Research Article |
Corresponding author: Andre Rodrigues ( andre.rodrigues@unesp.br ) Academic editor: Thorsten Lumbsch
© 2019 Quimi Vidaurre Montoya, Maria Jesus Sutta Martiarena, Danilo Augusto Polezel, Sérgio Kakazu, Andre Rodrigues.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Montoya QV, Martiarena MJS, Polezel DA, Kakazu S, Rodrigues A (2019) More pieces to a huge puzzle: Two new Escovopsis species from fungus gardens of attine ants. MycoKeys 46: 1-22. https://doi.org/10.3897/mycokeys.46.30951
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Escovopsis (Ascomycota: Hypocreales, Hypocreaceae) is the only known parasite of the mutualistic fungi cultivated by fungus-growing ants (Formicidae: Myrmicinae: Attini: Attina, the “attines”). Despite its ecological role, the taxonomy and systematics of Escovopsis have been poorly addressed. Here, based on morphological and phylogenetic analyses with three molecular markers (internal transcribed spacer, large subunit ribosomal RNA and the translation elongation factor 1-alpha), we describe Escovopsis clavatus and E. multiformis as new species isolated from fungus gardens of Apterostigma ant species. Our analysis shows that E. clavatus and E. multiformis belong to the most derived Escovopsis clade, whose main character is the presence of conidiophores with vesicles. Nevertheless, the most outstanding feature of both new species is the presence of a swollen region in the central hypha of the conidiophore named swollen cell, which is absent in all previously described Escovopsis species. The less derived Escovopsis clades lack vesicles and their phylogenetic position within the Hypocreaceae still remains unclear. Considering the high genetic diversity in Escovopsis, the description of these new species adds barely two pieces to a huge taxonomic puzzle; however, this discovery is an important piece for building the systematics of this group of fungi.
Hypocreales, Taxonomy, Phylogeny, Parasitic fungi, Symbiosis
Microorganisms play important roles in the stability of social insect colonies (
Attine ants are classified in two sister clades: the Palaeoattina and Neoattina (
The attine ant-fungus cultivar-Escovopsis symbiosis has been widely studied in leafcutter ants (
While Escovopsis species exploiting gardens of Atta, Acromyrmex, Trachymyrmex and Mycetophylax were formally described, the morphological characters of the species associated with Apterostigma are unknown. A previous study associated clades of the parasite with the colour pattern of Escovopsis colonies (brown, yellow, white and pink;
Five Escovopsis isolates were obtained from fungus gardens of five different colonies of Apterostigma spp. (Suppl. material
For fungal isolation, seven garden fragments (0.5–1 mm3) were inoculated on plates (three plates per colony) containing potato dextrose agar (PDA, Neogen Culture Media, Neogen) supplemented with chloramphenicol (150 µg mL-1, Sigma) and incubated at 25 °C in darkness. Plates were monitored daily for fungal growth and, when Escovopsis mycelia sprouted, they were transferred to new PDA plates. All isolates were prepared as axenic (monosporic) cultures and stored under sterile distilled water kept at 8 °C (
The morphological characters of the five isolates (LESF 847, LESF 853, LESF 854, LESF 855 and LESF 1136) were examined. Due to the lack of standardisation of culture conditions for Escovopsis, the macroscopic characters of the colonies, i.e. radial growth, mycelium colour, morphology and presence of soluble pigments, were evaluated on eight different media: PDA, malt agar 2% [MA2%: 20 g L-1 of malt extract (Neogen Culture Media) and 15 g L-1 of agar (Neogen Culture Media)], cornmeal agar (CMD, Neogen Culture Media), synthetic nutrient agar [SNA: 1 g L-1 of KH2PO4 (Labsynth), 1 g L-1 of KNO3 (Labsynth), 0.5 g L-1 of MgSO4(7H2O) (Labsynth), 0.5 g L-1 of KCl (Labsynth), 0.2 g L-1 of Glucose (Labsynth), 0.2 g L-1 of Sucrose (Labsynth) and 15 g L-1 of Agar (Neogen Culture Media)], oatmeal agar (OA), potato carrot agar (PCA, HiMedia), malt extract agar 2% [MEA: 30 g L-1 of malt extract (Neogen Culture Media), 5 g L-1 of bacteriological peptone (Neogen Culture Media), 20 g L-1 of glucose (Labsynth) and 15 g L-1of Agar (Neogen Culture Media)] and Czapek yeast extract agar [CYA; 30 g L-1 of Sucrose (Labsynth), 5 g L-1 of Yeast extract (Neogen Culture Media), 1 g L-1 of KH2PO4 (Labsynth), 0.3 g L-1 of NaNO3 (Synth), 0.05 g L-1 of KCl (Labsynth), 0.05 g L-1 of MgSO4(7H2O) (Labsynth), 0.001 g L-1 of FeSO4 (Labsynth), 0.001 g L-1 of ZnSO4 (Labsynth), 0.0005 g L-1 of CuSO4 (Labsynth), 15 g L-1 of Agar (Neogen Culture Media)] at five temperatures (10 °C, 20 °C, 25 °C, 30 °C and 35 °C). These temperatures correspond to the conditions used in previous studies that described Escovopsis species (
To examine the microscopic characters, i.e. the morphology, size, branching patterns, vesicles and swollen cells of the conidiophores, as well as phialides and conidia, slide cultures on PDA and MEA were performed. Briefly, we placed a 5 mm2 fragment of culture medium on a microscopic slide and then we inoculated the fungus at the centre of the fragment. Then, the inoculated medium was covered with a coverslip and incubated at 25 °C for 4–7 days in the dark. After that, the coverslips, where the fungus grew, were removed and placed in new slides with a drop of lactophenol. Finally, the slides were examined under a light microscope (DM750, Leica, Germany). Fungal microscopic structures were photographed and measured (with 30 measurements per structure) in LAS EZ v.4.0 (Leica Application Suite).
Microscopic structures were also examined under scanning electron microscopy (SEM). Fungal samples (five days old cultures on PDA) were fixed in osmium tetroxide vapour for 72 h. Then, samples were dehydrated using a series of acetone concentrations (50, 75, 90, 95 and 100%) and dried to critical point using liquid CO2 (Balzers CPD030). The dried material was sputtered with gold (Balzers SCD050) and examined under the scanning electron microscope (TM3000, Hitachi).
DNA extraction of the five strains was performed, following the steps published in
PCR and sequence reaction conditions followed the steps published in
To infer the phylogenetic position of the new species in the Escovopsis clade, sequences from previous studies were retrieved from the GenBank and aligned with our new sequences in a dataset for each marker (
“clavatus” in reference to the predominantly clavate shape of vesicles.
BRAZIL. Santa Catarina, Florianópolis, (27°44'39.6"S, 48°31'10.14"W), elev. 46 m, fungus garden, 08, 2015. A. Rodrigues. Holotype: CBS H-23845 (dried culture on PDA). Ex-type strain LESF 853 (= CBS 145326).
ITS (MH715096), tef1 (MH724270) and LSU (MH715110).
Colonies grow only at 20 and 25 °C (Fig.
Conidiophores arising from aerial hypha alternated or opposite (Fig.
Isolated from fungus gardens of Apterostigma sp.
BRAZIL. Santa Catarina, Florianópolis, (27°44'38.94"S, 48°31'9.3"W), elev. 32 m, fungus garden, 08, 2015. A. Rodrigues. LESF 854 (ITS – MH715097, tef1 – MH724271 and LSU – MH715111). Santa Catarina, Florianópolis, (27°44'39.49"S, 48°31'9.72"W), elev. 38 m, fungus garden, 08, 2015. A. Rodrigues. LESF 855 (ITS – MH71509, tef1 – MH724272 and LSU – MH715112).
Escovopsis clavatus is phylogenetically closely related to E. multiformis and its most distinctive characters are its growth temperatures, the conidiophore branching and the swollen cells. It grows at 20 and 25 °C; nevertheless, E. multiformis grows at 10, 20, 25 and 30 °C. The conidiophore of E. clavatus is larger and more branched than the conidiophore of E. multiformis. In addition, the swollen cells of E. clavatus are less frequent and shorter than in E. multiformis. The character distinguishing E. clavatus from other species of Escovopsis is the swollen cell on the conidiophores and because it is phylogenetically placed in a distinct clade.
“multiformis” in relation to the different vesicle shapes found in the same isolate.
BRAZIL. Santa Catarina, Florianópolis, (27°28'11.28"S, 48°22'39.48"W), elev. 119 m, fungus garden, 08, 2015. A. Rodrigues. Holotype: CBS H-23846 (dried culture on PDA). Ex-type strain LESF 847 (= CBS 145327).
ITS (MH715091), tef1 (MH724265) and LSU (MH715105).
Colonies grow at 10, 20, 25 and 30 °C (Fig.
Conidiophores arising from aerial hypha alternated or opposite (Fig.
Isolated from fungus garden of Apterostigma sp.
BRAZIL. Mato Grosso, Cotriguaçu, (09°49'22.74"S, 58°15'32.04"W), elev. 252 m, fungus garden, 10, 2017. Q. V. Montoya. LESF 1136 (ITS – MH715092, tef1 – MH724266 and LSU – MH715106).
Escovopsis multiformis is closely related to E. clavatus. Different from E. clavatus that grow at 20 and 25 °C, E. multiformis grow at 10, 20, 25 and 30 °C. The optimum growth temperature of E. multiformis is 30 °C and that of E. clavatus is 25 °C. The conidiophores of E. multiformis are smaller and less branched than E. clavatus and the swollen cells are more frequent and larger than those found in E. clavatus. E. multiformis differs from other described species by the presence of conidiophores with a swollen cell, the presence of different vesicles shapes and because it is phylogenetically placed in a distinct clade.
The isolates LESF 853 (Escovopsis clavatus, Figs
Escovopsis multiformis showed growth at wide ranges of temperature (from 10–30 °C); nonetheless, E. clavatus showed growth only at 20 and 25 °C (Fig.
All strains of both species have a unique type of conidiophore with a swollen cell, from which branches emerge (Figs
Separate phylogenetic analyses with the three molecular markers showed topological differences because of the incongruity placement of the formal described Escovopsis species and some strains that form new phylogenetic clades within the genus (Fig.
Phylogenetic position of Escovopsis clavatus and Escovopsis multiformis considering each molecular marker separately (ITS, LSU and tef1). The trees were reconstructed under Bayesian and Maximum Likelihood inferences. The numbers on branches indicate the posterior probabilities and the bootstrap support values, respectively. The seven Escovopsis ex-type strains are denoted in bold and the new species are highlighted in green (E. clavatus) and light brown (E. multiformis). The trees include a total of 46 Escovopsis sequences of each marker (ITS – 619 bp, LSU – 594 bp and tef1 – 758 bp) and Escovopsioides, Hypomyces, Sphaerostilbella, Trichoderma and Protocrea were included as the closest phylogenetic relatives of Escovopsis. Lecanicillium antillanum CBS 350.85 was used as the outgroup. ET: ex-type.
The combined analysis also confirmed E. multiformis and E. clavatus as two new phylogenetic species in Escovopsis (PP= 1; MLB= 100%, Fig.
Phylogenetic position of Escovopsis clavatus and Escovopsis multiformis. The phylogenetic analysis is based on the concatenated sequences of ITS, LSU and tef1; and the tree was reconstructed using Bayesian and Maximum Likelihood inferences. Numbers on branches indicate the posterior probabilities and the bootstrap support values, respectively. All Escovopsis species previously described are denoted in bold and the new species are highlighted in green (E. clavatus) and light brown (E. multiformis). The tree includes a total of 40 Escovopsis sequences with 1971 bp (ITS – 619 bp, LSU – 594 bp and tef1 – 758 bp). The data also included sequences from Escovopsioides, Hypomyces, Sphaerostilbella, Trichoderma and Protocrea as the closest phylogenetic relatives of the parasite. Lecanicillium antillanum CBS 350.85 was used as the outgroup. ET: ex-type strains. Bar: 0.04 substitutions per nucleotide position.
It is important to highlight that the concatenated analysis, as well as the trees inferred with ITS and LSU, showed the vesiculated Escovopsis (E. aspergilloides, E. clavatus, E. lentecrescens, E. microspora, E. moelleri, E. multiformis, E. weberi) as the most derived group, separated from the non-vesiculated Escovopsis (E. kreiselii and E. trichodermoides). In addition, both the combined and the analysis performed with ITS and tef1 showed some Escovopsis species (E. aspergilloides, E. kreiselii, E. lentecrescens and E. trichodermoides) often clustering with other Hypocreaceae genera or falling outside the Escovopsis clade, which reveals that Escovopsis is apparently paraphyletic (Figs
The attine ants have persisted for millions of years because of the biological relationships that these insects maintain with the beneficial microorganisms that inhabit their colonies. Several studies tried to understand how these biological relationships sculptured the evolutionary history of the attines (
Subsequent to the formal description of Escovopsis (
Recent attempts to expand the morphological concept of Escovopsis generated inconsistencies in the taxonomy and systematics of this genus (
Our study shows that the ex-type strains LESF 853 (E. clavatus) and LESF 847 (E. multiformis) form a monophyletic clade within most derived Escovopsis (vesiculated Escovopsis, PP = 1, BML = 100%). Most interesting, unlike the other Escovopsis species, the two new species present a unique type of conidiophore with a swollen cell, from which one to four branches arise. The newly described species also possess smooth conidia with slightly thickened walls. A recent study suggests the possibility that conidia ornamentation could be associated with the mechanism for horizontal transmission of Escovopsis between ant colonies and with the latency of the parasite conidia. This hypothesis was based on observations of some conidia adhering to the ant legs and in spore dormancy in vitro bioassays (
Considering the high genetic diversity of Escovopsis and the poor knowledge of its taxonomy, our study suggests that the fungus gardens of attine ants host a great diversity of Escovopsis that has yet to be discovered. Thus, the description of these new species are merely two small pieces of a complex puzzle. Nonetheless, our work should help future researchers to build the framework for the systematics of this parasitic fungus.
We are grateful to ‘‘Fundação de Amparo à Pesquisa do Estado de São Paulo’’ (FAPESP) for financial support (Grants # 2014/24298-1 and #2017/12689-4) conceded to AR and for the scholarship (# 2016/04955-3) to QVM. We also would like to thank Antônio Teruyoshi Yabuki (UNESP, Rio Claro) for SEM assistance, Lia Costa Pinto Wentzel, Rodolfo Bizarria Jr and Caitlin Conn for valuable comments on this manuscript. In addition, we are grateful to Dr. Huzefa Raja (reviewer #1), Keith Seifert (reviewer #2) and the editor Thorsten Lumbsch for providing constructive comments on this study.
Supplementary tables
Data type: phylogenetic data
Explanation note: Table S1. Escovopsis strains used in the phylogenetic analyses and their associated metadata. Table S2. Molecular markers, primers and PCR conditions used in this study.