Partner specificity is a well-known phenomenon in biotic interactions, but little is known about biotic and abiotic factors that determine specificity in plant-fungal associations. Using PacBio sequencing of soils from monospecific and mixed forest stands, we determined the predictors driving partner specificity in both ectomycorrhizal plants and fungi. Fungal guilds differed strongly in the patterns of partner preference and avoidance, and specificity to particular tree genera. Specialist ectomycorrhizal fungi dominated in belowground communities, and most species preferred one of their partner trees - mostly at the plant genus level. Furthermore, all tree genera (sometimes species) displayed preference towards certain fungal groups. Partner specificity was unrelated to rarity of fungi or plants or environmental conditions except soil pH. Depending on partner taxon, specificity in fungi tended to increase with dominance and optimal pH of the partner tree genus and stand age. Partner tree richness and increased evenness of ectomycorrhizal fungi in multi-host communities promotes species richness. However, mainly partner-generalist fungi contribute to the high diversity in mixed forests. Our results further suggest that reforestation with mixed tree species promotes soil biodiversity, and that besides conserving mixed forests, protection of old pure stands may be particularly important for conserving partner-specific ectomycorrhizal fungi.

Fungi are a key component of tropical biodiversity. Due to their inconspicuous and largely subterranean nature, they are however usually neglected in biodiversity inventories. The goal of this study was to identify the key determinants of fungal richness, community composition, and turnover in tropical rainforests. We tested specifically for the effect of soil properties, habitat, and locality in Amazonia. For these analyses, we used high-throughput sequencing data of short and long reads of fungal DNA present in soil and organic litter samples, combining existing and novel genomic data. Habitat type (phytophysiognomies) emerges as the strongest factor in explaining fungal community composition. Naturally open areas – campinas – are the richest habitat overall. Soil properties have different effects depending on the soil layer (litter or mineral soil) and the choice of genetic marker. We suggest that campinas could be a neglected hotspot of fungal diversity. An underlying cause for their rich diversity may be the overall low soil fertility, which increases the reliance on biotic interactions essential for nutrient absorption in these environments, notably ectomycorrhizal fungi–plant associations. Our results highlight the advantages of using both short and long DNA reads produced through high-throughput sequencing to characterize fungal diversity. While short-reads can suffice for diversity and community comparison, long-reads add taxonomic precision and have the potential to reveal population diversity.

The development of high-throughput sequencing (HTS) technologies has greatly improved our capacity to identify fungi and unveil their ecological roles across a variety of ecosystems. Here we provide an overview about current best practices in metabarcoding analysis of fungal communities, from experimental design through molecular and computational analyses. By re-analysing published datasets, we find that operational taxonomic units (OTUs) outperform amplified sequence variants (ASVs) in recovering fungal diversity, which is particularly evident for long markers. Additionally, analysis of the full-length ITS region allows more accurate taxonomic placement of fungi and other eukaryotes compared with the ITS2 subregion. We conclude that metabarcoding analyses of fungi are especially promising for co-analyses with the functional metagenomic or transcriptomic data, integrating fungi in the entire microbiome, recovery of novel fungal lineages and ancient organisms as well as barcoding of old specimens including type material.

Diatoms (Bacillariophyceae) are widely used as bioindicators of present and past water quality because they inhabit the vast majority of aquatic ecosystems, are very diverse, highly sensitive to a variety of environmental conditions, and are characterized by silicified cell walls that favor their long-term preservation in sediments. Alongside with traditional morphological analyses, metabarcoding has become a valuable tool to study the community structures of various organisms, including diatoms. Here, we aimed to test whether the quantity of sediment sample used for DNA extraction is affecting the results obtained from high-throughput sequencing (metabarcoding) of the diatom rbcL region by isolating DNA from 10 g and 0.5 g (wet weight) of lake surface sediment samples. Because bioinformatics processing of metabarcoding data may affect the outcome, we also tested the consistency of the results from three different pipelines. Additionally, the agreement between metabarcoding data and morphological inventories of corresponding samples were compared. Our results demonstrate highly uniform patterns between the diatom rbcL amplicons from 10 g and 0.5 g of DNA extracts (HTS 10 and HTS 0.5, respectively). Furthermore, metabarcoding results were highly consistent among the data sets produced by different bioinformatics pipelines. Comparing results from metabarcoding and microscopy, we identified some taxonomic mismatches, which are related to the common issue of incompleteness of the sequence databases, but also to inconsistencies in diatom taxonomy in general and potential dissolution effects of diatom valves caused by high alkalinity of the investigated lake waters. Nevertheless, multivariate community analysis demonstrated highly similar results between data sets identified by microscopy and metabarcoding, further confirming that metabarcoding is a viable alternative for identifying diatom-environment relationships.

In recent decades, multipartite mutualisms involving microorganisms such as fungi have been discovered in associations traditionally thought of as bipartite. Ant-plant mutualisms were long thought to be bipartite despite fungi being noticed in an epiphytic ant-plant over 100 years ago. We sequenced fungal DNA from the three distinct domatium chambers of the epiphytic ant-plant Myrmecodia beccarii Hook.f. to establish if fungal communities differ by chamber type across five locations spanning 675 km. The three chamber types serve different ant-associated functions including: ‘waste’ chambers, where ant workers deposit waste; ‘nursery’ chambers, where the brood are kept; and ‘ventilation’ chambers, that allow air into the domatium. Overall, fungi from the order Chaetothyriales dominated the chambers in terms of the proportion of OTUs (13.4%) and sequence abundances of OTUs (28% of the total), however a large portion of OTUs (28%) were unidentified at the order level. Notably, the fungal community in the waste chambers differed consistently from the nursery and ventilation chambers across all five locations. We identified 13 fungal OTUs as ‘common’ in the waste chambers that were rare or in very low sequence abundance in the other two chambers. Fungal communities in the nursery and ventilation chambers were also significantly different, but variation between these chambers was less pronounced. Differences in dominance of the common OTUs drive the observed patterns in the fungal communities for each of the chamber types. This suggests a multipartite mutualism involving fungi exists in this ant-plant and that the role of fungi differs among chamber types.