From the Tunnels into the Treetops: New Lineages of Black Yeasts from Biofilm in the Stockholm Metro System and Their Relatives among Ant-Associated Fungi in the Chaetothyriales

Abstract

Rock-inhabiting fungi harbour species-rich, poorly differentiated, extremophilic taxa of polyphyletic origin. Their closest relatives are often well-known species from various biotopes with significant pathogenic potential. Speleothems represent a unique rock-dwelling habitat, whose mycobiota are largely unexplored. Isolation of fungi from speleothem biofilm covering bare granite walls in the Kungsträdgården metro station in Stockholm yielded axenic cultures of two distinct black yeast morphotypes. Phylogenetic analyses of DNA sequences from six nuclear loci, ITS, nuc18S and nuc28S rDNA, rpb1, rpb2 and β-tubulin, support their placement in the Chaetothyriales (Ascomycota). They are described as a new genus Bacillicladium with the type species B. lobatum, and a new species Bradymyces graniticola. Bacillicladium is distantly related to the known five chaetothyrialean families and is unique in the Chaetothyriales by variable morphology showing hyphal, meristematic and yeast-like growth in vitro. The nearest relatives of Bacillicladium are recruited among fungi isolated from cardboard-like construction material produced by arboricolous non-attine ants. Their sister relationship is weakly supported by the Maximum likelihood analysis, but strongly supported by Bayesian inference. The genus Bradymyces is placed amidst members of the Trichomeriaceae and is ecologically undefined; it includes an opportunistic animal pathogen while two other species inhabit rock surfaces. ITS rDNA sequences of three species accepted in Bradymyces and other undescribed species and environmental samples were subjected to phylogenetic analysis and in-depth comparative analysis of ITS1 and ITS2 secondary structures in order to study their intraspecific variability. Compensatory base change criterion in the ITS2 secondary structure supported delimitation of species in Bradymyces, which manifest a limited number of phenotypic features useful for species recognition. The role of fungi in the speleothem biofilm and relationships of Bacillicladium and Bradymyces with other members of the Chaetothyriales are discussed.

Fig 1. An overview map of the Kungsträdgården metro station in Stockholm.

X marks the sample location.

Fig 2. The Kungsträdgården metro station in Stockholm.

Overview of the granite wall covered in fungal biofilm. Colorations represent wet and dry biofilm or mineral precipitates. Arrow indicates wall with wet biofilm and sample location. Bar = 1 m.

Fig 3. Phylogenetic analysis of the nucITS-nuc28S-β-tubulin sequences of the Chaetothyriales.

Branch support in nodes ≥ 70% maximum likelihood bootstrap support (ML BS) and ≥ 0.90 Bayesian posterior probability (PP) are indicated above or below branches. The asterisk (*) indicates nodes with 100% ML BS/1.0 PP. Taxa written in bold represent taxonomic novelties. ^T = ex-type.

Fig 4. Phylogenetic analysis of the combined nuc18S-nuc28S-rpb1-rpb2 sequences of the Chaetothyriomycetidae.

Details as in Fig 3.

Fig 5. Phylogenetic analysis of the ITS rDNA sequences of Bradymyces spp.

Groups of taxa with no CBC among them are labelled as CBC clades indicated by colour blocks. The non-CBCs and hCBCs substitutions identified in ITS1 and ITS2 2D structure are colour-coded as shown in the legend in the frame; respective internodes are labelled with them. The first base pair in the legend always refers to B. oncorhynchi. Details as in Fig 3.

Fig 6. ITS1 secondary structure of Bradymyces oncorhynchi (HG426062).

ITS1 helices are numbered I–V. All substitutions recorded among members of Bradymyces are mapped on the 2D model. The asterisk (*) marks the only hCBC between B. oncorhynchi and Bradymyces sp. 2. Legend to symbols and colours as in Fig 7.

Fig 7. ITS2 secondary structure of Bradymyces oncorhynchi (HG426062) and 5.8S-28S rRNA gene hybridization (proximal stem region).

ITS2 helices are numbered I−V. All substitutions recorded among members of Bradymyces are mapped on the 2D model. Identified substitutions are colour-coded: CBC (blue), hCBC (green) and non-CBC (red); single substitution (yellow). Parts of hairpin loops, junctions and a helix highlighted with grey colour represent regions with a variable number of nucleotides or sequence variation.

Fig 8. Macromorphology and micromorphology of Bacillicladium lobatum.

(A) Morphogenesis of colony growing on MEA at 25°C in 9 d, 2, 4 and 6 wks (left to right). (B, C) Phenotypic variability of 6-week-old colonies at 25°C on PDA (B) and PCA (C). (D‒I) Yeast-like state, budding (D, I), germinating by hyphae (E, G) or forming short chains (F‒I). (J‒L) Fungal elements from the inner parts of the colony with incrustations on their surface, occasionally proliferating. (M, N) Uni- or multicellular bodies, single or in chains. (O) Meristematic parenchyma-like structures formed in the inner parts of the colony (MEA). (P) Multicellular element released from the parenchymatous structure with roughened wall. Bar = 5 mm (A‒C), 5 μm (D‒P).

Fig 9. Hyphal state of Bacillicladium lobatum.

(A–M) Unbranched hyphae growing through blastic proliferation and covering surface of colonies on MEA and PDA: (A–F) hyphae of the first type, aseptate or ostensibly uniseptate, commonly constricted in the middle part; (G–M) hyphae of the second type with numerous cell wall reinforcements and multiseptate appearance. (N–W) Unbranched or sparsely branched vegetative hyphae from the deeper parts of the colonies, occasionally terminated by a swollen globose to ellipsoidal cell (N, O, R–T), sometimes with signs of budding (O, S, T), occasionally with uni- or bicellular chlamydospore-like hyphal swellings intercalary in position (U, V). Bar = 5 μm.

Fig 10. Morphology and variability of colonies and growth parameters of Bradymyces spp. and Bacillicladium lobatum after 6 wks of cultivation on MEA at three different temperatures.

(A) B. graniticola. (B) B. oncorhynchi. (C) B. alpinus. (D) Ba. lobatum. One ‘tick’ on the ruler in the upper part of the figure corresponds to 1 mm.

Fig 11. Macromorphology and micromorphology of Bradymyces graniticola.

(A) A 16-week-old colony on MEA at 17°C. (B) Phenotypic variability of 6-week-old colonies on PDA at 25°C. (C) Phenotypic variability of 6-week-old colonies on PCA at 25°C. (D–H) Endoconidia. (I–N) Moniliform hyphae proliferating at the apex. (O) Chlamydospore-like hyphal swellings intercalary in position. (P–S) Multicellular bodies released from meristematic part of colony. Bar = 5 mm (A‒C), 5 μm (D‒S).

Fig 12. Colony diameters of Bradymyces spp. and Bacillicladium lobatum after 6 wks of cultivation on different media and temperatures.

Isolates included: B. graniticola (CCF 5193−5197, CCF 5227); B. oncorhynchi (CCF 4369), B. alpinus (CCFEE 5493), Ba. lobatum (CCF 5199, CCF 5200). Each point represent mean of at least nine values per isolate; error bars represent standard deviations.

Fig 13. Biofilm on a granite wall.

Part of the wall with both dry and wet areas covered by biofilm; DB = dry biofilm, WB = wet biofilm. Bar = 5 cm.

Fig 14. Biofilm containing fungal hyphae.

(A) Removed biofilm containing fungal hyphae; note the tissue-like texture with protruding hyphae and white mineral precipitates. (B) Biofilm showing the fashion of fungal hyphae producing the tissue-like texture of the mat. (C) Fragment of meristematic tissue composed of isodiametric and angular cells. (D, E, H) Fragments of melanised monilioid hyphae. (F, G) Hyphae with endoconidia. Bar = 100 μm (A), 200 μm (B), 10 μm (C), 5 μm (D−H).