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.

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.

The Limited Establishment of Native Ectomycorrhizal Fungi in Exotic Eucalyptus spp. Stands in Japan.

Host specificity may potentially limit the distribution expansion of ectomycorrhizal (ECM) fungi into areas where their original host plants are absent. To test this hypothesis, we investigated whether populations of native ECM fungi may establish in stands of exotic host trees, namely those of the Eucalyptus species, in Japan. ECM fungal communities associated with eucalyptus and surrounding native host species (Pinus thunbergii and Fagaceae spp.) were investigated at two sites; one site in which eucalyptus and native trees were growing in isolation, and a second site in which these species were mixed. To identify fungal taxa, the nuclear ribosomal internal transcribed spacer region 1 was sequenced for the ECM fungi from the root tips and clustered into operational taxonomic units (OTUs). To confirm whether the retrieved OTUs were native to Japan, they were queried against the entire database of the National Center for Biotechnology Information, UNITE, and GlobalFungi, whereby sampling locations and associated hosts were obtained from sequences with ≥97% similarity. Eucalyptus trees were associated with seven and 12 ECM fungal OTUs, including putatively exotic OTUs in isolated and mixed sites, respectively. Among the 36 and 63 native ECM fungal OTUs detected from native hosts at isolated and mixed sites, only one OTU was shared with eucalyptus at the respective sites. This means that most native ECM fungi in Japan may be incapable of forming an association with exotic Eucalyptus spp. Notably, even ECM fungi associated with both Pinus and Quercus were not detected from eucalyptus, suggesting that host-fungus incompatibility is determined not only by host phylogenetic relatedness but also by host biogeographic affinities. Our findings show that the incompatibility with eucalyptus as well as dispersal limitation may prevent the distribution expansion of native ECM fungi in Japan into the distribution ranges of Eucalyptus spp., where the original hosts are absent.

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.

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.

Towards the conservation of ectomycorrhizal fungi on endangered trees: native fungal species on Pinus amamiana are rarely conserved in trees planted ex situ.

Ectomycorrhizal (ECM) symbiosis is essential for the survival of both host trees and associated ECM fungi. However, during conservation activities of endangered tree species, their ECM symbionts are largely ignored. Here, we investigated ECM fungi in ex situ populations established for the conservation of Pinus amamiana, an endangered species distributed on Yakushima Island, Japan. Our objective was to determine whether ECM fungi in natural forests are conserved in ex situ populations on the same island. In particular, we focused on the existence of Rhizopogon yakushimensis, which is specific to P. amamiana and the most dominant in natural P. amamiana forests. Molecular identification of ECM fungi in resident tree roots and soil propagule banks indicated that ECM fungal species native to natural forests were rarely conserved in ex situ populations. Furthermore, R. yakushimensis was not confirmed in any of the resident root or spore bioassay samples from the ex situ populations. Thus, ECM fungal spores may not be effectively dispersed from natural forests located on the same island. Instead, ECM fungi distributed in other geographical regions occurred more frequently in the ex situ populations, indicating unintentional introductions of non-native ECM fungi from the nurseries where seedlings were raised before transplanting. These findings imply that the current ex situ conservation practices of endangered tree do not work for the conservation of native ECM fungi, and instead may need modification to avoid the risk of introducing non-native ECM fungi near the endangered forest sites.