a site - showed stronger, more consistent responses across taxa to land use than widely used metrics of species richness, effective species numbers, and phylogenetic diversity. Overall, phylogenetic rarity explained the highest proportion of land use-related variance. Rarity declined from natural forest to planted forest, grassland, and perennial cropland for most soil invertebrate taxa, demonstrating pervasive impacts of agricultural land use on soil invertebrate communities. Commonly used diversity metrics may underestimate the impacts of land use on soil invertebrates, whereas rarity provides clearer and more consistent evidence of these impacts.

Living within the Earth's soil are millions of insects, worms and other invertebrates, which help keep the ground healthy and fertile. There is a growing concern that changing land-use habits, such as agriculture and urban development, are causing these populations of invertebrates to decline. However, to what extent different types of land use negatively impact soil invertebrates is not clear. Healthy habitats often have a greater variety of species. This biodiversity can be measured in a number of ways, ranging from counting the number of species, to more complex approaches that calculate a species' role in an ecosystem or how close it is to extinction. Finding a way to sensitively measure the biodiversity of soil invertebrates could further researcher's understanding of how different types of land use are affecting these communities. A new method known as DNA metabarcoding has made it easier to distinguish between different species and calculate the biodiversity of entire populations. Now, Dopheide et al. have used this technique to study invertebrate communities from 75 sites across New Zealand which have been impacted by different land-use habits. This revealed that the most reliable and consistent way to uncover how land use affects soil invertebrates was to measure the rarity of species (i.e. the number of unique species present at each site). Dopheide et al. found that agriculture negatively affected soil invertebrates and that most types of invertebrates responded in a similar way. Horticulture ­ such as orchards and vineyards ­ had the most severe impact, with the lowest variety of species compared to grassland or forest. Other measurements of biodiversity, such as the number of different species, may underestimate the negative impact agriculture is having on invertebrate communities. The findings of Dopheide et al. highlight why developing strategies to preserve and restore these communities is so important. However, more work is needed to understand what specifically is causing biodiversity to decline and how this effect can be reversed.

Rare species of wood-inhabiting fungi are not local.

Wood-inhabiting fungal communities are a diverse and ecologically critical part of forest ecosystems, yet the spatial structure of fungal biodiversity in these ecosystems is largely unknown. Legislation allowed harvesting of deadwood from temperate rainforests on conservation lands in New Zealand following Cyclone Ita in 2014. Harvesting guidelines specified widely spread harvesting, on the assumption that rare fungal species may be highly spatially restricted, but were not based on quantitative assessment. We sampled fungi in and on logs of Dacrydium cupressinum (Podocarpaceae) a long-lived, common, canopy tree in lowland New Zealand forests. DNA was extracted from 81 logs varying in decay state across a 40 km long region of West Coast (South Island) forests, and sequenced using general fungal primers for metabarcoding to identify OTUs (operational taxonomic units). We examined three axes of rarity: occupancy, dominance when present, and niche breadth (as spatial extent and decay state specialization). Low-occupancy fungi were common, including a group of infrequently occurring but dominant when present fungi, the majority of which were Ascomycota. Despite this, there was an overall positive relationship between occupancy and dominance. Widespread, dominant fungi were most commonly Basidiomycota. Testing all fungal OTUs, there were no more fungi with maximum range sizes < 4 km than would be expected at random. Of the 351 low-occupancy OTUs found two to four times, only 12 had maximum range sizes < 900 m, and there was no more spatial restriction at scales < 900 m than would be expected by random chance, although there was some evidence of niche breadth restriction based on decay state similarity. The results show that fungal communities in deadwood are highly diverse, and include many rare taxa. Nonetheless, the lack of fungal OTUs with spatial restriction at scales < 900 m suggests that spatially dispersed timber harvesting will not mitigate risks of harvesting to rare fungal biodiversity.

Community- and trophic-level responses of soil nematodes to removal of a non-native tree at different stages of invasion.

Success of invasive non-native plant species management is usually measured as changes in the abundance of the invasive plant species or native plant species following invader management, but more complex trophic responses to invader removal are often ignored or assumed. Moreover, the effects of invader removal at different stages of the invasion process is rarely evaluated, despite a growing recognition that invader impacts are density or stage-dependent. Therefore, the effectiveness of invasive species management for restoring community structure and function across trophic levels remains poorly understood. We determined how soil nematode diversity and community composition respond to removal of the globally invasive tree species Pinus contorta at different stages of invasion by reanalysing and expanding an earlier study including uninvaded vegetation (seedlings removed continuously), early invader removal (saplings removed), late removal (trees removed), and no removal (invaded). These treatments allowed us to evaluate the stage-dependent belowground trophic responses to biological invasion and removal. We found that invaded plots had half the nematode taxa richness compared to uninvaded plots, and that tree invasion altered the overall composition of the nematode community. Differences in nematode community composition between uninvaded nematode communities and those under the tree removal strategy tended to dilute higher up the food chain, whereas the composition of uninvaded vs. sapling removal strategies did not differ significantly. Conversely, the composition of invaded compared to uninvaded nematode communities differed across all trophic levels, altering the community structure and function. Specifically, invaded communities were structurally simplified compared to uninvaded communities, and had a higher proportion of short life cycle nematodes, characteristic of disturbed environments. We demonstrate that a shift in management strategies for a globally invasive tree species from removing trees to earlier removal of saplings is needed for maintaining the composition and structure of soil nematode communities to resemble uninvaded conditions.

Dual-mycorrhizal plants: their ecology and relevance.

Dual-mycorrhizal plants are capable of associating with fungi that form characteristic arbuscular mycorrhizal (AM) and ectomycorrhizal (EM) structures. Here, we address the following questions: (1) How many dual-mycorrhizal plant species are there? (2) What are the advantages for a plant to host two, rather than one, mycorrhizal types? (3) Which factors can provoke shifts in mycorrhizal dominance (i.e. mycorrhizal switching)? We identify a large number (89 genera within 32 families) of confirmed dual-mycorrhizal plants based on observing arbuscules or coils for AM status and Hartig net or similar structures for EM status within the same plant species. We then review the possible nutritional benefits and discuss the possible mechanisms leading to net costs and benefits. Cost and benefits of dual-mycorrhizal status appear to be context dependent, particularly with respect to the life stage of the host plant. Mycorrhizal switching occurs under a wide range of abiotic and biotic factors, including soil moisture and nutrient status. The relevance of dual-mycorrhizal plants in the ecological restoration of adverse sites where plants are not carbon limited is discussed. We conclude that dual-mycorrhizal plants are underutilized in ecophysiological-based experiments, yet are powerful model plant-fungal systems to better understand mycorrhizal symbioses without confounding host effects.

Land use is a determinant of plant pathogen alpha- but not beta-diversity.

Little is known about the diversity patterns of plant pathogens and how they change with land use at a broad scale. We employed DNA metabarcoding to describe the diversity and composition of putative plant pathogen communities in three substrates (soil, roots, and leaves) across five major land uses at a national scale. Almost all plant pathogen communities (fungi, oomycetes, and bacteria) showed strong responses to land use and substrate type. Land use category could explain up to 24% of the variance in composition between communities. Alpha-diversity (richness) of plant pathogens was consistently lower in natural forests than in agricultural systems. In planted forests, there was also generally low pathogen alpha-diversity in soil and roots, but alpha-diversity in leaves was high compared with most other land uses. In contrast to alpha-diversity, differences in within-land use beta-diversity of plant pathogens (the predictability of plant pathogen communities within land use) were subtle. Our results show that large-scale patterns and distributions of putative plant pathogens can be determined using metabarcoding, allowing some of the first landscape level insights into these critically important communities.

Contrasting responses of soil nematode communities to native and non-native woody plant expansion.

Woody plant expansion into grasslands is widespread, driven by both successions to dominance by native woody species or invasion by non-native woody species. These shifts from grass- to woody-dominated systems also have profound effects on both above- and belowground communities and ecosystem processes. Woody-plant expansion should also alter the functional composition of the soil biota, including that of nematodes, which are major drivers of soil food-web structure and belowground processes, but such belowground impacts are poorly understood. We determined whether succession by a widespread native (Kunzea ericoides) and invasion by a non-native woody species (Pinus nigra) into tussock grasslands affect the composition of nematode functional guilds and the structure of nematode-based food webs. Although increasing dominance by woody species in both systems altered the functional guild composition of the nematode community, we found contrasting responses of nematode functional guilds to the different dominant plant species. Specifically, nematode communities reflected conditions of resource enrichment with increasing K. ericoides tree cover, whereas communities became structurally simplified and dominated by stress-tolerant nematode families with increasing P. nigra tree cover. Because nematodes regulate both bacterial- and fungal-dominated food webs in soils, these shifts could in turn alter multiple ecosystem processes belowground such as nutrient cycling. Incorporating species' functional traits into the assessment of habitat-change impacts on communities can greatly improve our understanding of species responses to environmental changes and their consequences in ecosystems.

Biases in the metabarcoding of plant pathogens using rust fungi as a model system.

Plant pathogens such as rust fungi (Pucciniales) are of global economic and ecological importance. This means there is a critical need to reliably and cost-effectively detect, identify, and monitor these fungi at large scales. We investigated and analyzed the causes of differences between next-generation sequencing (NGS) metabarcoding approaches and traditional DNA cloning in the detection and quantification of recognized species of rust fungi from environmental samples. We found significant differences between observed and expected numbers of shared rust fungal operational taxonomic units (OTUs) among different methods. However, there was no significant difference in relative abundance of OTUs that all methods were capable of detecting. Differences among the methods were mainly driven by the method's ability to detect specific OTUs, likely caused by mismatches with the NGS metabarcoding primers to some Puccinia species. Furthermore, detection ability did not seem to be influenced by differences in sequence lengths among methods, the most appropriate bioinformatic pipeline used for each method, or the ability to detect rare species. Our findings are important to future metabarcoding studies, because they highlight the main sources of difference among methods, and rule out several mechanisms that could drive these differences. Furthermore, strong congruity among three fundamentally different and independent methods demonstrates the promising potential of NGS metabarcoding for tracking important taxa such as rust fungi from within larger NGS metabarcoding communities. Our results support the use of NGS metabarcoding for the large-scale detection and quantification of rust fungi, but not for confirming the absence of species.

Towards robust and repeatable sampling methods in eDNA-based studies.

DNA-based techniques are increasingly used for measuring the biodiversity (species presence, identity, abundance and community composition) of terrestrial and aquatic ecosystems. While there are numerous reviews of molecular methods and bioinformatic steps, there has been little consideration of the methods used to collect samples upon which these later steps are based. This represents a critical knowledge gap, as methodologically sound field sampling is the foundation for subsequent analyses. We reviewed field sampling methods used for metabarcoding studies of both terrestrial and freshwater ecosystem biodiversity over a nearly three-year period (n = 75). We found that 95% (n = 71) of these studies used subjective sampling methods and inappropriate field methods and/or failed to provide critical methodological information. It would be possible for researchers to replicate only 5% of the metabarcoding studies in our sample, a poorer level of reproducibility than for ecological studies in general. Our findings suggest greater attention to field sampling methods, and reporting is necessary in eDNA-based studies of biodiversity to ensure robust outcomes and future reproducibility. Methods must be fully and accurately reported, and protocols developed that minimize subjectivity. Standardization of sampling protocols would be one way to help to improve reproducibility and have additional benefits in allowing compilation and comparison of data from across studies.