Ant's Nest as a microenvironment: Distinct Mucoromycota (Fungi) community of the red wood ants' (Formica polyctena) mounds.

Many social insect species build nests, which differ from the surrounding environment and are often occupied by specific organismal communities. These organisms may interact mutualistically or parasitically with the nest-builders, or simply co-occur, being able to survive in these microenvironments. In temperate forests, red wood ants (e.g. Formica polyctena) are known to create distinct, highly developed nests, which consist of large, above-ground mounds, built primarily out of plant matter collected from the forest litter. The microorganismal communities of such mounds remain understudied. As representatives of Mucoromycota fungi commonly engage in the decomposition process of the forest litter, they would be expected to occur in the mounds. However, it is still not known whether the Mucoromycota community of these ants' nests differ from the one of the surrounding forest litter. In order to distinguish mound-associated taxa, we characterized Mucoromycota communities of Formica polyctena mounds and the surrounding forest litter. We sampled four sites, twice in a season. Sampled material was plated on agar media and emerging Mucoromycota colonies were identified based on their morphology. Fungal identification was further confirmed using DNA barcoding. In order to compare described communities, PERMANOVA test and non-metric multidimensional scaling ordinations were used. To distinguish taxa associated with the mounds, multilevel pattern analysis was performed. Our results show that the Mucoromycota community of Formica polyctena's mound differs from the community of the surrounding forest litter. While representatives of Entomortierella lignicola and Absidia cylindrospora clade were found to be associated with the mound environment, representatives of Umbelopsis curvata and Podila verticillata-humilis clade were associated with forest litter, and were rarely present in the mounds. Our findings strongly suggest that the red wood ants' nest is a specific microenvironment in the temperate forest floor, which is a preferred microhabitat for the mound-associated Mucoromycota, possibly adapted to live in proximity to ants.

Discovery of Formicomyces microglobosus gen. et sp. nov. strengthens the hypothesis of independent evolution of ant-associated fungi in Trichomeriaceae.

Different groups of fungi have been reported to interact with ants. Recent studies have shown that fungi of the order Chaetothyriales are important components of ant-fungus networks, including members of the family Trichomeriaceae, which is particularly rich in fungi isolated from carton ants nests. One of the still understudied ant-related environments are ants' infrabuccal pockets and pellets, which often contain fungal matter. The aim of this work was to determine the systematic and phylogenetic position of two slow growing strains of Trichomeriaceae isolated from infrabuccal pellets of Formica polyctena ants. Molecular analyses based on maximum likelihood and bayesian inference, using sequences of two ribosomal DNA markers: ITS and LSU have shown that the isolated strains form a monophyletic clade within the family Trichomeriaceae, sister to a clade formed by representatives of the genus Trichomerium. Morphological analyses additionally justified distinctiveness of the isolated strains, which have different morphology of conidia and conidiophores than Trichomerium representatives. Therefore, our results show that the isolated strains represent a new species within a not yet described fungal genus. Due to the strains' isolation source and their close relatedness to a fungal strain isolated from a carton nest of Lasius fuliginosus, we propose a name Formicomyces microglobosus Siedlecki & Piatek for this fungus. While our discovery strengthens a hypothesis of the multiple, independent evolution of ant-associated fungi in the family Trichomeriaceae, the ecology of F. microglobosus still remains to be characterized.

Chance or Necessity-The Fungi Co-Occurring with Formica polyctena Ants.

Studies on carton nesting ants and domatia-dwelling ants have shown that ant-fungi interactions may be much more common and widespread than previously thought. Until now, studies focused predominantly on parasitic and mutualistic fungi-ant interactions occurring mostly in the tropics, neglecting less-obvious interactions involving the fungi common in ants' surroundings in temperate climates. In our study, we characterized the mycobiota of the surroundings of Formica polyctena ants by identifying nearly 600 fungal colonies that were isolated externally from the bodies of F. polyctena workers. The ants were collected from mounds found in northern and central Poland. Isolated fungi were assigned to 20 genera via molecular identification (ITS rDNA barcoding). Among these, Penicillium strains were the most frequent, belonging to eight different taxonomic sections. Other common and widespread members of Eurotiales, such as Aspergillus spp., were isolated very rarely. In our study, we managed to characterize the genera of fungi commonly present on F. polyctena workers. Our results suggest that Penicillium, Trichoderma, Mucor, Schwanniomyces and Entomortierella are commonly present in F. polyctena surroundings. Additionally, the high diversity and high frequency of Penicillium colonies isolated from ants in this study suggest that representatives of this genus may be adapted to survive in ant nests environment better than the other fungal groups, or that they are preferentially sustained by the insects in nests.