Taxonomy and systematics of the fungus-growing ant associate Escovopsis (Hypocreaceae).

Escovopsis is a symbiont of fungus-growing ant colonies. Unstandardised taxonomy prevented the evaluation of the morphological diversity of Escovopsis for more than a century. The aim of this study is to create a standardised taxonomic framework to assess the morphological and phylogenetic diversity of Escovopsis. Therefore, to set the foundation for Escovopsis taxonomy and allow interspecific comparisons within the genus, we redescribe the ex-type cultures of Escovopsis aspergilloides, E. clavata, E. lentecrescens, E. microspora, E. moelleri, E. multiformis, and E. weberi. Thus, based on the parameters adopted in this study combined with phylogenetic analyses using five molecular markers, we synonymize E. microspora with E. weberi, and introduce 13 new species isolated from attine nests collected in Argentina, Brazil, Costa Rica, Mexico, and Panama: E. breviramosa, E. chlamydosporosa, E. diminuta, E. elongatistipitata, E. gracilis, E. maculosa, E. papillata, E. peniculiformis, E. phialicopiosa, E. pseudocylindrica, E. rectangula, E. rosisimilis, and E. spicaticlavata. Our results revealed a great interspecific morphological diversity throughout Escovopsis. Notwithstanding, colony growth rates at different temperatures, as well as vesicle shape, appear to be the most outstanding features distinguishing species in the genus. This study fills an important gap in the systematics of Escovopsis that will allow future researchers to unravel the genetic and morphological diversity and species diversification of these attine ant symbionts. Taxonomic novelties: New species: Escovopsis breviramosa Q.V. Montoya, M.J.S. Martiarena & A. Rodrigues, E. chlamydosporosa Q.V. Montoya, M.J.S. Martiarena & A. Rodrigues, E. diminuta Q.V. Montoya, M.J.S. Martiarena & A. Rodrigues, E. elongatistipitata Q.V. Montoya, M.J.S. Martiarena & A. Rodrigues, E. gracilis Q.V. Montoya, M.J.S. Martiarena & A. Rodrigues, E. maculosa Q.V. Montoya, M.J.S. Martiarena & A. Rodrigues, E. papillata Q.V. Montoya, M.J.S. Martiarena & A. Rodrigues, E. peniculiformis Q.V. Montoya, M.J.S. Martiarena & A. Rodrigues, E. phialicopiosa Q.V. Montoya, M.J.S. Martiarena & A. Rodrigues, E. pseudocylindrica Q.V. Montoya, M.J.S. Martiarena & A. Rodrigues, E. rectangula Q.V. Montoya, M.J.S. Martiarena & A. Rodrigues, E. rosisimilis Q.V. Montoya, M.J.S. Martiarena & A. Rodrigues, E. spicaticlavata Q.V. Montoya, M.J.S. Martiarena & A. Rodrigues. Citation: Montoya QV, Martiarena MJS, Rodrigues A (2023). Taxonomy and systematics of the fungus-growing ant associate Escovopsis (Hypocreaceae). Studies in Mycology 106: 349-397. doi: 10.3114/sim.2023.106.06.

Interactions among Escovopsis, Antagonistic Microfungi Associated with the Fungus-Growing Ant Symbiosis.

Fungi in the genus Escovopsis (Ascomycota: Hypocreales) are prevalent associates of the complex symbiosis between fungus-growing ants (Tribe Attini), the ants' cultivated basidiomycete fungi and a consortium of both beneficial and harmful microbes found within the ants' garden communities. Some Escovopsis spp. have been shown to attack the ants' cultivated fungi, and co-infections by multiple Escovopsis spp. are common in gardens in nature. Yet, little is known about how Escovopsis strains impact each other. Since microbe-microbe interactions play a central role in microbial ecology and evolution, we conducted experiments to assay the types of interactions that govern Escovopsis-Escovopsis relationships. We isolated Escovopsis strains from the gardens of 10 attine ant genera representing basal (lower) and derived groups in the attine ant phylogeny. We conducted in vitro experiments to determine the outcome of both intraclonal and interclonal Escovopsis confrontations. When paired with self (intraclonal interactions), Escovopsis isolated from lower attine colonies exhibited antagonistic (inhibitory) responses, while strains isolated from derived attine colonies exhibited neutral or mutualistic interactions, leading to a clear phylogenetic pattern of interaction outcome. Interclonal interactions were more varied, exhibiting less phylogenetic signal. These results can serve as the basis for future studies on the costs and benefits of Escovopsis coinfection, and on the genetic and chemical mechanisms that regulate the compatibility and incompatibility observed here.

Carbon dioxide levels and ventilation in Acromyrmex nests: significance and evolution of architectural innovations in leaf-cutting ants.

Leaf-cutting ant colonies largely differ in size, yet all consume O2 and produce CO2 in large amounts because of their underground fungus gardens. We have shown that in the Acromyrmex genus, three basic nest morphologies occur, and investigated the effects of architectural innovations on nest ventilation. We recognized (i) serial nests, similar to the ancestral type of the sister genus Trachymyrmex, with chambers excavated along a vertical tunnel connecting to the outside via a single opening, (ii) shallow nests, with one/few chambers extending shallowly with multiple connections to the outside, and (iii) thatched nests, with an above-ground fungus garden covered with plant material. Ventilation in shallow and thatched nests, but not in serial nests, occurred via wind-induced flows and thermal convection. CO2 concentrations were below the values known to affect the respiration of the symbiotic fungus, indicating that shallow and thatched nests are not constrained by harmful CO2 levels. Serial nests may be constrained depending on the soil CO2 levels. We suggest that in Acromyrmex, selective pressures acting on temperature and humidity control led to nesting habits closer to or above the soil surface and to the evolution of architectural innovations that improved gas exchanges.

Climate Change Influences Basidiome Emergence of Leaf-Cutting Ant Cultivars.

Maintaining symbiosis homeostasis is essential for mutualistic partners. Leaf-cutting ants evolved a long-term symbiotic mutualism with fungal cultivars for nourishment while using vertical asexual transmission across generations. Despite the ants' efforts to suppress fungal sexual reproduction, scattered occurrences of cultivar basidiomes have been reported. Here, we review the literature for basidiome occurrences and associated climate data. We hypothesized that more basidiome events could be expected in scenarios with an increase in temperature and precipitation. Our field observations and climate data analyses indeed suggest that Acromyrmex coronatus colonies are prone to basidiome occurrences in warmer and wetter seasons. Even though our study partly depended on historical records, occurrences have increased, correlating with climate change. A nest architecture with low (or even the lack of) insulation might be the cause of this phenomenon. The nature of basidiome occurrences in the A. coronatus-fungus mutualism can be useful to elucidate how resilient mutualistic symbioses are in light of climate change scenarios.

Fungi inhabiting attine ant colonies: reassessment of the genus Escovopsis and description of Luteomyces and Sympodiorosea gens. nov.

Escovopsis is a diverse group of fungi, which are considered specialized parasites of the fungal cultivars of fungus-growing ants. The lack of a suitable taxonomic framework and phylogenetic inconsistencies have long hampered Escovopsis research. The aim of this study is to reassess the genus Escovopsis using a taxonomic approach and a comprehensive multilocus phylogenetic analysis, in order to set the basis of the genus systematics and the stage for future Escovopsis research. Our results support the separation of Escovopsis into three distinct genera. In light of this, we redefine Escovopsis as a monophyletic clade whose main feature is to form terminal vesicles on conidiophores. Consequently, E. kreiselii and E. trichodermoides were recombined into two new genera, Sympodiorosea and Luteomyces, as S. kreiselii and L. trichodermoides, respectively. This study expands our understanding of the systematics of Escovopsis and related genera, thereby facilitating future research on the evolutionary history, taxonomic diversity, and ecological roles of these inhabitants of the attine ant colonies.

Antileishmanial macrolides from ant-associated Streptomyces sp. ISID311.

Three antifungal macrolides cyphomycin (1), caniferolide C (2) and GT-35 (3) were isolated from Streptomyces sp. ISID311, a bacterial symbiont associated with Cyphomyrmex fungus-growing ants. The planar structures of these compounds were established by 1 and 2D NMR data and MS analysis. The relative configurations of 1-3 were established using Kishi's universal NMR database method, NOE/ROE analysis and coupling constants analysis assisted by comparisons with NMR data of related compounds. Detailed bioinformatic analysis of cyphomycin biosynthetic gene cluster confirmed the stereochemical assignments. Compounds 1-3 displayed high antagonism against different strains of Escovopsis sp., pathogen fungi specialized to the fungus-growing ant system. Compounds 1-3 also exhibited potent antiprotozoal activity against intracellular amastigotes of the human parasite Leishmania donovani with IC50 values of 2.32, 0.091 and 0.073 µM, respectively, with high selectivity indexes.

Cryptic Diversity in Colombian Edible Leaf-Cutting Ants (Hymenoptera: Formicidae).

Leaf-cutting ants are often considered agricultural pests, but they can also benefit local people and serve important roles in ecosystems. Throughout their distribution, winged reproductive queens of leaf-cutting ants in the genus Atta Fabricius, 1804 are consumed as a protein-rich food source and sometimes used for medical purposes. Little is known, however, about the species identity of collected ants and the accuracy of identification when ants are sold, ambiguities that may impact the conservation status of Atta species as well as the nutritional value that they provide to consumers. Here, 21 samples of fried ants bought in San Gil, Colombia, were identified to species level using Cytochrome Oxidase I (COI) barcoding sequences. DNA was extracted from these fried samples using standard Chelex extraction methods, followed by phylogenetic analyses with an additional 52 new sequences from wild ant colonies collected in Panama and 251 publicly available sequences. Most analysed samples corresponded to Atta laevigata (Smith, 1858), even though one sample was identified as Atta colombica Guérin-Méneville, 1844 and another one formed a distinct branch on its own, more closely related to Atta texana (Buckley, 1860) and Atta mexicana (Smith, 1858). Analyses further confirm paraphyly within Atta sexdens (Linnaeus, 1758) and A. laevigata clades. Further research is needed to assess the nutritional value of the different species.

Cytogenetic data on six leafcutter ants of the genus Acromyrmex Mayr, 1865 (Hymenoptera, Formicidae, Myrmicinae): insights into chromosome evolution and taxonomic implications.

Cytogenetic data for the genus Acromyrmex Mayr, 1865 are available, to date, for a few species from Brazil and Uruguay, which have uniform chromosome numbers (2n = 38). The recent cytogenetic data of Acromyrmex striatus (Roger, 1863), including its banding patterns, showed a distinct karyotype (2n = 22), similar to earlier studied Atta Fabricius, 1804 species. Karyological data are still scarce for the leafcutter ants and many gaps are still present for a proper understanding of this group. Therefore, this study aimed at increasing cytogenetic knowledge of the genus through the characterization of other six species: Acromyrmex balzani (Emery, 1890), Acromyrmex coronatus Fabricius, 1804, Acromyrmex disciger (Mayr, 1887), Acromyrmex echinatior (Forel, 1899), Acromyrmex niger (Smith, 1858) and Acromyrmex rugosus (Smith, 1858), all of which were collected in Minas Gerais - Brazil, except for Acromyrmex echinatior which was collected in Barro Colorado - Panama. The number and morphology of the chromosomes were studied and the following banding techniques were applied: C-banding, fluorochromes CMA3 and DAPI, as well as the detection of 45S rDNA using FISH technique. All the six species had the same chromosome number observed for already studied species, i.e. 2n = 38. Acromyrmex balzani had a different karyotype compared with other species mainly due to the first metacentric pair. The heterochromatin distribution also showed interspecific variation. Nevertheless, all the studied species had a pair of bands in the short arm of the first subtelocentric pair. The fluorochrome CMA3 visualized bands in the short arm of the first subtelocentric pair for all the six species, while Acromyrmex rugosus and Acromyrmex niger also demonstrated in the other chromosomes. The AT-rich regions with differential staining using DAPI were not observed. 45S ribosomal genes were identified by FISH in the short arm of the first subtelocentric pair in Acromyrmex coronatus, Acromyrmex disciger and Acromyrmex niger. The uniform chromosome number in the genus Acromyrmex (2n = 38) suggests that Acromyrmex striatus (2n = 22) should be transferred to a new genus. Other aspects of the chromosome evolution in ants are also discussed.

A new species of the fungus-farming ant genus Mycetagroicus Brandão & Mayhé-Nunes (Hymenoptera, Formicidae, Attini) / Uma nova espécie de formiga cultivadora de fungo, do gênero Mycetagroicus Brandão & Mayhé-Nunes (Hymenoptera, Formicidae, Attini)

The fungus-farming ant genus Mycetagroicus Brandão & Mayhé-Nunes was proposed based on three species from the Brazilian "Cerrado": M. cerradensis, M. triangularis and M. urbanus. Here we describe a new species of Attini ant of the genus Mycetagroicus, M. inflatus n. sp., based on two workers collected in eastern Pará State, Brazil. A new key for species identification, comments on differences among species and new geographical distribution data are furnished.

O gênero de formigas cultivadoras de fungos, Mycetagroicus Brandão & Mayhé-Nunes, foi proposto com base em três espécies do Cerrado: M. cerradensis, M. triangularis e M. urbanus. Neste trabalho descrevemos uma nova espécie de Attini do gênero Mycetagroicus, M. inflatus n. sp., baseada em duas operárias coletadas no leste do Pará, Brasil. Apresentamos uma nova chave para a identificação das espécies, comentários sobre as diferenças entre as espécies e novos dados sobre a distribuição geográfica.

Seasonal Variations of Metapleural Secretion in the Leaf-Cutting ant Atta sexdens piriventris Santschi (Myrmicinae: Attini), and Lack of Fungicide Effect on Beauveria bassiana (Bals. ) Vuillemin / Variações Sazonais da Secreção Metapleural da Formiga Cortadeira Atta sexdens piriventris Santschi (Myrmicinae: Attini), e Ausência de Efeito Fungicida Sobre o Entomopatógeno Beauveria bassiana (Bals. ) Vuillemin

Os mecanismos envolvidos na manutenção das colônias de insetos sociais livres de patógenos são aspectos importantes relacionados à biologia evolutiva do grupo. No caso das formigas, tem sido sugerido que a secreção das glândulas metapleurais teria importante papel na assepsia da colônia. Neste trabalho, as diversas subcastas de operárias da formiga cortadeira Atta sexdens piriventris Santschi foram comparadas quanto à presença de secreção metapleural ao longo de um ano. Simultaneamente, a atividade tópica da secreção foi testada em operárias tratadas com o fungo entomopatogênico Beauveria bassiana (Bals.) Vuillemin. De acordo com a largura média da cápsula cefálica, foram definidas seis subcastas de operárias: jardineira, mínima, média, grande, máxima e soldado. Foi encontrada uma correlação positiva entre subcasta e comprimento do reservatório metapleural. As subcastas apresentaram diferenças significativas na presença de secreção metapleural ao longo do ano exceto nos meses de abril, maio, julho e novembro. Foi observada uma correlação positiva entre as temperaturas médias sazonais e presença de secreção metapleural. Foram registradas altas freqüências de operárias de todas subcastas infectadas por B. bassiana durante os bioensaios, indicando que a secreção metapleural não tem atividade fungicida contra esse patógeno. Os resultados sugerem que a secreção metapleural não está entre os principais mecanismos de assepsia das colônias de A. sexdens piriventris.

Mechanisms involved in maintaining colonies of social insects free of pathogens are among the main aspects of interest in the evolutionary biology of this group. For ants, it has been suggested that secretion from the metapleural glands play an important role in colony asepsis. In this study, different worker subcastes of the fungus-growing ant Atta sexdens piriventris Santschi were compared in relation to presence of metapleural secretion during a year. At the same time, the topical activity of the secretion was tested on workers treated with the entomopathogenic fungus Beauveria bassiana (Bals.) Vuillemin. Based on the mean width of the cephalic capsule, six worker subcastes were defined: gardener, minor, media, large, major and soldier. A positive correlation between worker subcaste and metapleural reservoir length was observed. Significant differences for presence of metapleural secretion were found throughout the year among subcastes, except in April, May, July and November. A positive correlation between seasonal mean temperature and presence of the metapleural secretion was observed. High frequencies of infected workers of all subcastes were observed during bioassays with inoculation of B. bassiana, indicating that metapleural secretion has no fungicidal activity against this pathogen. The results suggest that metapleural secretion of workers is not the main mechanism of A. sexdens piriventris colony asepsis.