The genus Purpureocillium was established in 2011 to accommodate Paecilomyces lilacinus, a medically and agriculturally important fungus, based on comparative analyses of ribosomal RNA gene fragments and translation elongation factor sequences. The genus belongs to the family Ophiocordycipitaceae within the order Hypocreales, a lineage famous for its entomopathogenic members, including the so-called zombie-ant fungi of the genus Ophiocordyceps. Purpureocillium species inhabit soil, insects, nematodes, and even marine sediments worldwide, and two species in particular, P. lilacinum and P. lavendulum, are prized as broad-spectrum biological control agents against plant-parasitic nematodes, whiteflies, thrips, aphids, spider mites, and leaf-cutter ants.

Yet species boundaries within the genus have remained incompletely resolved, and several new species have been described since 2024 as researchers begin to sample more intensively. Against this backdrop, a team led by Xiaoyan Liu, Hao Wang, Lijun Luo, Baomin Fan, and Hong Yu set out to reconstruct the phylogeny of Purpureocillium using a multilocus approach, combining five nuclear loci with mitochondrial gene sequences and comparative morphology. Their new species, isolated from an infected ant collected in September 2022 at an altitude of 1,380 meters, proved to be the centerpiece of the effort.

The researchers used a rigorous molecular toolkit. Nuclear phylogenies were built from concatenated sequences of the internal transcribed spacer region, the small and large ribosomal RNA subunits (18S and 28S), the translation elongation factor 1-alpha gene (TEF1-alpha), and the RNA polymerase II largest subunit gene (RPB1). Sequences were aligned with Clustal X and MEGA6, tested for incongruent phylogenetic signals with PAUP*, and analyzed under both maximum likelihood and Bayesian inference frameworks, with substitution models selected by ModelFinder under the Bayesian information criterion. Bootstrap support of 5,000 replicates in the maximum likelihood analysis and posterior probabilities in the Bayesian analysis provided convergent statistical validation of the resulting trees.

The nuclear analyses consistently and strongly placed the Yunnan isolate within the Purpureocillium clade as a distinct lineage. Pairwise comparisons revealed substantial divergence: 65 nucleotide differences in the ITS region and 44 differences in TEF1-alpha between the new species and P. lilacinum, and 69 and 44 to 45 differences, respectively, versus P. lavendulum. Divergence was lowest against P. atypicola, with only 14 differences in TEF1-alpha and 20 in RPB1, yet the combined multilocus dataset still recovered the new isolate as evolutionarily separate. The authors caution that the phylogenetic positions of recently described species such as P. roseum and P. sodanum remain provisional, because sequence data for them are limited to the ITS and 28S regions and show incongruence with other loci.

Morphology reinforced the molecular story. Under the microscope, P. xinfangense produces conidiophores bearing two or three whorls of phialides, the spore-forming cells, which are predominantly short and cylindrical with a pointed, mucronate apex. Its conidia are long and cylindrical, arranged in chains, measuring roughly 2.62 to 4.67 micrometers in length but only 0.65 to 1.59 micrometers wide. This is a striking contrast with its relatives: P. lilacinum produces subglobose to ellipsoidal conidia, P. takamizusanense produces ellipsoidal spores of intermediate length, and P. lavendulum produces subglobose to lemon-shaped conidia with an apiculate base. Colony growth on malt extract agar was also distinctive, reaching only 24 to 26 millimeters after fourteen days, with white fluffy mycelium and a pink-purple sporulation zone.

The mitochondrial genomes added an even more dramatic dimension. The team sequenced and compared the complete mitogenomes of four species: P. lilacinum at 23,498 base pairs, P. lavendulum at 23,475 base pairs, P. takamizusanense at 26,396 base pairs, and P. xinfangense at a towering 121,341 base pairs. All four genomes are closed circles and share the same core gene content: fifteen canonical protein-coding genes, including the ATP synthase subunits atp6, atp8, and atp9, cytochrome c oxidase subunits cox1 through cox3, seven NADH dehydrogenase subunits, the cytochrome b gene cob, and the ribosomal protein gene rps3, along with 25 transfer RNA genes and two ribosomal RNA genes. Among these genes, nad5 is the longest at 2,007 base pairs and atp8 the shortest at 147 base pairs, while tRNA genes range from 71 to 87 base pairs.

Why is the new species’ mitochondrial genome so enormous? The authors cannot yet say definitively, but they offer testable hypotheses grounded in prior work. A 112,465-base-pair mitogenome for P. atypicola is known to harbor numerous introns, and comparative analyses have shown that intron gain and loss are the primary drivers of mitochondrial genome size divergence within Purpureocillium, with the core gene order largely conserved. Similar cases exist across the fungal kingdom: intron invasion expanded the mitogenomes of Rhynchosporium species, and the 135-kilobase mitochondrial genome of the cultivated mushroom Agaricus bisporus is packed with group I and group II introns and plasmid-related sequences. Kingdom-wide surveys likewise link fungal mitogenome size variation to accessory elements such as introns, homing endonuclease genes, and unidentified open reading frames. Whether intron proliferation, repeat accumulation, or extra open reading frames account for the expansion in P. xinfangense awaits detailed repeat annotation and intron mapping.

Nucleotide composition offered a further signature. GC skew was positive in all four genomes, but AT skew differed sharply: it was slightly negative in P. lilacinum, P. lavendulum, and P. takamizusanense, yet positive in P. xinfangense. Such strand-specific compositional asymmetry is thought to reflect asymmetric mutational pressures during mitochondrial replication and transcription, so the compositional fingerprint of the new species differs from its congeners in a measurable way. Mitochondrial phylogenetic analysis of fourteen concatenated protein-coding genes across 52 hypocrealean taxa placed P. xinfangense in a phylogenetically isolated position, sister to all other Purpureocillium species, with high topological stability and strong support for the monophyly of all six sampled families within Hypocreales.

The discovery carries ecological intrigue as well. The type specimen came from a naturally infected ant cadaver associated with an Ophiocordyceps unilateralis species complex, neither of which could be identified to species level. This suggests a possible hyperparasitic association, in which one fungus colonizes another, but the authors emphasize that the exact relationship remains uncertain and will require direct observation, infection assays, and genomic analyses to confirm. Although the new species is currently known from a single collection, the combination of distinctive morphology, a rare ant-associated substrate, and concordant support from both nuclear and mitochondrial datasets justified its formal description. With five new Purpureocillium species described since 2024, including taxa associated with spiders in Brazil and karst landscapes in China, the genus is clearly far more diverse than its two well-studied biocontrol representatives suggest, and the authors call for broader taxonomic sampling to resolve species boundaries and unlock the biocontrol potential of these enigmatic purple-spored fungi.

Subject of Research: Phylogenetics and mitochondrial genomics of the fungal genus Purpureocillium and description of a new species from southwestern China

Article Title: Phylogenetic reconstruction of the genus Purpureocillium based on multilocus nuclear and mitochondrial DNA sequences, with the description of Purpureocillium xinfangense sp. nov. (Ophiocordycipitaceae, Hypocreales) from Southwestern China

Article References: Liu, X., Wang, H., Luo, L., Liu, Z., Yu, Y., Fan, B., & Yu, H. (2026). Phylogenetic reconstruction of the genus Purpureocillium based on multilocus nuclear and mitochondrial DNA sequences, with the description of Purpureocillium xinfangense sp. nov. (Ophiocordycipitaceae, Hypocreales) from Southwestern China. Frontiers in Fungal Biology, 7, Article 1972355. https://doi.org/10.3389/ffunb.2026.1972355

Image Credits: AI Generated

DOI: 10.3389/ffunb.2026.1972355

Keywords: Purpureocillium, Purpureocillium xinfangense, Ophiocordycipitaceae, Hypocreales, mitochondrial genome, phylogenetics, fungal taxonomy, entomopathogenic fungi, Yunnan China, multilocus phylogeny, biocontrol fungi, species discovery