Long amplicon metabarcoding has opened the door for phylogenetic analysis of the largely unknown communities of microeukaryotes in soil. Here, we amplified and sequenced the ITS and LSU regions of the rDNA operon (around 1500 bp) from grassland soils using PacBio SMRT sequencing. We tested how three different methods for generation of operational taxonomic units (OTUs) effected estimated richness and identified taxa, and how well large‐scale ecological patterns associated with shifting environmental conditions were recovered in data from the three methods. The field site at Kungsängen Nature Reserve has drawn frequent visitors since Linnaeus's time, and its species rich vegetation includes the largest population of Fritillaria meleagris in Sweden. To test the effect of different OTU generation methods, we sampled soils across an abrupt moisture transition that divides the meadow community into a Carex acuta dominated plant community with low species richness in the wetter part, which is visually distinct from the mesic‐dry part that has a species rich grass‐dominated plant community including a high frequency of F. meleagris. We used the moisture and plant community transition as a framework to investigate how detected belowground microeukaryotic community composition was influenced by OTU generation methods. Soil communities in both moisture regimes were dominated by protists, a large fraction of which were taxonomically assigned to Ciliophora (Alveolata) while 30%–40% of all reads were assigned to kingdom Fungi. Ecological patterns were consistently recovered irrespective of OTU generation method used. However, different methods strongly affect richness estimates and the taxonomic and phylogenetic resolution of the characterized community with implications for how well members of the microeukaryotic communities can be recognized in the data.

Only a small fraction of the global fungal diversity is described, and a large number of fungal sequences from environmental DNA (eDNA) lack relevant reference sequences for taxonomic identification in metabarcoding studies. Among these are several deeply divergent fungal lineages, hypothesized to be class- and order-level lineages, currently known only by eDNA sequences. Here, we formally describe the first representative of one such lineage, previously referred to as Clade GS25, now captured in culture. We use a phylogenomic approach to test the phylogenetic placement and taxonomic rank hypothesized for this lineage and present the formally described Semicentenialea rex sp. nov . as the first known species in the novel class Semicentenialomycetes ( Pucciniomycotina , Basidiomycota ). Semicentenialea rex sp. nov . was isolated from pine roots taken from a podzol soil profile in a pine forest in central Sweden and shows an affinity to mineral soil horizons in this habitat. The same species was isolated independently from pine roots collected in central Finland. Additionally, we provide the first phylogenomic resolution of Pucciniomycotina , using reference genomes from all described classes with the exception of Cryptomycocolacomycetes . In addition to expanding our knowledge about the diversity of Pucciniomycotina , our study highlights the importance of continued culturing efforts in combination with eDNA studies to uncover hidden fungal diversity. Furthermore, our data provide insights into the strengths and limitations associated with amplicon sequence variants, different similarity cut-offs, and genome-derived ribosomal operons.