The frequency and the geographical extent of symbiotic associations between ants and fungi of the order Chaetothyriales have been highlighted only recently. Using a phylogenetic approach based on seven molecular markers, we showed that ant-associated Chaetothyriales are scattered through the phylogeny of this order. There was no clustering according to geographical origin or to the taxonomy of the ant host. However, strains tended to be clustered according to the type of association with ants: strains from ant-made carton and strains from plant cavities occupied by ants (‘domatia’) rarely clustered together. Defining molecular operational taxonomic units (MOTUs) with an internal transcribed spacer sequence similarity cut-off of 99% revealed that a single MOTU could be composed of strains collected from various ant species and from several continents. Some ant-associated MOTUs also contained strains isolated from habitats other than ant-associated structures. Altogether, our results suggest that the degree of specialization of the interactions between ants and their fungal partners is highly variable. A better knowledge of the ecology of these interactions and a more comprehensive sampling of the fungal order are needed to elucidate the evolutionary history of mutualistic symbioses between ants and Chaetothyriales.

Ascomycete fungi in the nests of ants inhabiting plants (= myrmecophytes) are very often cultivated by the ants in small patches and used as food source. Where these fungi come from is not known yet. Two scenarios of fungus recruitment are possible: (1) random infection through spores or hyphal fragments from the environment, or (2) transmission from mother to daughter colonies by the foundress queen. It is also not known at which stage of the colony life cycle fungiculture is initiated, and whether the- symbiont fungi serve as food for the ant queen. To clarify these questions, we investigated four Azteca ant species inhabiting three different Cecropia species (C. insignis, C. obtusifolia, and C. peltata). We analysed an rRNA gene fragment from 52 fungal patches produced by founding queens and compared them with those from established Azteca colonies (n = 54). The infrabuccal pockets of winged queens were dissected to investigate whether young queens carry fungi from their mother colony. Additionally, 15N labelling experiments were done to verify whether the queen feeds on the patches until she is nourished by her first worker offspring. We infer from the results that the fungi cultivated in hollow plant structures are transferred from the parental colony of the young queen. First, fungal genotypes/OTU diversity was not significantly different between foundress queen patches and established colonies, and second, hyphal parts were discovered in the infrabuccal pockets of female alates. We could show that fungiculture already starts before queens lay their eggs, and that the queens do not feed on fungal patch material but feed it to the larvae. Our findings suggest that fungiculture may be crucial for successful colony founding of arboreal ants in the tropics.

Five Cecropia tree species occupied by four Azteca ant species from Costa Rica and French Guiana were investigated to assess the diversity and host specificity of chaetothyrialean fungal symbionts. The ITS rDNA region of the symbiotic fungi was sequenced either from pure culture isolation, or from environmental samples obtained from ant colonies nesting in hollow stems of the Cecropia host plants. The investigation revealed six closely related OTUs of Chaetothyriales. Neither the four Azteca species nor the six fungal OTUs were associated with specific Cecropia species. In contrast, ants and fungi showed an association. Azteca alfari was associated with a particular OTU, and often contained only one. Azteca coeruleipennis, A. constructor and A. xanthochroa were associated with a different set of OTUs and often had multiple OTUs within colonies. Possible reasons for these differences and the role of the fungi for the Azteca-Cecropia symbiosis are discussed.

New associations have recently been discovered between arboreal ants that live on myrmecophytic plants, and different groups of fungi. Most of the – usually undescribed – fungi cultured by the ants belong to the order Chaetothyriales (Ascomycetes). Chaetothyriales occur in the nesting spaces provided by the host plant, and form a major part of the cardboard-like material produced by the ants for constructing nests and runway galleries. Until now, the fungi have been considered specific to each ant species. We focus on the three-way association between the plant Tetrathylacium macrophyllum (Salicaceae), the ant Azteca brevis (Formicidae: Dolichoderinae) and various chaetothyrialean fungi. Azteca brevis builds extensive runway galleries along branches of T. macrophyllum. The carton of the gallery walls consists of masticated plant material densely pervaded by chaetothyrialean hyphae. In order to characterise the specificity of the ant–fungus association, fungi from the runway galleries of 19 ant colonies were grown as pure cultures and analyzed using partial SSU, complete ITS, 5.8S and partial LSU rDNA sequences. This gave 128 different fungal genotypes, 78% of which were clustered into three monophyletic groups. The most common fungus (either genotype or approximate species-level OTU) was found in the runway galleries of 63% of the investigated ant colonies. This indicates that there can be a dominant fungus but, in general, a wider guild of chaetothyrialean fungi share the same ant mutualist in Azteca brevis.

Diazotrophic microorganisms introduce biologically available nitrogen (N) to the global N cycle through the activity of the nitrogenase enzyme. The genetically conserved dinitrogenase reductase (nifH) gene is phylogenetically distributed across four clusters (I–IV) and is widely used as a marker gene for N2 fixation, permitting investigators to study the genetic diversity of diazotrophs in nature and target potential participants in N2 fixation. To date there have been limited, standardized pipelines for analyzing the nifH functional gene, which is in stark contrast to the 16S rRNA gene. Here we present a bioinformatics pipeline for processing nifH amplicon datasets – NifMAP (“NifH MiSeq Illumina Amplicon Analysis Pipeline”), which as a novel aspect uses Hidden-Markov Models to filter out homologous genes to nifH. By using this pipeline, we evaluated the broadly inclusive primer pairs (Ueda19F–R6, IGK3–DVV, and F2–R6) that target the nifH gene. To evaluate any systematic biases, the nifH gene was amplified with the aforementioned primer pairs in a diverse collection of environmental samples (soils, rhizosphere and roots samples, biological soil crusts and estuarine samples), in addition to a nifH mock community consisting of six phylogenetically diverse members. We noted that all primer pairs co-amplified nifH homologs to varying degrees; up to 90% of the amplicons were nifH homologs with IGK3–DVV in some samples (rhizosphere and roots from tall oat-grass). In regards to specificity, we observed some degree of bias across the primer pairs. For example, primer pair F2–R6 discriminated against cyanobacteria (amongst others), yet captured many sequences from subclusters IIIE and IIIL-N. These aforementioned subclusters were largely missing by the primer pair IGK3–DVV, which also tended to discriminate against Alphaproteobacteria, but amplified sequences within clusters IIIC (affiliated with Clostridia) and clusters IVB and IVC. Primer pair Ueda19F–R6 exhibited the least bias and successfully captured diazotrophs in cluster I and subclusters IIIE, IIIL, IIIM, and IIIN, but tended to discriminate against Firmicutes and subcluster IIIC. Taken together, our newly established bioinformatics pipeline, NifMAP, along with our systematic evaluations of nifH primer pairs permit more robust, high-throughput investigations of diazotrophs in diverse environments.