Evolutionary dependence of host type and chasmothecial appendage morphology in obligate plant parasites belonging to Erysipheae (powdery mildew, Erysiphaceae).

Evolutionary relationships between the morphological and ecological traits of fungi are poorly understood. The appendages of chasmothecia, which are sexual reproductive organs of Erysiphaceae, are considered to play a crucial role in the overwintering strategies of these fungi on host plants. Previous studies suggested that both the host type and appendage morphology evolved at the same nodes and transitioned from complex appendages on deciduous hosts to simple appendages on herb/evergreen hosts. However, the evolutionary dependence between host type and appendage morphology remains unproven owing to the limited species data used in analyses. To elucidate the evolutionary relationship between host type and appendage morphology, we used phylogenetic comparative methods (PCMs) to investigate the state transition, ancestral state, evolutionary dependence, and contingent evolution within Erysipheae, the largest and most diverse tribe in Erysiphaceae. Our PCMs, based on a comprehensive data set of Erysipheae, revealed that the most ancestral states were deciduous host types and complex appendages. From these ancestral states, convergent evolution toward the herb/evergreen host types and simple appendages occurred multiple times at the same nodes. For the first time in Erysiphaceae, we detected an evolutionary dependence between host type and appendage morphology. This is one of the few examples in which evolutionary dependence between host phenology and morphological traits in plant-parasitic fungi was demonstrated using PCMs. Appendage simplification on herb/evergreen hosts and complications on deciduous hosts can be reasonably explained by the functional advantages of each appendage type in different overwintering strategies. These expected appendage functions can explain approximately 90% of host type and appendage morphology combinations observed in the analyzed taxa. However, our results also highlighted the occurrence of evolutionary shifts that deviate from the expected advantages of each appendage morphology. These seemingly irrational shifts might be interpretable from the flexibility of overwintering strategies and quantification of appendage functions.

Long-term Consequences on Soil Fungal Community Structure: Monoculture Planting and Natural Regeneration.

Understanding the regeneration and succession of belowground communities, particularly in forests, is vital for maintaining ecosystem health. Despite its importance, there is limited knowledge regarding how fungal communities change over time during ecosystem development, especially under different forest restoration strategies. In this study, we focused on two restoration methods used in northern Japan: monoculture planting and natural regeneration. We examined the responses of the fungal community to monoculture plantations (active tree planting) and naturally regenerated (passive regeneration) forests over a 50-year chronosequence, using natural forests as a reference. Based on DNA metabarcoding, we assessed the richness of fungal Operational Taxonomic Units (OTUs) and their dissimilarity. Our findings revealed that soil fungal richness remained stable after natural regeneration but declined in monoculture plantations, from 354 to 247 OTUs. While the compositional dissimilarity of fungal assemblages between monoculture plantations and natural forests remained consistent regardless of the time since tree planting, it significantly decreased after natural regeneration, suggesting recovery to a state close to the reference level. Notably, the composition of key functional fungal groups-saprotrophic and ectomycorrhizal- has increasingly mirrored that of natural forests over time following passive natural regeneration. In summary, our study suggests that monoculture plantations may not be effective for long-term ecosystem function and service recovery because of their limited support for soil fungal diversity. These results underscore the importance of natural regeneration in forest restoration and management strategies.

Synergistic effects of succession and microtopography of moraine on the fungal spatial diversity in a glacier forefield.

Primary succession and microtopography result in environmental changes and are important processes influencing the community assembly of soil fungi in the Arctic region. In glacier forefields that contain a series of moraine ridges, both processes contribute synchronously to fungal spatial diversity. To reveal the synergistic effects of succession and microtopography, we investigated the fungal community structure and environmental variables in the moraines of the Arklio Glacier, Ellesmere Island. The study sites were established at four locations from the top to the bottom of the ridge slope within each of the three moraine ridges of different post-glacial ages. The location-dependent community composition was equally diverse in both the initial and later stages of succession, suggesting that successional time could alter the effects of microtopography on the fungal community. Moreover, our results suggest that fungal communities at different locations follow different successional trajectories, even if they have passed through the same time lapse. Such a synergistic effect of succession and microtopography of moraines does not allow for parallel changes in fungal communities among moraines or among locations, suggesting that the moraine series contributes substantially to fungal spatial diversity in the glacier forefield.

Discrepancies of fungi and plants in the pattern of beta-diversity with environmental gradient imply a comprehensive community assembly rule.

Beta-diversity partitioning has shown that the nestedness component is developed with environmental stress in a variety of taxa. However, soil fungal community may maintain its turnover components in contrast to the development of plants' nestedness component, and the potential causes remain unclear. To investigate the process of species turnover of soil fungi along a stress gradient in the Arctic, we divided species turnover component into sub-components: ßsim_hete and ßsim_homo representing species turnover with and without a change in the guilds, respectively. The results indicate that fungal communities maintain their turnover components, unlike plant communities; however, their ßsim_hete increased under stressful conditions. Additionally, GDM analysis showed that ßsim_hete was mainly explained by stress gradient and plant nestedness, suggesting that the functionality of soil fungi was ecologically filtered by environmental stress and plant community structure. The discordant trend of beta-diversity values between plant and fungi (i.e. development of plant nestedness and maintenance of fungal turnover) is possibly not caused by different assembly rules working in parallel on the two taxa, but according to an ecological rule that reflects plant-fungal interaction.

Influence of DNA extraction kits on freshwater fungal DNA metabarcoding.

Background: Environmental DNA (eDNA) metabarcoding is a common technique for efficient biodiversity monitoring, especially of microbes. Recently, the usefulness of aquatic eDNA in monitoring the diversity of both terrestrial and aquatic fungi has been suggested. In eDNA studies, different experimental factors, such as DNA extraction kits or methods, can affect the subsequent analyses and the results of DNA metabarcoding. However, few methodological studies have been carried out on eDNA of fungi, and little is known about how experimental procedures can affect the results of biodiversity analysis. In this study, we focused on the effect of DNA extraction method on fungal DNA metabarcoding using freshwater samples obtained from rivers and lakes. Methods: DNA was extracted from freshwater samples using the DNeasy PowerSoil kit, which is mainly used to extractmicrobial DNA from soil, and the DNeasy Blood & Tissue kit, which is commonly used for eDNA studies on animals. We then compared PCR inhibition and fungal DNA metabarcoding results; i.e., operational taxonomic unit (OTU) number and composition of the extracted samples. Results: No PCR inhibition was detected in any of the samples, and no significant differences in the number of OTUs and OTU compositions were detected between the samples processed using different kits. These results indicate that both DNA extraction kits may provide similar diversity results for the river and lake samples evaluated in this study. Therefore, it may be possible to evaluate the diversity of fungi using a unified experimental method, even with samples obtained for diversity studies on other taxa such as those of animals.

Evolutionary patterns of host type and chasmothecial appendage morphology in obligate plant parasites belonging to Cystotheceae (powdery mildew, Erysiphaceae).

The chasmothecial appendages of Erysiphaceae are considered to function in the overwintering strategy and evolve morphologically in line with transitions of different host type. However, the evolutionary patterns and relationships of these traits have not yet been verified using statistical models based on phylogenetic information. We aimed to clarify the evolutionary process of host type and appendage morphology in Cystotheceae using phylogenetic comparative methods (PCMs) and to evaluate the evolutionary relationship of these traits. The ancestral state estimation of host types showed that the deciduous type is the most ancestral in Cystotheceae, and the herb or evergreen types evolved secondarily four times and twice, respectively. Branched- or circinate-type appendages were estimated to be the most ancestral, and the mycelioid and rudimentary types evolved secondarily thrice and once, respectively. The results of the random forest analysis showed that the host type was predictable from the phylogeny and appendage morphology. The ancestral state estimation suggested that simultaneous transitions of the host type and appendage morphology occurred at several ancestral nodes. These results suggest some functional relationships between host type and appendage morphology, but there was no statistical support for an overall trend in evolutionary dependence between these traits. Our results demonstrate the utility of PCMs in the study of trait evolution in Cystotheceae, which can be applied to a broader phylogeny of powdery mildews to elucidate the evolutionary relationship and functional causality of phenotypic traits.

Functionally explicit partitioning of plant ß-diversity reveal soil fungal assembly in the subarctic tundra.

Metabarcoding technologies for soil fungal DNA pools have enabled to capture the diversity of fungal community and the agreement of their ß-diversity with plant ß-diversity. However, processes underlying the synchrony of the aboveground-belowground biodiversity is still unclear. By using partitioning methods for plant ß-diversity, this study explored the process driving synchrony in tundra ecosystems, in which drastic vegetation shifts are observed with climate warming. Our methods based on Baselga's partitioning enabled the division of plant ß-diversity into two phenomena and three functional components. Correlation of fungal ß-diversity with the components of plant ß-diversity showed that the spatial replacement of fungi was promoted by plant species turnover, in particular, plant species turnover with functional exchange. In addition, spatial variety of graminoid or forbs species, rather than shrubs, enhanced fungal ß-diversity. These results suggest the importance of small-scale factors such as plant-fungal interactions or local environments modified by plants for the fungal community assemblage. The process-based understanding of community dynamics of plants and fungi allows us to predict the ongoing shrub encroachment in the Arctic region, which could weaken the aboveground-belowground synchrony.

Illumina iSeq 100 and MiSeq exhibit similar performance in freshwater fish environmental DNA metabarcoding.

Environmental DNA (eDNA) analysis is a method of detecting DNA from environmental samples and is used as a biomonitoring tool. In recent studies, Illumina MiSeq has been the most extensively used tool for eDNA metabarcoding. The Illumina iSeq 100 (hereafter, iSeq), one of the high-throughput sequencers (HTS), has a relatively simple workflow and is potentially more affordable than other HTS. However, its utility in eDNA metabarcoding has still not been investigated. In the present study, we applied fish eDNA metabarcoding to 40 water samples from river and lake ecosystems to assess the difference in species detectability and composition between iSeq and MiSeq. To check differences in sequence quality and errors, we also assessed differences in read changes between the two HTS. There were similar sequence qualities between iSeq and MiSeq. Significant difference was observed in the number of species between two HTS, but no difference was observed in species composition between the two HTS. Additionally, the species compositions in common with the conventional method were the same between the two HTS. According to the results, using the same amplicon library for sequencing, two HTS would exhibit a similar performance of fish species detection using eDNA metabarcoding.

Evaluation of seasonal dynamics of fungal DNA assemblages in a flow-regulated stream in a restored forest using eDNA metabarcoding.

Investigation of seasonal variation in fungal communities is essential for understanding biodiversity and ecosystem functions. However, the conventional sampling method, with substrate removal and high spatial heterogeneity of community composition, makes surveying the seasonality of fungal communities challenging. Recently, water environmental DNA (eDNA) analysis has been explored for its utility in biodiversity surveys. In this study, we assessed whether the seasonality of fungal communities can be detected by monitoring eDNA in a forest stream. We conducted monthly water sampling in a forest stream over 2 years and used DNA metabarcoding to identify fungal eDNA. The stream water contained DNA from functionally diverse aquatic and terrestrial fungi, such as plant decomposers, parasites and mutualists. The variation in the fungal assemblage showed a regular annual periodicity, meaning that the assemblages in a given season were similar, irrespective of the year or sampling. Furthermore, the strength of the annual periodicity varied among functional groups. Our results suggest that forest streams may act as a 'trap' for terrestrial fungal DNA derived from different habitats, allowing the analysis of fungal DNA in stream water to provide information about the temporal variation in fungal communities in both the aquatic and the surrounding terrestrial ecosystems.

Variability of decomposing ability among fungi associated with the bleaching of subtropical leaf litter.

A diverse array of fungi take part in decomposition, but the variability of their decomposing ability is not fully understood. A total of 49 isolates of Ascomycota and Basidiomycota were obtained from bleached areas of leaf litter in a subtropical forest in southern Japan, and their ability to decompose leaves and recalcitrant compounds was quantified in pure culture. Mass loss of leaves of Castanopsis sieboldii (Fagaceae) and Schima wallichii (Theaceae) was evaluated at 20 C for 12 wk in vitro. Fungi caused a loss of -0.4% to 34.3% of the original leaf mass. The greatest mass loss was caused by four isolates of Mycena (Tricholomataceae). Isolates of Lachnocladiaceae caused greater mass loss of recalcitrant compounds, registered as acid-unhydrolyzable residue (AUR), which includes lignin, relative to Xylariaceae. Isolates of Rhytismataceae, Lachnocladiaceae, Marasmiaceae, and Omphalotaceae exhibited selective decomposition of AUR, Mycena exhibited simultaneous decomposition of AUR and components other than AUR, and Xylariaceae exhibited selective decomposition of components other than AUR. Mass losses of leaves and AUR caused by each fungal isolate were similar for C. sieboldii and S. wallichii. The Mycena isolates obtained from C. sieboldii caused greater mass losses of leaves and AUR than those obtained from the other tree species, suggesting a "home-field advantage" of decomposing potentialities, with implications for natural interactions beyond the in vitro system evaluated here.

Mycorrhizal communities of two closely related species, Pyrola subaphylla and P. japonica, with contrasting degrees of mycoheterotrophy in a sympatric habitat.

Mycoheterotrophic plants typically form associations with a narrow range of mycorrhizal fungi. Consequently, mycorrhizal specialization is often considered to be an important step in mycoheterotrophic evolution. However, it remains unclear whether such specialization is likely to occur in plants of the genus Pyrola, which are generally associated with fungi in multiple ectomycorrhizal families. Here, we investigated the mycorrhizal communities of a nearly fully mycoheterotrophic Pyrola species (Pyrola subaphylla), a closely related partially mycoheterotrophic Pyrola species (Pyrola japonica), and a co-occurring autotrophic ectomycorrhizal tree, Quercus crispula, which is their potential carbon source, in a cool-temperate Japanese forest. High-throughput DNA sequencing revealed that numerous common ectomycorrhizal OTUs interact with the two Pyrola species and Q. crispula, thereby providing an opportunity to exploit a certain amount of carbon from common mycorrhizal networks. In addition, not only P. japonica but also P. subaphylla exhibited exceptionally high alpha mycobiont diversity, with 52 ectomycorrhizal OTUs belonging to 12 families being identified as P. subaphylla mycobionts and 69 ectomycorrhizal OTUs in 18 families being detected as P. japonica mycobionts. Nonetheless, the beta mycobiont diversity of P. subaphylla and P. japonica individuals was significantly lower than that of Q. crispula. Moreover, the beta mycobiont diversity of P. subaphylla was found to be significantly lower than that of P. japonica. Therefore, despite their seemingly broad mycorrhizal interactions, the two Pyrola species (particularly P. subaphylla) showed consistent fungal associations, suggesting that mycorrhizal specialization may have developed during the course of mycoheterotrophic evolution within the genus Pyrola.

Diversity and Geographic Distribution of Ligninolytic Fungi Associated With Castanopsis sieboldii Leaf Litter in Japan.

The diversity and geographic pattern of ligninolytic fungi were investigated within the distribution range of an evergreen tree, Castanopsis sieboldii (Fagaceae), in Japan. Fungal isolates obtained from 18 sites in subtropical and temperate regions in Japan were classified into 50 operational taxonomic units in Ascomycota and Basidiomycota according to the base sequence of the rDNA internal transcribed spacer region. Ordination by nonmetric multidimensional scaling showed the separation of fungal compositions between the study sites which was significantly related to the latitude, longitude, and mean annual temperature (MAT) of the study sites. We applied variation partitioning to separate the magnitude of the climatic, spatial, and leaf property factors and found the roles of MAT and spatial factors in structuring fungal assemblages, suggesting the importance of both niche processes and such non-niche processes as priority effect and dispersal limitation. The bleached area on leaf litter was greater at sites with higher MAT and precipitation located at lower latitudes and at sites where some major ligninolytic fungi occurred at greater relative frequencies, indicating that not only the climatic conditions but also the biogeographic patterns of distribution of ligninolytic fungi influence the decomposition of lignin in leaf litter.

Two-years of investigation revealed the inconsistency of seasonal dynamics of an ectomycorrhizal fungal community in Japanese cool-temperate forest across years.

Ectomycorrhizal (ECM) fungal communities show temporal dynamics. Such dynamics have been mainly assessed with 1 year of investigations and have been related to the seasonal changes in environment. Recent study in sub-tropical region has revealed that stochastic temporal-based process can affect ECM fungal community, making the community of the same season different between years. The different community structures across years have also been observed in the Arctic region with a grass host. Nevertheless, in temperate zones, the effect of temporal-based processes and the consistency of seasonal dynamics have never been investigated. We conducted a 2-year root sampling in a cool temperate Fagus crenata forest to test whether the temporal variation of ECM fungal community composition could be explained by season. The explanation powers of temporal distance and environmental factors for the temporal dynamics of ECM fungal community were simultaneously evaluated. The variation in community structure was significantly explained by year but not by season, indicating that seasonal community structure differed between years. This difference in the community structure across years was partly explained by temporal factors. Our study implies that the temporal dynamics of ECM fungal communities in temperate forests are affected by temporal-based factors and can vary across years.

Evaluation of host effects on ectomycorrhizal fungal community compositions in a forested landscape in northern Japan.

Community compositions of ectomycorrhizal (ECM) fungi are similar within the same host taxa. However, careful interpretation is required to determine whether the combination of ECM fungi and plants is explained by the host preference for ECM fungi, or by the influence of neighbouring heterospecific hosts. In the present study, we aimed to evaluate the effects of host species on the ECM community compositions in a forested landscape (approx. 10 km) where monodominant forest stands of six ECM host species belonging to three families were patchily distributed. A total of 180 ECM operational taxonomic units (OTUs) were detected with DNA metabarcoding. Quantitative multivariate analyses revealed that the ECM community compositions were primarily structured by host species and families, regardless of the soil environments and spatial arrangements of the sampling plots. In addition, 38 ECM OTUs were only detected from particular host tree species. Furthermore, the neighbouring plots harboured similar fungal compositions, although the host species were different. The relative effect of the spatial factors on the ECM compositions was weaker than that of host species. Our results suggest that the host preference for ECM fungi is the primary determinant of ECM fungal compositions in the forested landscape.

Complementary molecular methods reveal comprehensive phylogenetic diversity integrating inconspicuous lineages of early-diverged wood-decaying mushrooms.

Among terrestrial microorganisms, mushroom-forming fungi have been relatively well investigated, however the inconspicuous strains may be overlooked by conventional visual investigations causing underestimation of their phylogenetic diversity. Herein, we sought to obtain a comprehensive phylogenetic diversity profile for the early-diverging wood-decaying mushrooms Dacrymycetes, using an approach that combines fruiting-body collection, culture isolation, and environmental DNA (eDNA) metabarcoding of decaying branches. Among the 28 operational taxonomic units (OTUs) detected during a three-year investigation, 10 each were from fruiting bodies and cultured mycelia and 27 were detected as eDNA sequences. eDNA metabarcoding revealed various lineages across the Dacrymycetes phylogeny. Alternatively, fruiting-body and culture surveys uncovered only ~50% of the OTUs detected through eDNA metabarcoding, suggesting that several inconspicuous or difficult-to-isolate strains are latent in the environment. Further, eDNA and culture surveys revealed early-diverging clades that were not identified in the fruiting-body survey. Thus, eDNA and culture-based techniques can uncover inconspicuous yet phylogenetically important mushroom lineages that may otherwise be overlooked via typical visual investigations.

Biogeographic Patterns of Ectomycorrhizal Fungal Communities Associated With Castanopsis sieboldii Across the Japanese Archipelago.

Biogeographic patterns in ectomycorrhizal (ECM) fungal communities and their drivers have been elucidated, including effects of host tree species and abiotic (climatic and edaphic) conditions. At these geographic scales, genotypic diversity and composition of single host tree species change with spatial and environmental gradients, reflecting their historical dispersal events. However, whether the host genotypes can be associated with the biogeographic patterns of ECM communities remains unclear. We investigated the biogeographic pattern of ECM fungal community associated with the single host species Castanopsis sieboldii (Fagaceae), whose genotypic diversity and composition across the Japanese archipelago has already been evaluated. ECM communities were investigated in 12 mature Castanopsis-dominated forests covering almost the entire distribution range of C. sieboldii, and we quantified the effect of host genotypes on the biogeographic pattern of ECM fungal communities. Richness and community composition of ECM fungi changed with latitude and longitude; these biogeographic changes of ECM community were significantly correlated with host genotypic variables. Quantitative analyses showed a higher relative explanatory power of climatic and spatial variables than that of host genotypic variables for the biogeographic patterns in the ECM community. Our results suggest historical events of host dispersal can affect the biogeographic patterns of the ECM fungal community, while their explanation power was lower than that for climatic filtering and/or fungal dispersal.

Environmental DNA metabarcoding for fish community analysis in backwater lakes: A comparison of capture methods.

The use of environmental DNA (eDNA) methods for community analysis has recently been developed. High-throughput parallel DNA sequencing (HTS), called eDNA metabarcoding, has been increasingly used in eDNA studies to examine multiple species. However, eDNA metabarcoding methodology requires validation based on traditional methods in all natural ecosystems before a reliable method can be established. To date, relatively few studies have performed eDNA metabarcoding of fishes in aquatic environments where fish communities were intensively surveyed using multiple traditional methods. Here, we have compared fish communities' data from eDNA metabarcoding with seven conventional multiple capture methods in 31 backwater lakes in Hokkaido, Japan. We found that capture and field surveys of fishes were often interrupted by macrophytes and muddy sediments in the 31 lakes. We sampled 1 L of the surface water and analyzed eDNA using HTS. We also surveyed the fish communities using seven different capture methods, including various types of nets and electrofishing. At some sites, we could not detect any eDNA, presumably because of the polymerase chain reaction (PCR) inhibition. We also detected the marine fish species as sewage-derived eDNA. Comparisons of eDNA metabarcoding and capture methods showed that the detected fish communities were similar between the two methods, with an overlap of 70%. Thus, our study suggests that to detect fish communities in backwater lakes, the performance of eDNA metabarcoding with the use of 1 L surface water sampling is similar to that of capturing methods. Therefore, eDNA metabarcoding can be used for fish community analysis but environmental factors that can cause PCR inhibition, should be considered in eDNA applications.

Temporal distance decay of similarity of ectomycorrhizal fungal community composition in a subtropical evergreen forest in Japan.

Community compositions of ectomycorrhizal (ECM) fungi are known to show spatial distance decay of similarity, which arises from both deterministic niche-based processes and stochastic spatial-based processes (e.g. dispersal limitation). Recent studies have highlighted the importance of incorporating the spatial-based processes in the study of community ecology of ECM fungi. However, few studies have investigated the temporal distance decay of similarity of ECM fungal communities. More specifically, the role of stochastic temporal-based processes, which could drive the temporal distance decay of similarity independently of niche-based processes, in the temporal variation of the communities remains unclear. Here we investigated ECM fungi associated with roots of Castanopsis sieboldii at 3-month intervals over a 2-year period. We found that dissimilarity of the ECM fungal community composition was significantly correlated with temporal distance but not with environmental distance among sampling dates. Both climatic and temporal variables significantly explained the temporal variation of the community composition. These results suggest that temporal variations of ECM fungi can be affected not only by niche-based processes but also by temporal-based processes. Our findings imply that priority effects may play important roles in the temporal turnover of ECM fungal community at the site.

Disentangling the relative importance of host tree community, abiotic environment and spatial factors on ectomycorrhizal fungal assemblages along an elevation gradient.

Recent studies have shown that changes in community compositions of ectomycorrhizal (ECM) fungi along elevation gradients are mainly affected by changes in host tree communities and/or in abiotic environments. However, few studies have taken the effects of processes related to fungal dispersal (i.e. spatial processes) into account and distinguished the effects of host community, abiotic environment and spatial processes on community composition along elevation gradients. This has left unclear the relative importance of these factors in structuring the ECM community assemblages. To address this, we investigated the community composition of ECM fungi along an elevation gradient in northern Japan with 454 meta-barcoding. We found that the community composition of ECM fungi changed along the elevation and that all three factors jointly affected the compositional changes. We separated the magnitude of importance of the three factors in structuring ECM fungal communities and found that most of the spatial variation in ECM fungal community was explained by host communities and abiotic environments. Our results suggest that while biotic and/or abiotic environments can be important factors in determining the ECM fungal community composition along an elevation gradient, spatial processes may also be a primary determinant.

Low multifunctional redundancy of soil fungal diversity at multiple scales.

Theory suggests that biodiversity might help sustain multiple ecosystem functions. To evaluate possible biodiversity-multifunctionality relationships in a natural setting, we considered different spatial scales of diversity metrics for soil fungi in the northern forests of Japan. We found that multifunctionality increased with increasing local species richness, suggesting a limited degree of multifunctional redundancy. This diversity-multifunctionality relationship was independent of the compositional uniqueness of each community. However, we still found the importance of community composition, because there was a positive correlation between community dissimilarity and multifunctional dissimilarity across the landscape. This result suggests that functional redundancy can further decrease when spatial variations in identities of both species and functions are simultaneously considered at larger spatial scales. We speculate that different scales of diversity could provide multiple levels of insurance against the loss of functioning if high-levels of local species diversity and compositional variation across locations are both maintained. Alternatively, making species assemblages depauperate may result in the loss of multifunctionality.