Discussion

In this study, three new species belonging to two genera, Arachnidicola and Simplicillium, are described based on morphological characteristics, multi-locus phylogenetic analyses, and PHI tests. The robust phylogenetic placement of these taxa, coupled with morphological distinctions and recombination tests, provides compelling evidence for their recognition as novel species.

The genus Arachnidicola, established by Khonsanit et al. (2024) with A. sulphurea as the type species, currently comprises 16 species (Chen et al. 2025b; Chang et al. 2026). A striking feature of this genus is its apparent near-exclusive association with spiders; to date, almost all known Arachnidicola species have been isolated from arachnid hosts. This strong host fidelity raises critical questions regarding the degree of host specialization within the genus. While our combined analysis supports the delineation of A. fodingshanensis as a distinct taxon, the available data do not reveal an obvious pattern of strict co-evolution with specific spider families. Instead, the host association appears to be ecologically driven – spiders as a group may provide a consistent microhabitat (e.g., cuticular chemistry, humidity, or nutrient profile) that favors the proliferation of this fungal lineage (Chang et al. 2026). This observation suggests that what we observe may be ecological host fidelity rather than strict phylogenetic host specialization, though a comprehensive survey of spider-pathogenic fungi across diverse arachnid families is needed to test this hypothesis.

It should be noted that the phylogenetic relationship between Arachnidicola fodingshanensis and its close relatives is not fully resolved, and the nucleotide divergence between A. fodingshanensis and A. kanyawimiae appears relatively limited (see Suppl. material 1). Nevertheless, the recognition of A. fodingshanensis as a novel species is supported by multiple independent lines of evidence – including morphology and the PHI test – rather than by any single dataset. While the limited sequence divergence may partly reflect the substantially shorter sequences available for A. kanyawimiae at several loci, which could underestimate the true genetic distance, the combined evidence collectively justifies the proposal of this new species. Future studies with more complete sequence data for A. kanyawimiae would be valuable for further clarifying the deeper relationships within this species complex.

In China, nine species have been reported, five of which originated from Guizhou Province, and all five Guizhou collections were derived from forest habitats, with one notable exception from a Tiankeng niche. The discovery of A. fodingshanensis from a similar forest environment reinforces the notion that Guizhou’s forest ecosystems harbor a significant, yet-to-be-discovered diversity of this genus, particularly in underexplored micro-niches such as leaf litter, understory vegetation, and rock crevices.

In contrast, the genus Simplicillium exhibits a broader ecological amplitude. Established with S. lanosoniveum as the type species by Zare and Gams (2001), it currently encompasses 36 species, with only five previously reported from spider hosts (Chen et al. 2025b; Chang et al. 2026). The two novel Simplicillium species described herein – one associated with a spider (S. shiqianense) and the other with a lepidopteran cocoon (S. fodingshanense)—provide a unique opportunity to explore the dynamics of host divergence within this genus. Chen et al. (2022b) demonstrated that host jumping is a widespread phenomenon within Simplicillium, suggesting that S. araneae likely originated from an insect host and underwent an interkingdom jump to spiders. Expanding on this concept, our findings offer a comparative perspective on host transitions within different ecological settings. The spider-associated S. shiqianense, collected from a forest habitat, may represent an independent lineage that similarly underwent an insect-to-spider transition, though its phylogenetic proximity to soil-borne S. subtropicum raises an alternative hypothesis: that spider pathogenicity might have arisen from saprobic or soil-dwelling ancestors through host availability and ecological opportunity.

More intriguingly, the discovery of S. fodingshanense from a lepidopteran cocoon – a substrate functionally and biologically distinct from a spider host – raises new questions about the ecological plasticity of this genus. Cocoons represent a nutrient-rich, protected microhabitat that may be exploited by opportunistic fungi. The isolate could be a true pathogen of the pupa, a saprobe colonizing the cocoon after host death, or even a mycoparasite. Its phylogenetic closeness to S. shiqianense (from spider) and S. subtropicum (from soil) suggests that the lineage encompassing these three species may have undergone multiple host shifts. The co-occurrence of these two novel species in the same reserve, albeit from different hosts and microhabitats (spiders vs. cocoons), highlights the potential role of resource partitioning and host availability in driving speciation events within Simplicillium. Future experimental studies, including cross-infection assays and genomic comparisons, would be essential to determine the host range and pathogenic potential of these fungi.

In addition, Akanthomyces muscarius (Petch) Spatafora et al. and Beauveria bassiana (Bals.-Criv.) Vuill. have previously been reported from soil environments in the Fodingshan Nature Reserve (Hu 2021). The addition of three new species from this single study underscores that the documented fungal diversity in this protected area is merely a fraction of the actual species richness. As highlighted by Wijayawardene et al. (2021), karst habitats, with their complex microclimates and isolated niches, are biodiversity hotspots that warrant significantly greater attention from mycologists. The Fodingshan Nature Reserve, characterized by its forest-covered karst landscape, likely harbors a wealth of undescribed entomopathogenic and arthropod-associated fungi. The present results, combined with the unique host associations observed, provide a strong impetus for future investigations. Future research should prioritize systematic surveys across different seasons, altitudes, and microhabitats (e.g., forest floor, valley, and cavernous niches) to fully assess the species richness of entomopathogenic fungi in this reserve. Furthermore, in-depth genomic and ecological studies are urgently needed to unravel the molecular mechanisms underlying host specificity and the adaptive advantages conferring host jumping in Simplicillium, as well as to elucidate the precise ecological role of these fungi in their respective host life cycles.