Soil Micro-eukaryotic Diversity Patterns Along Elevation Gradient Are Best Estimated by Increasing the Number of Elevation Steps Rather than Within Elevation Band Replication.

The development of high-throughput sequencing (HTS) of environmental DNA (eDNA) has stimulated the study of soil microbial diversity patterns and drivers at all scales. However, given the heterogeneity of soils, a challenge is to define effective and efficient sampling protocols that allow sound comparison with other records, especially vegetation. In studies of elevational diversity pattern, a trade-off is choosing between replication within elevation bands vs. sampling more elevation bands. We addressed this question for soil protists along an elevation gradient on Mt. Asahi, Hokkaido, Japan. We compared two sampling approaches: (1) the replicate strategy (five replicates at six elevational bands, total = 30) and (2) the transect strategy (one sample in each of 16 different elevational bands). Despite a nearly twofold lower sampling effort, the transect strategy yielded congruent results compared to the replicate strategy for the estimation of elevational alpha diversity pattern: the regression coefficients between diversity indices and elevation did not differ between the two options. Furthermore, for a given total number of samples, gamma diversity estimated across the entire transect was higher when sampling more elevational bands as compared to replication from fewer elevational bands. Beta diversity (community composition turnover) was lower within a given elevational band than between adjacent bands and increased with elevation distance. In redundancy analyses, soil organic matter-related variable (the first principal component of soil organic matter, water content, total organic carbon, and nitrogen by whom were highly correlated) and elevation best explained elevational beta diversity pattern for both sampling approaches. Taken together, our results suggest that sampling a single plot per elevation band will be sufficient to obtain a good estimate of soil micro-eukaryotic diversity patterns along elevation gradients. This study demonstrated the effectiveness of the transect strategy in estimating diversity patterns along elevation gradients which is instructive for future environmental or even experimental studies. While not advocating for completely replacing replication-based sampling practices, it is important to note that both replicate and transect strategies have their merits and can be employed based on specific research goals and resource limitations.

Supervised machine learning is superior to indicator value inference in monitoring the environmental impacts of salmon aquaculture using eDNA metabarcodes.

Increasing anthropogenic impact and global change effects on natural ecosystems has prompted the development of less expensive and more efficient bioassessments methodologies. One promising approach is the integration of DNA metabarcoding in environmental monitoring. A critical step in this process is the inference of ecological quality (EQ) status from identified molecular bioindicator signatures that mirror environmental classification based on standard macroinvertebrate surveys. The most promising approaches to infer EQ from biotic indices (BI) are supervised machine learning (SML) and the calculation of indicator values (IndVal). In this study we compared the performance of both approaches using DNA metabarcodes of bacteria and ciliates as bioindicators obtained from 152 samples collected from seven Norwegian salmon farms. Results from standard macroinvertebrate-monitoring of the same samples were used as reference to compare the accuracy of both approaches. First, SML outperformed the IndVal approach to infer EQ from eDNA metabarcodes. The Random Forest (RF) algorithm appeared to be less sensitive to noisy data (a typical feature of massive environmental sequence data sets) and uneven data coverage across EQ classes (a typical feature of environmental compliance monitoring scheme) compared to a widely used method to infer IndVals for the calculation of a BI. Second, bacteria allowed for a more accurate EQ assessment than ciliate eDNA metabarcodes. For the implementation of DNA metabarcoding into routine monitoring programmes to assess EQ around salmon aquaculture cages, we therefore recommend bacterial DNA metabarcodes in combination with SML to classify EQ categories based on molecular signatures.

Improving eDNA-based protist diversity assessments using networks of amplicon sequence variants.

Effective and precise grouping of highly similar sequences remains a major bottleneck in the evaluation of high-throughput sequencing datasets. Amplicon sequence variants (ASVs) offer a promising alternative that may supersede the widely used operational taxonomic units (OTUs) in environmental sequencing studies. We compared the performance of a recently developed pipeline based on the algorithm DADA2 for obtaining ASVs against a pipeline based on the algorithm SWARM for obtaining OTUs. Illumina-sequencing of 29 individual ciliate species resulted in up to 11 ASVs per species, while SWARM produced up to 19 OTUs per species. To improve the congruency between species diversity and molecular diversity, we applied sequence similarity networks (SSNs) for second-level sequence grouping into network sequence clusters (NSCs). At 100% sequence similarity in SWARM-SSNs, NSC numbers decreased from 7.9-fold overestimation without abundance filter, to 4.5-fold overestimation when an abundance filter was applied. For the DADA2-SSN approach, NSC numbers decreased from 3.5-fold to 3-fold overestimation. Rand index cluster analyses predicted best binning results between 97% and 94% sequence similarity for both DADA2-SSNs and SWARM-SSNs. Depending on the ecological questions addressed in an environmental sequencing study with protists we recommend ASVs as replacement for OTUs, best in combination with SSNs.

Correction to: A comprehensive fungi-specific 18S rRNA gene sequence primer toolkit suited for diverse research issues and sequencing platforms.

Following publication of the original article [1], we have been notified that three of the primer names identified as most promising candidates for fungal community surveys were incorrectly renamed following the primer nomenclature system proposed by Gargas & DePriest [2].

Protist Biodiversity and Biogeography in Lakes From Four Brazilian River-Floodplain Systems.

The biodiversity and biogeography of protists inhabiting many ecosystems have been intensely studied using different sequencing approaches, but tropical ecosystems are relatively under-studied. Here, we sampled planktonic waters from 32 lakes associated with four different river-floodplains systems in Brazil, and sequenced the DNA using a metabarcoding approach with general eukaryotic primers. The lakes were dominated by the largely free-living Discoba (mostly the Euglenida), Ciliophora, and Ochrophyta. There was low community similarity between lakes even within the same river-floodplain. The protists inhabiting these floodplain systems comprise part of the large and relatively undiscovered diversity in the tropics.

Increasing N deposition impacts neither diversity nor functions of deadwood-inhabiting fungal communities, but adaptation and functional redundancy ensure ecosystem function.

Nitrogen deposition can strongly affect biodiversity, but its specific effects on terrestrial microbial communities and their roles for ecosystem functions and processes are still unclear. Here, we investigated the impacts of N deposition on wood-inhabiting fungi (WIF) and their related ecological functions and processes in a highly N-limited deadwood habitat. Based on high-throughput sequencing, enzymatic activity assay and measurements of wood decomposition rates, we show that N addition has no significant effect on the overall WIF community composition or on related ecosystem functions and processes in this habitat. Nevertheless, we detected several switches in presence/absence (gain/loss) of wood-inhabiting fungal OTUs due to the effect of N addition. The responses of WIF differed from previous studies carried out with fungi living in soil and leaf-litter, which represent less N-limited fungal habitats. Our results suggest that adaptation at different levels of organization and functional redundancy may explain this buffered response and the resistant microbial-mediated ecosystem function and processes against N deposition in highly N-limited habitats.

A comprehensive fungi-specific 18S rRNA gene sequence primer toolkit suited for diverse research issues and sequencing platforms.

BACKGROUND: Several fungi-specific primers target the 18S rRNA gene sequence, one of the prominent markers for fungal classification. The design of most primers goes back to the last decades. Since then, the number of sequences in public databases increased leading to the discovery of new fungal groups and changes in fungal taxonomy. However, no reevaluation of primers was carried out and relevant information on most primers is missing. With this study, we aimed to develop an 18S rRNA gene sequence primer toolkit allowing an easy selection of the best primer pair appropriate for different sequencing platforms, research aims (biodiversity assessment versus isolate classification) and target groups. RESULTS: We performed an intensive literature research, reshuffled existing primers into new pairs, designed new Illumina-primers, and annealing blocking oligonucleotides. A final number of 439 primer pairs were subjected to in silico PCRs. Best primer pairs were selected and experimentally tested. The most promising primer pair with a small amplicon size, nu-SSU-1333-5'/nu-SSU-1647-3' (FF390/FR-1), was successful in describing fungal communities by Illumina sequencing. Results were confirmed by a simultaneous metagenomics and eukaryote-specific primer approach. Co-amplification occurred in all sample types but was effectively reduced by blocking oligonucleotides. CONCLUSIONS: The compiled data revealed the presence of an enormous diversity of fungal 18S rRNA gene primer pairs in terms of fungal coverage, phylum spectrum and co-amplification. Therefore, the primer pair has to be carefully selected to fulfill the requirements of the individual research projects. The presented primer toolkit offers comprehensive lists of 164 primers, 439 primer combinations, 4 blocking oligonucleotides, and top primer pairs holding all relevant information including primer's characteristics and performance to facilitate primer pair selection.

Consistent patterns of high alpha and low beta diversity in tropical parasitic and free-living protists.

Tropical animals and plants are known to have high alpha diversity within forests, but low beta diversity between forests. By contrast, it is unknown whether microbes inhabiting the same ecosystems exhibit similar biogeographic patterns. To evaluate the biogeographies of tropical protists, we used metabarcoding data of species sampled in the soils of three lowland Neotropical rainforests. Taxa-area and distance-decay relationships for three of the dominant protist taxa and their subtaxa were estimated at both the OTU and phylogenetic levels, with presence-absence and abundance-based measures. These estimates were compared to null models. High local alpha and low regional beta diversity patterns were consistently found for both the parasitic Apicomplexa and the largely free-living Cercozoa and Ciliophora. Similar to animals and plants, the protists showed spatial structures between forests at the OTU and phylogenetic levels, and only at the phylogenetic level within forests. These results suggest that the biogeographies of macro- and micro-organismal eukaryotes in lowland Neotropical rainforests are partially structured by the same general processes. However, and unlike the animals and plants, the protist OTUs did not exhibit spatial structures within forests, which hinders our ability to estimate the local and regional diversity of protists in tropical forests.

Life in leaf litter: novel insights into community dynamics of bacteria and fungi during litter decomposition.

Microorganisms play a crucial role in the biological decomposition of plant litter in terrestrial ecosystems. Due to the permanently changing litter quality during decomposition, studies of both fungi and bacteria at a fine taxonomic resolution are required during the whole process. Here we investigated microbial community succession in decomposing leaf litter of temperate beech forest using pyrotag sequencing of the bacterial 16S and the fungal internal transcribed spacer (ITS) rRNA genes. Our results reveal that both communities underwent rapid changes. Proteobacteria, Actinobacteria and Bacteroidetes dominated over the entire study period, but their taxonomic composition and abundances changed markedly among sampling dates. The fungal community also changed dynamically as decomposition progressed, with ascomycete fungi being increasingly replaced by basidiomycetes. We found a consistent and highly significant correlation between bacterial richness and fungal richness (R = 0.76, P < 0.001) and community structure (RM antel  = 0.85, P < 0.001), providing evidence of coupled dynamics in the fungal and bacterial communities. A network analysis highlighted nonrandom co-occurrences among bacterial and fungal taxa as well as a shift in the cross-kingdom co-occurrence pattern of their communities from the early to the later stages of decomposition. During this process, macronutrients, micronutrients, C:N ratio and pH were significantly correlated with the fungal and bacterial communities, while bacterial richness positively correlated with three hydrolytic enzymes important for C, N and P acquisition. Overall, we provide evidence that the complex litter decay is the result of a dynamic cross-kingdom functional succession.

Effects of long-term differential fertilization on eukaryotic microbial communities in an arable soil: a multiple barcoding approach.

To understand the fine-scale effects of changes in nutrient availability on eukaryotic soil microorganisms communities, a multiple barcoding approach was used to analyse soil samples from four different treatments in a long-term fertilization experiment. We performed PCR amplification on soil DNA with primer pairs specifically targeting the 18S rRNA genes of all eukaryotes and three protist groups (Cercozoa, Chrysophyceae-Synurophyceae and Kinetoplastida) as well as the ITS gene of fungi and the 23S plastid rRNA gene of photoautotrophic microorganisms. Amplicons were pyrosequenced, and a total of 88,706 quality filtered reads were clustered into 1232 operational taxonomic units (OTU) across the six data sets. Comparisons of the taxonomic coverage achieved based on overlapping assignment of OTUs revealed that half of the eukaryotic taxa identified were missed by the universal eukaryotic barcoding marker. There were only little differences in OTU richness observed between organic- (farmyard manure), mineral- and nonfertilized soils. However, the community compositions appeared to be strongly structured by organic fertilization in all data sets other than that generated using the universal eukaryotic 18S rRNA gene primers, whereas mineral fertilization had only a minor effect. In addition, a co-occurrence based network analysis revealed complex potential interaction patterns between OTUs from different trophic levels, for example between fungivorous flagellates and fungi. Our results demonstrate that changes in pH, moisture and organic nutrients availability caused shifts in the composition of eukaryotic microbial communities at multiple trophic levels.

The symbiotic alga Trebouxia fuels a coherent soil ecosystem on the landscape scale in the Atacama Desert.

Biocrusts represent associations of lichens, green algae, cyanobacteria, fungi and other microorganisms, colonizing soils in varying proportions of principally arid biomes. The so-called grit crust represents a recently discovered type of biocrust situated in the Coastal Range of the Atacama Desert (Chile) made of microorganisms growing on and in granitoid pebbles, resulting in a checkerboard pattern visible to the naked eye on the landscape scale. This specific microbiome fulfills a broad range of ecosystem services, all probably driven by fog and dew-induced photosynthetic activity of mainly micro-lichens. To understand its biodiversity and impact, we applied a polyphasic approach on the phototrophic microbiome of this biocrust, combining isolation and characterization of the lichen photobionts, multi-gene phylogeny of the photobionts and mycobionts based on a direct sequencing and microphotography approach, metabarcoding and determination of chlorophylla+b contents. Metabarcoding showed that yet undescribed lichens within the Caliciaceae dominated the biocrust together with Trebouxia as the most abundant eukaryote in all plots. Together with high mean chlorophylla+b contents exceeding 410 mg m-2, this distinguished the symbiotic algae Trebouxia as the main driver of the grit crust ecosystem. The trebouxioid photobionts could be assigned to the I (T. impressa/gelatinosa) and A (T. arboricola) clades and represented several lineages containing five potential species candidates, which were identified based on the unique phylogenetic position, morphological features, and developmental cycles of the corresponding isolates. These results designate the grit crust as the only known coherent soil layer with significant landscape covering impact of at least 440 km2, predominantly ruled by a single symbiotic algal genus.

Metabarcoding Approaches for Soil Eukaryotes, Protists, and Microfauna.

There have been major developments in the molecular characterization of soil protist and micrometazoan diversity, leading to a better understanding of these minute soil eukaryotes. Like in all newly developing research fields, several approaches are currently used in parallel to study these organisms. Here, we synthesize these various approaches and propose a best practice manual that should help researchers to efficiently target soil eukaryotic diversity as a whole. We cover the whole working pipeline, ranging from sampling to nucleic acids extraction to bioinformatic processing and sequence identification. Synchronous approaches to molecularly survey microbial-sized eukaryotes and other soil biodiversity groups are needed in order to provide a cumulative knowledge of soil biodiversity, as here shown for the soil eukaryome. This will be crucial in understanding the important ecosystem functions provided by soil biodiversity.

Soil filtration-sedimentation improves shelled protist recovery in eukaryotic eDNA surveys.

A large part of the soil protist diversity is missed in metabarcoding studies based on 0.25 g of soil environmental DNA (eDNA) and universal primers due to ca. 80% co-amplification of non-target plants, animals and fungi. To overcome this problem, enrichment of the substrate used for eDNA extraction is an easily implemented option but its effect has not yet been tested. In this study, we evaluated the effect of a 150 µm mesh size filtration and sedimentation method to improve the recovery of protist eDNA, while reducing the co-extraction of plant, animal and fungal eDNA, using a set of contrasted forest and alpine soils from La Réunion, Japan, Spain and Switzerland. Total eukaryotic diversity was estimated by V4 18S rRNA metabarcoding and classical amplicon sequence variant calling. A 2- to 3-fold enrichment in shelled protists (Euglyphida, Arcellinida and Chrysophyceae) was observed at the sample level with the proposed method, with, at the same time, a 2-fold depletion of Fungi and a 3-fold depletion of Embryophyceae. Protist alpha diversity was slightly lower in filtered samples due to reduced coverage in Variosea and Sarcomonadea, but significant differences were observed in only one region. Beta diversity varied mostly between regions and habitats, which explained the same proportion of variance in bulk soil and filtered samples. The increased resolution in soil protist diversity estimates provided by the filtration-sedimentation method is a strong argument in favour of including it in the standard protocol for soil protist eDNA metabarcoding studies.

Changes in Phylogenetic and Functional Diversity of Ciliates along the Course of a Mediterranean Karstic River.

Ciliates are a group of phagotrophic protists found in a wide variety of ecosystems. This study builds on recent studies of ciliates in the Krka river and investigates changes in the phylogenetic and functional diversity of ciliates in biofilm to predict the phylogenetic and functional structure of ciliates in other karstic rivers. Biofilm samples were collected from four representative locations: upstream (Krka spring), midstream (Marasovine), and downstream (Roski slap, Skradinski buk) of the Krka river to test for differences in phylogenetic and functional diversity of ciliates in relation to location and positioning on tufa stones (light/dark-exposed side of tufa stone). Our results showed that Krka spring had higher phylogenetic species variability, lower phylogenetic diversity, and lower functional richness than Skradinski buk, suggesting phylogenetic overdispersal at Krka spring. This could be due to environmental filtering, competitive exclusion, or a combination of these factors. As the first study of its kind in the Mediterranean, our results shed light on the phylogenetic and functional diversity of ciliates in karst ecosystems and provide a basis for future ecological and conservation efforts.

DNA Metabarcoding for the Characterization of Terrestrial Microbiota-Pitfalls and Solutions.

Soil-borne microbes are major ecological players in terrestrial environments since they cycle organic matter, channel nutrients across trophic levels and influence plant growth and health. Therefore, the identification, taxonomic characterization and determination of the ecological role of members of soil microbial communities have become major topics of interest. The development and continuous improvement of high-throughput sequencing platforms have further stimulated the study of complex microbiota in soils and plants. The most frequently used approach to study microbiota composition, diversity and dynamics is polymerase chain reaction (PCR), amplifying specific taxonomically informative gene markers with the subsequent sequencing of the amplicons. This methodological approach is called DNA metabarcoding. Over the last decade, DNA metabarcoding has rapidly emerged as a powerful and cost-effective method for the description of microbiota in environmental samples. However, this approach involves several processing steps, each of which might introduce significant biases that can considerably compromise the reliability of the metabarcoding output. The aim of this review is to provide state-of-the-art background knowledge needed to make appropriate decisions at each step of a DNA metabarcoding workflow, highlighting crucial steps that, if considered, ensures an accurate and standardized characterization of microbiota in environmental studies.

Protist taxonomic and functional diversity in soil, freshwater and marine ecosystems.

Protists dominate eukaryotic diversity and play key functional roles in all ecosystems, particularly by catalyzing carbon and nutrient cycling. To date, however, a comparative analysis of their taxonomic and functional diversity that compares the major ecosystems on Earth (soil, freshwater and marine systems) is missing. Here, we present a comparison of protist diversity based on standardized high throughput 18S rRNA gene sequencing of soil, freshwater and marine environmental DNA. Soil and freshwater protist communities were more similar to each other than to marine protist communities, with virtually no overlap of Operational Taxonomic Units (OTUs) between terrestrial and marine habitats. Soil protists showed higher γ diversity than aquatic samples. Differences in taxonomic composition of the communities led to changes in a functional diversity among ecosystems, as expressed in relative abundance of consumers, phototrophs and parasites. Phototrophs (eukaryotic algae) dominated freshwater systems (49% of the sequences) and consumers soil and marine ecosystems (59% and 48%, respectively). The individual functional groups were composed of ecosystem- specific taxonomic groups. Parasites were equally common in all ecosystems, yet, terrestrial systems hosted more OTUs assigned to parasites of macro-organisms while aquatic systems contained mostly microbial parasitoids. Together, we show biogeographic patterns of protist diversity across major ecosystems on Earth, preparing the way for more focused studies that will help understanding the multiple roles of protists in the biosphere.

Urban areas as hotspots for bees and pollination but not a panacea for all insects.

Urbanisation is an important global driver of biodiversity change, negatively impacting some species groups whilst providing opportunities for others. Yet its impact on ecosystem services is poorly investigated. Here, using a replicated experimental design, we test how Central European cities impact flying insects and the ecosystem service of pollination. City sites have lower insect species richness, particularly of Diptera and Lepidoptera, than neighbouring rural sites. In contrast, Hymenoptera, especially bees, show higher species richness and flower visitation rates in cities, where our experimentally derived measure of pollination is correspondingly higher. As well as revealing facets of biodiversity (e.g. phylogenetic diversity) that correlate well with pollination, we also find that ecotones in insect-friendly green cover surrounding both urban and rural sites boost pollination. Appropriately managed cities could enhance the conservation of Hymenoptera and thereby act as hotspots for pollination services that bees provide to wild flowers and crops grown in urban settings.

Experimental Evidence of Functional Group-Dependent Effects of Tree Diversity on Soil Fungi in Subtropical Forests.

Deconvoluting the relative contributions made by specific biotic and abiotic drivers to soil fungal community compositions facilitates predictions about the functional responses of ecosystems to environmental changes, such as losses of plant diversity, but it is hindered by the complex interactions involved. Experimental assembly of tree species allows separation of the respective effects of plant community composition (biotic components) and soil properties (abiotic components), enabling much greater statistical power than can be achieved in observational studies. We therefore analyzed these contributions by assessing, via pyrotag sequencing of the internal transcribed spacer (ITS2) rDNA region, fungal communities in young subtropical forest plots included in a large experiment on the effects of tree species richness. Spatial variables and soil properties were the main drivers of soil fungal alpha and beta-diversity, implying strong early-stage environmental filtering and dispersal limitation. Tree related variables, such as tree community composition, significantly affected arbuscular mycorrhizal and pathogen fungal community structure, while differences in tree host species and host abundance affected ectomycorrhizal fungal community composition. At this early stage of the experiment, only a limited amount of carbon inputs (rhizodeposits and leaf litter) was being provided to the ecosystem due to the size of the tree saplings, and persisting legacy effects were observed. We thus expect to find increasing tree related effects on fungal community composition as forest development proceeds.

Land-Use Intensity Rather Than Plant Functional Identity Shapes Bacterial and Fungal Rhizosphere Communities.

The rhizosphere encompasses the soil surrounding the surface of plants' fine roots. Accordingly, the microbiome present is influenced by both soil type and plant species. Furthermore, soil microbial communities respond to land-use intensity due to the effects on soil conditions and plant performance. However, there is limited knowledge about the impact of grassland management practices under field conditions on the composition of both bacteria and fungi in the rhizosphere of different plant functional groups. In spring 2014 we planted four phytometer species, two forbs (Plantago lanceolata, Achillea millefolium) and two grasses (Dactylis glomerata, Arrhenatherum elatius) into 13 permanent experimental grassland plots, differing in management. After 6 months, rhizosphere and bulk soil associated with the phytometer plants were sampled, microbial genomic DNA was extracted and bacterial 16S and fungal ITS rDNA were sequenced using Illumina MiSeq. Our study revealed that the rhizosphere microbial community was more diverse than the bulk soil community. There were no differences in microbial community composition between the two plant functional groups, but a clear impact of root traits and edaphic conditions. Land-use intensity strongly affected plant productivity, neighboring plant richness and edaphic conditions, especially soil C/N ratio, which in turn had a strong influence on root traits and thereby explained to large extent microbial community composition. Rhizosphere microbes were mainly affected by abiotic factors, in particular by land-use intensity, while plant functional type had only subordinate effects. Our study provides novel insights into the assembly of rhizosphere bacterial and fungal communities in response to land-use intensity and plant functional groups in managed grassland ecosystems.

Determinants of Deadwood-Inhabiting Fungal Communities in Temperate Forests: Molecular Evidence From a Large Scale Deadwood Decomposition Experiment.

Despite the important role of wood-inhabiting fungi (WIF) in deadwood decomposition, our knowledge of the factors shaping the dynamics of their species richness and community composition is scarce. This is due to limitations regarding the resolution of classical methods used for characterizing WIF communities and to a lack of well-replicated long-term experiments with sufficient numbers of tree species. Here, we used a large scale experiment with logs of 11 tree species at an early stage of decomposition, distributed across three regions of Germany, to identify the factors shaping WIF community composition and Operational Taxonomic Unit (OTU) richness using next generation sequencing. We found that tree species identity was the most significant factor, corresponding to (P < 0.001) and explaining 10% (representing 48% of the explainable variance) of the overall WIF community composition. The next important group of variables were wood-physicochemical properties, of which wood pH was the only factor that consistently corresponded to WIF community composition. For overall WIF richness patterns, we found that approximately 20% of the total variance was explained by wood N content, location, tree species identity and wood density. It is noteworthy that the importance of determinants of WIF community composition and richness appeared to depend greatly on tree species group (broadleaved vs. coniferous) and it differed between the fungal phyla Ascomycota and Basidiomycota.