A new family of ant-associated fungi in Chaetothyriales

Abstract

The order Chaetothyriales comprises the black yeasts and relatives, of which numerous species are prevalent as opportunists on human hosts. The present paper introduces a clade of species that live in ant nests inside hollow structures of tropical plants (so-called domatia) and their closest relatives. To clarify the evolutionary trajectory of the domatia-associated clade, molecular, morphological, and physiological data were analysed. The position of the domatia clade within the Chaetothyriales was assessed by phylogenetic analysis of ITS and LSU. Species delimitations were calculated and genealogical concordance performed with a dataset including the gene of the ribosomal operon, β-tubulin (BT2) and RNA polymerase II largest subunit (RBP1). Genome sequencing allowed additional analysis of mating types, mitochondrial genomes, and estimation of a species tree based on the proteins of 770 single copy orthologous genes. A new family with two new genera in Chaetothyriales was introduced to accommodate the taxa from ant-inhabited domatia and a related clade of plant- and rock-colonizing species. The family is monophyletic and has strong statistical support. Although species delimitation criteria suggested the separation of more than 10 species in the domatia-clade, genealogical concordance of ribosomal and housekeeping gene markers indicated genetic exchange. Seven new species were delineated, with species also being characterized by phenotypic features of fungal colony morphology, micromorphology, physiology and ecology. However, intra-specific variability remained exceptionally large and did not always match with ecological and geographic data. It is hypothesized that the high degrees of intra- and interspecific variability of some of the clades acknowledged as separate species might be related to extended periods of molecular evolution. The newly described species seem to have their preferred habitat in tropical ant nests, and they have adapted to this specific environment. Ant-domatia provide a remarkable habitat rich in volatile chemicals, which could be tolerated by the fungi under study. The family is distantly related to the family Herpotrichiellaceae comprising numerous human-opportunistic species, where hydrocarbon tolerance has been hypothesized to play a role in black yeast evolution. Taxonomic novelties: New family: Domatiomycetaceae Meizhu Wang, Voglmayr, V.E. Mayer, S.A. Ahmed & de Hoog. New genera: Domatiomyces Meizhu Wang, Voglmayr, V.E. Mayer, S.A. Ahmed & de Hoog, Lapsomyces Meizhu Wang, S.A. Ahmed & de Hoog. New species: Domatiomyces globalis Meizhu Wang, Voglmayr, V.E. Mayer, S.A. Ahmed & de Hoog, Domatiomyces clavatus Meizhu Wang, Voglmayr, V.E. Mayer, S.A. Ahmed & de Hoog, Domatiomyces catenatus Meizhu Wang, Voglmayr, V.E. Mayer, S.A. Ahmed & de Hoog, Domatiomyces disarticulatus Meizhu Wang, Voglmayr, V.E. Mayer, S.A. Ahmed & de Hoog, Domatiomyces pauciseptatus Meizhu Wang, Voglmayr, V.E. Mayer, S.A. Ahmed & de Hoog, Domatiomyces germinans Meizhu Wang, Voglmayr, V.E. Mayer, S.A. Ahmed & de Hoog, Lapsomyces furvus Meizhu Wang, S.A. Ahmed & de Hoog. New combinations: Lapsomyces behniae (Crous) Meizhu Wang, S.A. Ahmed & de Hoog, Lapsomyces hostae (Crous) Meizhu Wang, S.A. Ahmed & de Hoog, Lapsomyces scillae (Deighton) Meizhu Wang, S.A. Ahmed & de Hoog. Citation: Wang MZ, Belmonte-Lopes R, Pan T, Ahmed SA, Rodrigues Lustosa BP, Quan Y, Al-Hatmi AMS, Mayer VE, Voglmayr H, Grisolia ME, de Souza Lima BBJF, Vicente VA, Zhou SQ, Cao Y, Kang YQ, de Hoog GS (2025). A new family of ant-associated fungi in Chaetothyriales. Studies in Mycology 110: 111-143. doi: 10.3114/sim.2024.110.02.

Keywords: Chaetothyriales; ant-association; black yeast; ecology; evolution; myrmecophily; new taxa.

Fig. 1

Domatia (hollow structures in plants) inhabited by ants. A. Founding queen of Cladomyrma petalae (Formicinae) gnawing an entrance hole to enter the domatium of a Saraca thaipingensis (Fabaceae) stem in Borneo. B. Opened stem of a young Saraca shoot with larvae and workers of C. petalae ants. C. Stem domatium of the climbing shrub Keetia hispida (Rubiaceae) inhabited by Crematogaster margaritae (Myrmicinae) in Cameroon. Domatiomyces species are localized in the blackish area. D. Opened branch of Barteria fistulosa (Passifloraceae) from Cameroon inhabited by Tetraponera aethiops (Pseudomyrmecinae). The Domatiomyces species were isolated from the blackish area. E. Pseudomyrmex penetrator (Pseudomyrmecinae) in a branch of Tachigali sp. (Fabaceae) from French Guyana. The white spots in the blackish area with Domatiomyces species are accumulations of bacterivorous nematodes (Rhabditidae) which are regularly found in this habitat. F. Brood chamber in the hollow stem of a young Cecropia peltata inhabited by Azteca constructor (Dolichoderinae) in Costa Rica. The Domatiomyces species are located in the black spots. Photo credits: A, B: J. Moog, C, D, E: R. Blatrix, F: V. Mayer.

Fig. 2

Physiological tests of species of Domatiomyces. A. Growth of type strains of species of Domatiomyces on MEA incubated at temperatures ranging from 6–40 °C. Colony diam (mm). B. Histograms with standard deviation of triplicate tests of colony diam of strains of Domatiomyces incubated on MEA at with additional glucose or NaCl at 28 °C recorded after 14 d incubation.

Fig. 3

Phylogenetic tree of Chaetothyriales based on ITS and LSU sequences, obtained by Bayesian analysis and maximum likelihood (values of ≥ 0.9 for Bayesian probability or ≥ 70 % for maximum likelihood shown). Chaetothyriales sp. Pet5a = CBS 135087 was used as outgroup. Blue branches denote isolates / clades of Domatiomycetaceae, red branches from ant-made carton, green branches from Attine ants.

Fig. 4

Concatenated tree of the genes ITS, LSU, BT2, and RPB1 from the domatia lineages estimated using MrBayes, with node values representing posterior probabilities (left side), and results of the species delimitation’s obtained for the different genes and methods (ABGD, ASAP, PTP, bPTP, and GMYC). Equal colours in the columns indicate that strains belong to the same delimited clade, while equal colours in the lines indicates that the results of the different delimitation methods were the same. Numbers at the bottom of the right side indicates the number of clades delimited by each gene and method. White coloured areas represent missing data.

Fig. 5

Tanglegram comparisons of analysed sequences of LSU, ITS, BT2 and RPB1.

Fig. 6

Alignment of the BT2 gene indicating exons, introns, mutations in relation to the consensus sequence, and for the exons, the proteins generated in each codon are presented below the nucleotide sequence.

Fig. 7

Haplotype networks using the geneHapR R library with calculations of haplotype and nucleotide diversity encountered in ITS (A), LSU (B), BT2 (C), RPB1 (D) sequences of the 6 species of Domatiomyces, encoded as different colours. The different colours indicate the six species of Domatiomyces. The translucent circles indicate the number of haplotypes for the different genes.

Fig. 8

Pairwise genetic distances for the ITS gene of the members of Domatiomyces. The black line indicates the mean value found between the proposed species, and the red lines indicate its 95 % confidence interval. Start of the gaps between the groups is indicated by different colours and symbols. The Y-axis indicates the lineage that is compared to all others, while the coloured dots indicate the group of the second sample based on the 0.05 value.

Fig. 9

Species tree obtained from the orthologous 770 single-copy orthologous protein sequences using Orthofinder2. Support values represent the proportion of single-locus gene trees supporting each bipartition of the species tree. Branch block colours represent the clades evidenced in Fig. 4. dH 24566 = CBS 143850.

Fig. 10

Order of the mitochondrial genes in the Chaetothyriales (the cox1 gene was set arbitrarily as the origin). Genomes available at GenBank are indicated by accession numbers, whereas the ones from MycoCosm are indicated by “v1.0” after the species and strain information. The scale indicates the size of the genomes (in base pairs), and the colours the genes (see legend).

Fig. 11

Domatiomyces globalis, CBS 134916. A, B. Colonies on MEA and OA, respectively, after 4 wk incubation. C–J. Sympodial conidiogenous cells (red arrows). Black arrows showing ramoconidia. Scale bars = 10 μm.

Fig. 12

Domatiomyces globalis, Lineage 3, CBS 135086. A, B. Colonies on PDA and OA, respectively, after 4 wk incubation. C–J. Sympodial conidiogenous cells. K, L. Catenate conidia. Scale bars = 10 μm.

Fig. 13

Domatiomyces globalis, Lineage 4, CBS 129053. A, B. Colonies on PDA and OA, respectively, after 4 wk incubation. C, D, J. Catenate conidia. E–G. Sympodial conidiogenous cells. H. Sympodial cell on torulose hypha. K. Spiral hypha. Scale bars = 10 μm.

Fig. 14

Domatiomyces globalis, Lineage 6, CBS 143848. A, B. Colonies on PDA and OA, respectively, after 4 wk incubation. C–J. Sympodial cells with conidial chains rapidly falling apart into separate cells. Scale bars = 10 μm.

Fig. 15

Domatiomyces globalis, Lineage 7, CBS 129057. A, B. Colonies on PDA and OA, respectively, after 4 wk incubation. C–J. Sympodial conidiogenous cells. Scale bars = 10 μm.

Fig. 16

Domatiomyces globalis, Lineage 8, CBS 132038. A, B. Colonies on PDA and OA, respectively, after 4 wk incubation. C–J. Sympodial conidiogenous cells. Scale bars = 10 μm.

Fig. 17

Domatiomyces clavatus, CBS 134923. A, B. Colonies on PDA and OA, respectively, after 4 wk incubation. C–J. Sympodial conidiogenous cells. Scale bars = 10 μm.

Fig. 18

Domatiomyces catenatus, CBS 134920. A, B. Colonies on PDA and OA, respectively, after 4 wk incubation. C–J. Sympodial conidiogenous cells, conidia with catenate extension. Scale bars = 10 μm.

Fig. 19

Domatiomyces disarticulatus, CBS 128963. A, B. Colonies on PDA and OA, respectively, after 4 wk incubation. C–L. Sympodial conidiogenous cells. Scale bars = 10 μm.

Fig. 20

Domatiomyces pauciseptatus, CBS 128966. A, B. Colonies on PDA and OA, respectively, after 4 wk incubation. C–L. Sympodial conidiogenous cells. Scale bars = 10 μm.

Fig. 21

Domatiomyces germinans, CBS 134922. A, B. Colonies on PDA and OA, respectively, after 4 wk incubation. C–G. Conidiogenesis with conidia rapidly falling apart into separate cells. Scale bars = 10 μm.

Fig. 22

Lapsomyces furvus, CBS 118278. A, B. Colonies on MEA and OA, respectively, after 4 wk incubation. C–H. Catenate conidiogenesis. Scale bars = 10 μm.