The discovery, published in the journal IMA Fungus, addresses one of the most persistent frustrations in modern mycology. Estimates of global fungal diversity run into the millions of species, yet only around 150,000 have been formally described. The vast majority of what ecologists detect in soil, air, and water samples is environmental DNA, or eDNA — genetic material amplified from mixed samples without ever isolating the organism itself. A large share of these sequences cannot be matched to any known reference, and some of the same unidentified sequences, dubbed ‘dark taxa’, recur in study after study from across the globe. Some have even earned a place on an informal list of the ’50 most wanted fungi’. Without names and classification, these organisms remain ecological ghosts: their roles in nutrient cycling, plant health, and soil processes cannot be assessed.

The trail to Semicentenialea rex began, somewhat indirectly, with a different hunt. At the Ivantjärnsheden Field Station in central Sweden, researchers had been searching for Archaeorhizomycetes, another famous dark taxon once known as ‘Soil Clone Group 1’ that was finally described in 2011. In that effort, thousands of surface-sterilized pine root tips were plated onto nutrient media, yielding 160 fungal cultures. Barcode sequencing of the internal transcribed spacer (ITS) or large subunit (LSU) ribosomal regions showed that none of the cultures belonged to the target class. Years later, however, a search against the UNITE sequence database revealed that several of the unidentified isolates matched Clade GS25, a lineage hypothesized in 2017 to represent a class-level branch within the subphylum Pucciniomycotina.

That placement is significant. The Pucciniomycotina, best known for the rust fungi that devastate crops as obligate plant pathogens, is considered the sister lineage to all other Basidiomycota — the broad group that also includes mushrooms and smuts. The subphylum spans ten previously described classes encompassing endophytes, orchid mycorrhizal partners, animal associates, and mycoparasites. Yet relationships among its major lineages have stubbornly resisted resolution in earlier multi-locus analyses, and at least three novel class-level lineages, including GS25, had been flagged from eDNA alone.

To pin down where the new fungus belongs, the team turned to phylogenomics — comparing not one or a few genes but hundreds of conserved genes across whole genomes. They assembled a dataset of 16 genomes representing every described class of Pucciniomycotina except Cryptomycocolacomycetes, for which no cultured representative is known, plus eight outgroup taxa. From these they extracted 287 single-copy orthologous genes using the BUSCO pipeline, aligned and trimmed the sequences, and inferred trees under maximum likelihood with IQ-TREE and under a coalescent model with ASTRAL. The result was unambiguous: the GS25 lineage sits within Pucciniomycotina as the sister group to Classiculomycetes, a small class of aquatic fungi, with maximal statistical support. This contradicted an earlier hypothesis, based on eDNA alone, that GS25 was sister to all other Pucciniomycotina — a reminder of how much phylogenomic data can revise conclusions drawn from short barcode fragments.

The genome of the type isolate, HU4064, was sequenced at the U.S. Department of Energy Joint Genome Institute as part of the 1000 Fungal Genomes Project using long-read PacBio technology. The final assembly spans roughly 54 million base pairs across 405 scaffolds with about 92 percent gene completeness. Notably, the assembly pipeline had separated out a second fungal genome — a contaminant from the Helotiales — which the authors traced to handling of the DNA extract rather than an impure culture, verified by re-sequencing and microscopy. The study also delivered the first phylogenomic resolution of the Pucciniomycotina backbone, confirming several class-level groupings with strong support while showing that many deeper nodes remain uncertain, a sign that substantial undescribed diversity still lurks within the subphylum.

Under the microscope, Semicentenialea rex proved to be a dimorphic fungus, growing both as hyphae and as budding yeast-like cells. Its hyphae are hyaline, septate, and primarily binucleate, with clamp connections at some septa — a hallmark of basidiomycetes. Colonies creep along the agar at just 0.3 to 0.4 millimeters per day, forming pale ochre, leathery growth after eight weeks. Confocal laser scanning microscopy, with nuclei stained by propidium iodide and cell walls by Calcofluor White, revealed three types of swellings that often co-occur: obovoid basidium-like cells subtended by clamp connections, thick-walled probasidium- or teliospore-like cells that can detach and germinate into knobby promycelium-like hyphae, and intercalary inflated cells resembling chlamydospores. Spore-like cells with two or three nuclei form inside the thick-walled swellings and are released as their walls degrade. The precise function of these structures, and where in the life cycle karyogamy — the fusion of nuclei — occurs, remain open questions the authors hope future time-lapse and electron microscopy will answer.

Ecologically, the species shows a clear affinity for mineral soil. At the Swedish site, its sequences appeared in 22 of 24 mineral soil samples from the E and B horizons of podzol profiles, but only trace amounts in organic layers. It was isolated from pine roots in Sweden and, independently, from ectomycorrhizal roots of Scots pine at two field sites in central Finland, with cultures now deposited in collections including CBS, DSMZ, and HAMBI. Yet environmental records suggest pine is not the whole story: the dominant species hypothesis in UNITE, SH1268087.09FU, encompasses more than 2,600 ITS sequences from every continent except Africa and Antarctica, mostly from soils but also from larch and oak leaves and tanoak root tips, hinting that the fungus may live as a general plant endophyte.

Beyond naming a new class, order, family, genus, and species — all in honor of the golden jubilee of Sweden’s King Carl XVI Gustaf, a longtime advocate of boreal forest conservation — the study delivers a pointed methodological message. The exact ribosomal sequences recovered from the cultures were not identical to any of the amplicon sequence variants (ASVs) from eDNA at the same site; similarities across the ITS2 region ranged from 97.4 to 98.7 percent, likely reflecting PCR and sequencing-technology artifacts rather than biology. Meanwhile, the 2,497 GS25 sequences in public databases clustered into 19 species hypotheses at a 1.5 percent dissimilarity threshold, but phylogenetic analysis showed these collapse into at least three true species. The authors argue that percent-similarity cut-offs are poor proxies for species and advocate using ASVs combined with phylogenetic analysis and diagnostic sequence motifs — such as a 21-base diagnostic region that cleanly separates S. rex from its unnamed relatives.

Perhaps the most sobering technical lesson came from the genome itself. Recovering a complete ribosomal operon from the assembly proved assembler-dependent: the standard pipeline captured only a partial ITS, even though raw reads clearly contained the full region, and different assemblers yielded varying copy numbers and similarity levels. That echoes a recent survey of over 34,000 eukaryote genomes in which full-length operons could be retrieved from only 34 percent of assemblies. Because the ribosomal barcode is the bridge linking a genome to the vast metadata of eDNA studies, this fragility matters. The authors close with a call to action: pair eDNA surveys with explorative culturing, barcode the unidentified isolates already sitting in culture collections, and include phylogenetically informative genes like 18S and 28S in metabarcoding studies. Many dark taxa, they suggest, may already be growing quietly in laboratories around the world, waiting for someone to look.

Subject of Research: Description of a new fungal class, Semicentenialomycetes, and its first cultured species from pine roots

Article Title: Another dark taxon comes to light: Semicentenialomycetes , a new class within the Pucciniomycotina ( Basidiomycota ) , and its first described representative, Semicentenialea rex

Article References: Nogerius, V. T., Sánchez-García, M., Kluting, K., Heinonsalo, J., Ryberg, M., Toome, M., Haridas, S., Mondo, S., LaButti, K., Nolan, M., Lipzen, A., Koriabine, M., Bauer, D., Barry, K., Grigoriev, I. V., Aime, M. C., & Rosling, A. (2026). Another dark taxon comes to light: Semicentenialomycetes, a new class within the Pucciniomycotina (Basidiomycota), and its first described representative, Semicentenialea rex. IMA Fungus, 17, Article e189848. https://doi.org/10.3897/imafungus.17.189848

Image Credits: AI Generated

DOI: 10.3897/imafungus.17.189848

Keywords: mycology, dark taxa, environmental DNA, Pucciniomycotina, Semicentenialomycetes, phylogenomics, Basidiomycota, soil fungi, taxonomy, Scots pine, UNITE, ribosomal ITS