Coprophilous fungi, as dung-dwelling species are known, occupy one of the most competitive and ephemeral niches in biology. A pile of herbivore dung is a nutrient-rich island that appears suddenly, teems with bacterial and fungal rivals, invertebrate grazers, and protists, and then dries out or is dispersed. Before a fungus can even begin growing, its spores must survive the harsh passage through a mammalian digestive tract. Ecologists have long hypothesized that this brutal combination of competition and disturbance favors the evolution of distinctive secondary metabolites, the small molecules fungi use as chemical weapons and signals. Yet standard isolation techniques tend to recover fast-growing, ubiquitous species that quickly overgrow petri dishes, leaving the more unusual dung specialists largely unstudied.

The new study began with a deliberately patient approach. Dung samples from cows and goats were collected in late 2021 and placed in moist chambers on filter paper at room temperature. For two months, the researchers examined the samples every few days under a stereomicroscope, waiting for the ornate, necked fruiting bodies characteristic of the order Sordariales to mature. Spores were then picked individually with a sterile needle and transferred to agar plates dosed with penicillin, streptomycin, and ivermectin to suppress bacteria, mites, and nematodes. From one such isolate, strain CCF 6931, the team grew cultures on four different media and profiled growth across temperatures ranging from 5 to 40 degrees Celsius, finding an optimum at 25 degrees and a maximum of just 28 degrees.

Confirming that the isolate was genuinely new required molecular evidence. The researchers sequenced four genetic loci: the internal transcribed spacer region, the large subunit of the nuclear ribosomal RNA gene cluster, and fragments of the RNA polymerase II subunit rpb2 and beta-tubulin genes. The combined alignment spanned 3,101 base pairs and included type and reference strains from across the family Schizotheciaceae. Both maximum likelihood and Bayesian phylogenetic analyses placed the Kenyan strain firmly within Schizothecium, in a clade with 100 percent bootstrap support and a posterior probability of 0.98, but far enough from every accepted species to justify formal description. The closest relative was S. conicum, one of the most common dung fungi known, but the new species can be distinguished by its longer agglutinated hairs on the fruiting bodies, reaching up to 160 micrometers versus 120 in S. conicum, and a longer lower ascospore cell.

With the taxonomy settled, the chemistry began. The fungus was fermented for two weeks on a solid brown rice medium across thirty flasks, and the resulting material was extracted with acetone and partitioned into ethyl acetate, yielding 2.23 grams of crude extract. Flash chromatography and successive rounds of preparative high-performance liquid chromatography then teased apart the mixture, ultimately delivering seven pure compounds in milligram quantities. The most intriguing was a white amorphous solid the team named pyrrospirone Z, whose molecular formula of C32H41NO6 was established by high-resolution mass spectrometry from a protonated ion at m/z 536.2987.

Structural elucidation of pyrrospirone Z relied on a battery of nuclear magnetic resonance experiments recorded in acetone-d6 at 500, 600, and 700 megahertz, including HSQC, COSY, HMBC, and NOESY spectra. The proton spectrum revealed two methyl doublets, three methyl singlets, and four aromatic protons consistent with a para-substituted benzene ring, hallmarks of the decahydrofluorene class of alkaloids. Key long-range correlations linked the aromatic ring to a gamma-lactam unit and anchored it to the tricyclic decahydrofluorene core, while a distinctive proton signal at 5.71 parts per million pointed to an oxepan-4-one, a seven-membered cyclic ether ketone. A hemiacetal carbon resonating far downfield at 76.3 parts per million, close to that of the recently reported pyrrospirone S, confirmed the seven-membered ring. NOESY correlations then pinned down the relative stereochemistry, showing that protons H-13, H-7, H-9, H-17, and methyl group 32 all face the same direction, while H-8 and methyl 30 point the opposite way.

Decahydrofluorene alkaloids are a structurally elaborate family of fungal metabolites built around a tricyclic core, a large ether ring, a para-phenyl ether, and either a gamma-lactam or succinimide group. Until now they had been divided into four groups, the GKK1032s, pyrrocidines, hirsutellones, and pyrrospirones, and had only ever been reported from marine-derived and endophytic fungal strains. Finding them in a coprophilous species dramatically extends both the taxonomic and ecological range of the class. The other compounds recovered from S. keniense included pyrrospirone F, pyrrospirone M, GKK1032A2, daldinol, orthosporin, and a trihydroxynaphthalenone, several of which have also been documented in related ascomycetes, hinting at conserved or convergent biosynthetic machinery across these lineages.

The biological screening produced the study’s most striking numbers. Against the Gram-positive bacterium Bacillus subtilis, GKK1032A2 showed a minimum inhibitory concentration of 4.2 micrograms per milliliter and pyrrospirone F came in at 8.3, both outperforming the oxytetracycline control at 16.6 micrograms per milliliter. Both compounds also inhibited Staphylococcus aureus at 8.3 micrograms per milliliter and showed mild activity against the filamentous fungus Mucor hiemalis at 33.3 micrograms per milliliter. In cytotoxicity assays across five human cancer cell lines, including prostate, ovarian, breast, epidermoid, and lung carcinomas, GKK1032A2 was the standout, with IC50 values ranging from 2.0 to 8.1 micromolar. Pyrrospirone F and pyrrospirone M showed moderate activity, while the new pyrrospirone Z was only weakly cytotoxic, a difference the authors attribute to structural features of the decahydrofluorene core, since prior work has shown that disrupting the carbon bond between positions C-10 and C-1 sharply reduces antibacterial potency in this class.

The researchers caution that detecting these metabolites in laboratory culture does not prove the fungus produces them on actual dung in nature, and in situ metabolomic studies will be needed to confirm that. Even so, the implications are considerable. A genus never before chemically examined has turned out to harbor a molecule new to science and a suite of alkaloids with clinically relevant activity, all from a substrate most people would rather not touch. The finding reinforces the argument that dung-inhabiting fungi, and biodiversity hotspots like Kenya more broadly, deserve a far larger share of attention in the global search for new antibiotics and anticancer leads, at a moment when resistance to existing drugs continues to climb.

Subject of Research: Discovery of the new coprophilous fungus Schizothecium keniense and its bioactive decahydrofluorene alkaloids

Article Title: Pyrrospirone Z and related decahydrofluorene alkaloids produced by the new coprophilous species Schizothecium keniense ( Schizotheciaceae , Sordariales )

Article References: Agudelo-Restrepo, M., Dibouloul, P., Harms, K., Tchamgoue, J., Stadler, M., Matasyoh, J., Kouam, S. F., & Marin-Felix, Y. (2026). Pyrrospirone Z and related decahydrofluorene alkaloids produced by the new coprophilous species Schizothecium keniense (Schizotheciaceae, Sordariales). MycoKeys, 136, 139-159. https://doi.org/10.3897/mycokeys.136.191424

Image Credits: AI Generated

DOI: 10.3897/mycokeys.136.191424

Keywords: Schizothecium keniense, coprophilous fungi, pyrrospirone Z, decahydrofluorene alkaloids, natural products, antibacterial activity, cytotoxicity, Sordariales, Kenya, secondary metabolites, drug discovery, mycology