Taxonomy and systematics of the fungus-growing ant associate Escovopsis (Hypocreaceae) (2024)

Abstract: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 Mycology106: 349–397. doi: 10.3114/sim.2023.106.06

Sampling and strains

We used 138 Escovopsis strains, including ex-type cultures of E. aspergilloides, E. clavata, E. lentecrescens, E. microspora, E. moelleri, E. multiformis, and E. weberi (Table 1). The ex-type material of the five species from Argentina described by Marfetán et al. (2019) were unavailable and we did not obtain any isolates that we could identify as these species, so it was not possible to include them in our study. Of the 138 isolates, 86 were obtained from previous studies (Currie et al. 2003, Augustin et al. 2013, Meirelles et al. 2015a, b, Montoya et al. 2019, 2021) whereas the remaining 52 isolates were obtained from attine nests collected in Argentina, Brazil, Costa Rica, Mexico, and Panama (Table 1). For isolating cultures in this study, we followed the methods published in Montoya et al. (2019). Briefly, ant garden fragments (0.5–1 mm³) were inoculated onto PDA (Neogen^® Culture Media, Lansing, USA) supplemented with 150 μg/mL of chloramphenicol (Sigma-Aldrich, St. Louis, USA). We inoculated three plates for each ant fungus garden and seven garden fragments per plate. The plates were incubated at 25 °C in darkness and monitored daily for seven days. When Escovopsis mycelia were grown, they were transferred to new PDA plates without chloramphenicol and finally axenic cultures were obtained by single conidial isolation.

All Escovopsis isolates used in this study (except those that have the specimen vouchers QVM281–QVM289) are stored at the Laboratory of Fungal Ecology and Systematics [LESF– Department of General and Applied Biology, São Paulo State University (UNESP), Rio Claro, SP, Brazil] in sterile distilled water at 8–10 °C (Castellani 1963), in 10 % aqueous solution of glycerol at -80 °C (cryopreservation), and as freeze-dried in 10 % Skim Milk. Isolates that have the specimen vouchers QVM281–QVM289 were sequenced while still viable, but later they lost viability, so we were unable to preserve them (Table 1). Holotypes (metabolically inactive, freeze-dried cultures) and the ex-type cultures of the new species were also deposited at the culture collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands (CBS) (Table 1).

Adjusting the parameters to evaluate the morphology of Escovopsis

To enable reliable comparison of cultural and micromorphological characters, we selected a set of media and optimal cultivation conditions for Escovopsis species. To do so, we recorded characters of colonies in culture, i.e., mycelium colour, morphology and presence of soluble pigments, in 21 isolates of seven Escovopsis species (including the ex-type cultures), i.e., E. aspergilloides (n = 1), E. clavata (n = 3), E. lentecrescens (n = 1), E. microspora (n = 1), E. moelleri (n = 1), E. multiformis (n = 4), and E. weberi (n = 10), using the combination of all conditions, i.e., eight media [cornmeal agar dextrose (CMD), CYA, PDA, malt agar 2 % (MA2 %), MEA, oatmeal agar (OA), potato carrot agar (PCA), and synthetic nutrient-poor agar (SNA) — Supplementary Table S1] and five temperatures (10, 20, 25, 30, 35 °C), used in previous studies (Seifert et al. 1995, Augustin et al. 2013, Masiulionis et al. 2015, Meirelles et al. 2015a, b, Montoya et al. 2019).

To standardize the inoculum (make the number of conidia in all inoculum approximately the same), we hom*ogeneously spread 200 μL of 10⁶ conidia/mL (from 7-d-old colonies), on Petri dishes (90 × 15 mm) with water agar (WA) (Montoya et al. 2019). These Petri dishes were incubated for 7 d at 25 °C in darkness (Montoya et al. 2019). An agar plug (ca. 5 mm diam × 5 mm height) of WA with mycelium was cut and inoculated in the centre of Petri dishes (90 × 15 mm) containing each test medium, and incubated at 10, 20, 25, 30, and 35 °C. All Petri dishes were incubated unsealed to allow air exchange and better development of fungal colonies (Montoya et al. 2019). We performed three replicates for each ex-type culture at each media and temperature. Morphological characters were examined every 24 h for 14 d. From this experiment, we selected CMD (Neogen^® Culture Media, Lansing, USA), MEA [30 g/L of malt extract (Neogen^® Culture Media, Lansing, USA), 5 g/L of bacteriological peptone (Neogen^® Culture Media, Lansing, USA), 20 g/L of glucose (Labsynth, Diadema, Brazil), and 15 g/L of Agar (Neogen^® Culture Media, Lansing, USA], and PDA as the most suitable media to evaluate the macroscopic features of Escovopsis species. These media were selected based on the (i) ease to evaluate the growth rate; (ii) expression of unique phenotypic characters of each species, (iii) feasibility of comparison of morphological features of Escovopsis with other genera in the Hypocreaceae, and (iv) ease of access to media in most laboratories. To describe the colony colours, we used the standard names and codes provided by Ridgway (1912). Likewise, the optimal time for measuring the growth and evaluating the macroscopic characters of colonies were standardised based on the point at which we were able to observe the most significant differences in growth rate and morphological characters between species.

To evaluate the growth rate of the Escovopsis ex-type cultures, and the new described species, on the selected media, we grew the colonies, in quadruplicate, on each medium at 20, 25 and 30 °C, on four separate time periods for 1 wk. Measurements of the colony radius (Supplementary Table S2) were carried out on the fourth day (see the results section for details on the selected temperatures and time for growth measurements). The growth measurements shown in the descriptions of the species (taxonomy section) represent the minimum and maximum values observed among the 16 values obtained. Statistical analyses were performed in R Studio using one-way ANOVA, followed by Duncan’s multiple range test. Differences were considered significant when P ≤ 0.05.

The microscopic structures of all Escovopsis ex-type cultures, and our newly described species, i.e., conidiophores, branches of conidiophores, swollen cells of conidiophores (formed at the apex of the conidiophore and from which branches are formed), vesicles, conidiogenous cells, conidia and chlamydospores (sensuAugustin et al. 2013), their shape, size, colour, and pattern of formation and aggregation (Montoya et al. 2021), were evaluated on PDA following the method used by Montoya et al. (2019). Briefly, we carried out slide culture preparations using plugs from PDA 5 mm diam × 5 mm height, placed on microscopic slides. Then, the plugs were inoculated with conidia, covered with coverslips, and incubated at 25 °C for 4–7 d in the dark. Finally, the coverslips were removed and placed on new slides with a drop of lactophenol. The slides were examined using compound light microscope (DM750, Leica, Wetzlar), and microscopic structures were photographed and 30 measurements recorded for each structure using LAS EZ v. 4.0 (Leica Application Suite) and ImageJ2 v. 2.3.0 in Fiji (Schindelin et al. 2012). The measurements of the microscopic structures shown in the descriptions of the species (taxonomy section) represent the minimum and maximum values observed among the 30 measurements obtained.

DNA extraction, PCR and sequencing

We used five molecular markers in this study, i.e., the internal transcribed spacers (ITS), the large subunit nuclear ribosomal RNA gene (28S), the translation elongation factor 1-alpha (tef1), and the RNA polymerase II protein-coding largest and second largest subunit genes rpb1 and rpb2 (Supplementary Table S3). Of the 138 isolates used in this study, 64 were previously sequenced for these five markers in other studies (Augustin et al. 2013, Meirelles et al. 2015a, b, Montoya et al. 2019, 2021; see Table 1). The ITS and tef1 regions of 22 isolates were previously sequenced by Meirelles et al. (2015b). These were supplemented with our sequences of the 28S, rpb1, and rpb2 genes. All five markers were sequenced for the remaining 52 isolates (Table 1).

For the isolates sequenced in this study, we first extracted the genomic DNA using a modified CTAB method (Möller et al. 1992). Briefly, aerial mycelium, grown for 7 d at 25 °C on PDA, was crushed with the aid of glass beads (Sigma) in a lysis solution. Five μL of Proteinase K were added to this solution and incubated at 65 °C for 30 min. Then, the organic phase of the solution was separated by centrifugation at 10 000 g for 10 min, using chloroformisoamyl alcohol (24: 1). Four hundred μL of the supernatant were collected, and the genomic DNA was precipitated with 3 M sodium acetate and isopropanol. The genomic DNA was purified with two successive washes of 70 % ethanol and left at room temperature to dry overnight. Finally, the DNA was suspended in 30 μL of Tris-EDTA solution and stored at -20 °C.

Amplification reactions for ITS, 28S, tef1, rpb1 and rpb2 regions were carried out using the primers and conditions published by Meirelles et al. (2015a, b), Augustin et al. (2013), and Montoya et al. (2021) (summarized in Supplementary Table S3). Amplicons were purified with the Wizard SV Gel and PCR Clean-up System (Promega, Madison) following the manufacturer’s protocol and sequenced (forward and reverse sequencies) on an ABI 3500 Genetic Analyzer (Life Technologies). Consensus sequences were assembled using Geneious v. 6.0 (Kearse et al. 2012) and deposited in GenBank (see Table 1 for accession numbers).

Phylogenetic analyses

We performed a multilocus analysis combining the five molecular markers. First, the data sets were aligned separately for each marker in MAFFT v. 7 (). The nucleotide substitution model for each alignment was calculated in jModelTest v. 2 (Darriba et al. 2012) using the Akaike Information Criterion (AIC) with 95 % confidence intervals. To determine if all Escovopsis species clades remained constant (monophyletic) considering the GCPSR concept (Taylor et al. 2000), a phylogenetic tree was inferred using each molecular marker separately (Supplementary Fig. S1). Finally, the datasets were concatenated using Winclada v. 1.00.08 (Nixon 2002). The final data set contained a total of 139 sequences 3 700 bp long [ITS (570 bp), 28S (591 bp), rpb1 (751 bp), rpb2 (1 030 bp), and tef1 (758 bp)]. Escovopsis species described by Marfetán et al. (2019) were not included in the multilocus analyses of this study because most of the sequences of these species are unavailable (Montoya et al. 2021). However, 21 available 28S sequences of those species were combined with our 28S data to reveal their phylogenetic relationships (Supplementary Table S4 and Supplementary Fig. S2).

We reconstructed the final phylogenetic trees using Bayesian Inference (BI) in MrBayes v. 3.2.2 (Ronquist et al. 2012) and Maximum Likelihood (ML) in RAxML v. 8 (Stamatakis 2014). For the BI analysis, we carried out two separate runs (each consisting of three hot chains and one cold chain) using the GTR model for each partition independently. Two million generations of the Markov Chain Monte Carlo (MCMC) were enough to reach convergence (standard deviation of split frequencies < 0.01). The first 25 % of trees were discarded as burn-in to generate the best BI tree. For the ML analysis, we estimated 1 000 independent trees and performed 1 000 bootstrap replicates using the GTR model. The final tree was visualized in FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/) and edited in Adobe Illustrator CC v. 17.1. The alignments and resulting trees were deposited in TreeBASE (Study Accession URL: http://purl.org/phylo/treebase/phylows/study/TB2:S30754).

Taxonomic key to Escovopsis species

Sixty-eight morphological features from species of Escovopsis (Supplementary Table S5) were analysed using the “rpart” library () in R v. 3.6.3. Nineteen out of the 68 characters were selected using a recursive partitioning algorithm (with the information gain as a measure for deciding between alternative splits) as the most informative features to build the dichotomous key (Williams 2011). Finally, a dichotomous key (in cladogram format) was reconstructed using a decision tree that started with a single node root that split into 18 (Supplementary Fig. S3; numbered in red) branches to end in the leaves corresponding to each species (Supplementary Fig. S3). The final cladogram was manually edited using Adobe Illustrator CC v. 17.1, and the information on branches was used to construct the taxonomic key. The Escovopsis species described by Marfetán et al. (2019) could not be included in this key because the morphological features of those species had been described using conditions different to those used in this study.

Adjusting the parameters to evaluate the morphology of Escovopsis

Colony growth of Escovopsis species was observed on different media at temperatures between 10 and 30 °C. Escovopsis weberi, E. clavata, E. microspora, and E. moelleri were able to grow at 10 °C, but their growth was limited and inconspicuous. Thus, 10 °C was selected as the minimum test temperature and growth of all strains at this temperature is reported as present or absent. The seven Escovopsis ex-type cultures grew well at 20, 25 and 30 °C, although at 30 °C, colony growth of E. aspergilloides, E. clavata, E. lentecrescens, E. multiformis was much slower than that of E. microspora, E. moelleri, and E. weberi. Therefore, we consider these temperatures most appropriate for evaluating macroscopic characters. At temperatures 20, 25 and 30 °C, colonies started to grow after the first (Escovopsis microspora, E. moelleri, and E. weberi) or the second day (E. aspergilloides, E. clavata, E. lentecrescens, E. multiformis). Some fast-growing species (E. microspora, E. moelleri, and E. weberi) covered the diameter of the Petri dishes between the third and the fourth day. Therefore, we selected the fourth day as the best time to measure growth radius at 20, 25, and 30 °C.

Evaluation of colony growth on the eight agar media tested (Fig. 1) demonstrated that some media did not yield informative colony characters while some species exhibited similar morphological traits on different media. For instance, growth on all media resulted in a similar morphology for E. microspora and E. weberi. Likewise, on MA2 % colonies showed similar growth patterns to those on CYA, MEA, OA, as well as on PDA (E. moelleri), on CYA (E. aspergilloides), and on PCA and PDA (E. clavata, E. lentecrescens, and E. multiformis). By contrast, none of the isolates grew well on SNA (all strains exhibited inconspicuous aerial mycelia, and the morphological patterns were difficult to observe). Oatmeal Agar was also not ideal because the growth was difficult to measure due to the medium’s opacity. On the other hand, except for E. aspergilloides, all isolates exhibited vigorous growth and notable expression of colony colour on MEA. Differences in colony growth rate were most apparent on CMD, and, on this medium, some species (e.g., E. microspora and E. weberi) produced pustule-like hyphal structures on which conidiophores were often produced, similar to the sporulating tufts produced by Trichoderma. Based on these results, we selected CMD, MEA, and PDA as the most suitable media to evaluate macroscopic features of Escovopsis species. These media are also used for the culture assessments of many other genera in the Hypocreaceae, which facilitates comparisons across the family.

Colonies of most Escovopsis species grew better (colonies develop faster and form more mycelium and conidia) at 25 °C on CMD, MEA, and PDA (Fig. 2), and the differences in macroscopic characters, especially colony colours, were most apparent on the seventh day. For instance, White (LIII73(10)) to Colonial Buff (XXX21′′d) and Light Brownish Olive (XXX19′′k) (E. clavata, E. lentecrescens, E. microspora, E. moelleri, E. multiformis, and E. weberi), and Light Yellow-Green (VI31d) to *Olive-Yellow (XXX23′′) colours (E. aspergilloides) were clearly distinguishable on the seventh day. Although less clear, other colours [Ecru-Olive (XXX21′′i), *Vinaceous-Cinnamon (XXIX13′′b), and Deep Colonial Buff (XXX21′′b)] were observed on colonies of E. weberi and E. microspora on PDA and MEA. Between the seventh and tenth day, most species became Light Brownish Olive (XXX19′′k) on the three media, thus, the distinction between them became less apparent (except for E. lentecrescens). After the 10^th day, the aerial mycelium of all species started to deteriorate (collapse). Therefore, 7 d of growth at 25 °C appears optimal to evaluate the macroscopic characters of Escovopsis species. Supplementary Table S6 shows the conditions, adopted in this study, for macroscopic characters evaluation of the colonies.

The most informative microscopic structures for distinguishing Escovopsis species are: conidiophores, conidiophore branching, presence and shape of conidiophore swollen cells, vesicles, phialides, conidia, and chlamydospores. A complete micromorphological description should consider the following: (i) Conidiophores: The number of vesicles (mono-vesiculate, poly-vesiculate conidiophore), length, stipe (length, septum, distance of septum from the foot cell), shape (pyramidal, irregular, etc.), cell wall (smooth or rough), arrangement (alternate, opposite, in verticils, etc.); (ii) Conidiophore branches: length, arrangement, shape, levels of branching, stipe; (iii) Vesicle: length and width, shapes, presence or absence of septum, stipe; (vi) Phialide: where is it formed (vesicles, aerial mycelium), shape, total length and dimensions (length and width) of the base, swollen section and neck; (v) Conidia: formed in chains or solitary, shape, length, width, colour (individually and in mass), presence or absence of ornamentation; (vi) Chlamydospores: where is it formed (on aerial or submerged mycelium), arrangement (intercalary or terminal), shape, colour, length and width. Supplementary Table S7 shows the characters and parameters, selected in this study, for the evaluation of the microscopic morphology.

Escovopsis phylogeny

The analysis resulting from the combination of the five molecular markers showed that the 138 Escovopsis isolates distributed among 19 well supported monophyletic groups across the genus phylogeny (Fig. 3). Six out of the 19 clades correspond to the previously described species: Escovopsis aspergilloides, E. clavata, E. lentecrescens, E. moelleri, E. multiformis, and E. weberi. The ex-type strain of E. microspora was placed in the same clade with strains of E. weberi [Posterior Probability (PP) = 1; Maximum likelihood bootstrap (MLB) = 100 %, Fig. 3]. The remaining 13 clades correspond to the new species described in this study, i.e., 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, (Fig. 3).

In total, five major clades can be differentiated in the multilocus phylogeny of Escovopsis (Fig. 3). Clade I comprises E. breviramosa, E. gracilis, E. peniculiformis, and E. weberi. Clade II comprises E. chlamydosporosa and E. rectangula. Clade III comprises by E. elongatistipitata, E. moelleri, E. phialicopiosa, E. pseudocylindrica, and E. spicaticlavata. Clade IV comprises E. aspergilloides, E. diminuta, E. maculosa, E. lentecrescens, and E. rosisimilis. Lastly, clade V comprises E. clavata, E. multiformis, and E. papillata. Species in Clades I, II and III, grow faster and over wider temperature ranges, and form mostly cylindrical vesicles, while species in clades IV and V grow slowly, at narrow temperature ranges, and form globose vesicles (Figs 2, ​,33).

In the analyses performed with the molecular markers separately, we observed that the phylogenetic placement of the 19 Escovopsis species may vary depending on the marker used (Supplementary Fig. S1). The rpb2 and tef1 genes, for example, showed each of the 19 Escovopsis species forming well-supported monophyletic clades (Supplementary Fig. S1A, B). The topology of these trees differs slightly from that of the tree with all the markers combined (Figs 3, S1F). In the rpb2 gene tree, E. chlamydosporosa and E. rectangula do not share the same common ancestor (Fig. S1A). In the case of the tef1 gene, E. breviramosa is more closely related to E. weberi than to E. peniculiformis; E. papillata is more closely related to clade IV, and E. multiformis and E. clavata do not share the same common ancestor, as seen in the tree based on the five markers (Fig. S1B). On the other hand, most of the species form well-supported monophyletic groups in the trees reconstructed with the ITS or rpb1 markers (Fig. S1C, D). However, the clades E. breviramosa, E. chlamydosporosa (ITS tree, Fig. S1C), and E. phialicopiosa (rpb1 tree, Fig. S1D) were unresolved. Likewise, in the rpb1 tree, E. peniculiformis is placed within E. weberi (Fig. S1D). Finally, the 28S marker was the one with the lowest resolution to separate Escovopsis species (Fig. S1E). While E. breviramosa, E. clavata, E. lentecrescens, E. maculosa, E. lower whiskers the lowest and the highest values, respectively (without considering outliers). Boxes marked with the same letter indicate species for which the mean growth rates did not differ significantly according to the Duncan’s Multiple Range Test. multiformis, E. papillata, E. rectangula, and E. rosisimilis formed well-supported monophyletic groups, their phylogenetic placement, as well as the monophyly of the other species, are not well-resolved (Fig. S1E).

Morphological diversity Escovopsis

While species in the five main clades observed in the Escovopsis phylogeny share some morphological characters, each also has unique character states that differentiate them from one another (Fig. 3). Clades I and II form pyramidal conidiophores mostly producing cylindrical vesicles and smooth-walled conidia. However, species in Clade I (i.e., E. breviramosa, E. gracilis, and E. peniculiformis) usually have septate vesicles and phialides produced both on vesicles and aerial mycelium (although the latter is less frequent). In contrast, species in Clade II have shorter and less frequently septate vesicles without phialides formed in the aerial mycelium. In addition, some species in Clade II (e.g., E. chlamydosporosa), usually form chlamydospores which are rare in species in Clade I.

In contrast, species in Clades III, IV and V form irregular-shaped conidiophores, differing among each other in the type of vesicle and conidia. Species in Clade III form clavate, cymbiform, subulate, and lanceolate vesicles, and conidia with thickened walls and ornamentation, except for E. phialicopiosa, in which conidia have inconspicuous ornamentation. Species in Clade IV form globose, subglobose, and capitate vesicles, and smooth-walled conidia. Species in this clade are usually slow growing, with the striking example provided by E. lentecrescens, the slowest growing species in the genus.

Finally, distinct from all other clades, species in Clade V have the most variable vesicle shapes, ranging from globose, subglobose, capitate, obovoid, prolate, spatulate, clavate, cymbiform, lanceolate to subulate. Also, in contrast to species in other clades, most species in Clade V can only grow between 20 and 25 °C, except for some isolates of E. multiformis that can also grow at 10 and 30 °C.

Taxonomy

Our taxonomic protocol is applied below to provide standardised morphological descriptions of the know species of Escovopsis, excluding the five species described by Marfetán et al. (2019) for which no cultures were available. We re-described the extype cultures of E. aspergilloides, E. clavata, E. lentecrescens, E. moelleri, E. multiformis, and E. weberi (Figs 4, ​,7,7, ​,11,11, ​,13,13, ​,14,14, ​,22).22). The re-description of these species provided the basis for the morphological analysis of the genus. Furthermore, based on the similarity of their sequence and morphological characters, we synonymize E. microspora with E. weberi. Finally, we describe 13 new species obtained from our field work in Argentina, Brazil, Costa Rica, Mexico, and Panama.

Escovopsis aspergilloides K.A. Seifert et al., Mycologia 87: 408. 1995. MycoBank MB 413060. Fig. 4.

Diagnosis: Escovopsis aspergilloides forms colonies with diffuse pale-yellow to yellowish-brown colours and conidiophores with globose vesicles.

Typus: Trinidad and Tobago, near ASA Wright Nature Centre, isolated from a nest of Trachymyrmex ruthae (collected 15–20 cm below the soil surface in a wet, dense, secondary tropical rainforest by T. R. Schultz, 9 Nov. 1992), in Ithaca, New York by I.H. Chapela, no. 92110905C [holotype DAOM 216382 (dried agar culture), ex-type culture CBS 423.93].

Description: Conidiophores forming 2–15 vesicles, hyaline, irregular shape, smooth-walled, alternate or opposite, formed on aerial hyphae. Mono-vesiculate conidiophores 40–72 μm, polyvesiculate 80–300 μm long. Conidiophore stipes 24–100 × 5–6 μm, with a septum 4–6 μm from the foot cell. Conidiophore branches 32–160 μm long, formed in one to three levels, in almost right angles, alternate. Second branching level usually longer than other branching levels. Stipes on branches 10–49 μm long, with a septum 2–4 μm from conidiophore axis. Vesicles of various shapes, i.e., globose, sub-globose, capitate, obovoid, prolate and spatulate, 13–29 × 10–24 μm, aseptate, formed on the tips of conidiophore and branches. Vesicle stipe 10–40 μm long, with one or two septa. Phialides formed on vesicles, 6–10 μm long, ampulliform, 1–2 × 0.5–1.5 μm at the base, 3.5–4 × 2–3 μm at the swollen section and 4 × 2 μm at the neck. Conidia formed in chains, globose to oblong, 2.5–3 × 2–2.5 μm, Olive-Ochre (XXX21′′), with smooth and slightly thickened walls. Chlamydospores intercalary, hyaline, 11–22 × 8–14 μm.

Culture characteristics: Colonies growing at 20 and 25 °C on CMD, PDA, and MEA. At 20 °C, growth starts on second day on PDA, on third day on MEA, and on fourth day on CMD. At 25 °C, growth starts on second day on MEA and PDA, and on third day on CMD. Colony radius after 4 d at 20 °C: 0–1 mm on CMD, 2–5 mm on MEA and 5–8 mm on PDA; at 25 °C: 1–3 mm on CMD, 5–7 mm on MEA and 5–10 mm on PDA. Colony morphology — CMD 25 °C, 7 d: colonies with diffuse aerial mycelium, spread by stolons, Light Yellow-Green (VI31d) to Olive-Ochre (XXX21′′). MEA 25 °C, 7 d: colonies with submerged mycelium forming dense circular zones, diffuse aerial mycelium forming concentric rings, White (LIII73(10)) to Picnic Yellow (IV23d) and *Olive-Yellow (XXX23′′) (*Olive-Yellow (XXX23′′) at centre and White (LIII73(10)) at margin). PDA 25 °C, 7 d: colonies with abundant aerial mycelium, spread by stolons, Picnic Yellow (IV23d) to *Olive-Yellow (XXX23′′) and Light Yellow-Green (VI31d) to Colonial buff (XXX21′′d). Pustule-like structures and soluble pigments absent.

Ecology: Unknown, but this species has only been found in a nest of Trachymyrmex ruthae in a rain forest. Distribution: Trinidad.

Notes: Escovopsis aspergilloides is closely related to E. maculosa. However, E. aspergilloides grows slower and forms longer and more branched conidiophores. Unlike E. maculosa, which has mainly globose vesicles, E. aspergilloides forms vesicles of various shapes, i.e., globose, sub-globose, capitate, obovoid, prolate and spathulate.

Escovopsis breviramosa Q.V. Montoya, M.J.S. Martiarena & A. Rodrigues, sp. nov. Mycobank MB 847805. Fig. 5.

Etymology: “breviramosa” (brevi = short, ramose = branches) in reference to the short branches formed on the conidiophores of this species.

Diagnosis: Escovopsis breviramosa frequently has mono-vesiculate conidiophores and their polyvesiculate conidiophores have branches comprised mainly of a sessile vesicle.

Typus: Brazil, Bahia, Camacan, Serra Bonita, 15°23’43.0’’S, 39°33’49.1’’W, fungus garden of Acromyrmex sp., May 2015, A. Rodrigues, LESF 055 (holotype CBS 149741 preserved as metabolically inactive culture, extype culture CBS 149741).

Description: Conidiophores forming 2–33 vesicles, hyaline, usually pyramidal, smooth-walled, alternate or less frequent opposite, formed on aerial hyphae. Mono-vesiculate conidiophores 17–55 μm, polyvesiculate 36–430 μm long. Conidiophore stipes 3.5–130 μm × 3.5–5 μm, with a septum 1.5–28.5 μm from the foot cell. Conidiophore branches 20.5–190 μm long, formed in one or three levels, at almost right angles, alternate. Stipes on branches 1–24.5 μm long, with a septum 0.5–13 μm from conidiophore axis. Vesicles cylindrical, 29.5–60 × 3.5–9.5 μm, predominantly aseptate, less frequently septate (1–2 septa), formed on conidiophore axis or on the axis of branches. Vesicle stipe 1–6 μm long, with one septum, rarely with two septa. Phialides predominantly formed on vesicles, less frequently on aerial mycelia, 4.5–12 μm long, lageniform, 0–1.5 × 0.5–2 μm at the base, 2.5–4 × 2–3 μm at the swollen section and 1–9.5 × 0.5 μm at the neck. Conidia formed in chains, subglobose, 1–3 × 1–2 μm, Olive-Ochre (XXX21′′), with smooth and thick wall. Chlamydospores absent.

Culture characteristics: Colonies growing at 10, 20, 25, and 30 °C on CMD, PDA, and MEA. At 10 °C, growth starts between the first and second day, and at 20, 25, and 30 °C growth starts at the first day, on all media. Colony radius, after 4 d at 10 °C: Inconspicuous growth (the colony barely grows on the inoculum); at 20 °C: 7–12 mm on CMD, 10–40 mm on MEA and 40 mm on PDA; at 25 °C: 15–18 mm on CMD and 40 mm on MEA and PDA; at 30 °C: 15–18 mm on CMD, 15–33 mm on MEA and 40 mm on PDA. Colony morphology — CMD 25 °C, 7 d: Colonies with diffuse aerial mycelium, spread by stolons, abundant pustule-like formations, White (LIII73(10)) to Olive-Ochre (XXX21′′). MEA 25 °C, 7 d: Colonies with aerial mycelium at centre, dense submerged mycelium at margin, pustule-like formations, White (LIII73(10)) to Olive-Ochre (XXX21′′). PDA 25 °C, 7 d: Colonies with abundant aerial mycelium, spread by stolons, without pustule-like formations, White (LIII73(10)) to Olive-Ochre (XXX21′′) (White (LIII73(10)) at centre and Olive-Ochre (XXX21′′) at margin. Soluble pigments absent.

Ecology: Unknown.

Distribution: This species is found in different regions in Brazil and Panama in fungus gardens of the attine ant genera Atta, Acromyrmex, Apterostigma, and Mycetomoellerius.

Additional materials examined: Brazil, Rio Grande do Sul, Nova Petrópolis, grape orchard, 29°22’38.2’’S, 50°57’18.1’’W, fungus garden of Acromyrmex ambiguus, Jun. 2004, A. Rodrigues, LESF 039; São Paulo, Rio Claro, São Paulo State University (UNESP), 22°23’46.0”S, 47°32’43.2”W, fungus garden of Mycetomoellerius sp., unknown date, A. Rodrigues, LESF 316.

Notes: Escovopsis breviramosa is closely related to E. gracilis and E. peniculiformis. Unlike strains of E. peniculiformis, E. breviramosa grows at 10 °C. Conidiophores of E. breviramosa are usually shorter, more branched, and have shorter and broader terminal vesicles than those of E. peniculiformis. Unlike E. gracilis, strains of E. breviramosa grow at 10 and 30 °C. In addition, E. breviramosa forms wider and branched conidiophores, and wider and longer vesicles than E. gracilis.

Escovopsis chlamydosporosa Q.V. Montoya, M.J.S. Martiarena & A. Rodrigues, sp. nov. MycoBank MB 847806. Fig. 6.

Etymology: “chlamydosporosa” (osa = Lat feminine indicating abundance) in reference to the abundant chlamydospores formed by isolates of this species.

Diagnosis: Escovopsis chlamydosporosa forms chlamydospores (sensuAugustin et al. 2013) more frequently and abundantly than any other known Escovopsis species; these structures are rare or absent in most species of this genus.

Typus: Brazil, Amazonas, Novo Airão, Parque Nacional de Anavilhanas, 2°31’25.6”S, 60°49’32.4”W, fungus garden of Trachymyrmex sp. sensu lato, Jan. 2017, Q.V. Montoya, LESF 984 (holotype CBS 149748 preserved as metabolically inactive culture, ex-type culture CBS 149748).

Description: Conidiophores forming 2–26 vesicles, scarce, thin, hyaline, irregular shaped, smooth-walled, alternate, formed on aerial hyphae. Mono-vesiculate conidiophores 1.5–65.5 μm, polyvesiculate 45.5–380 μm long. Conidiophore stipes 4–170 μm × 3.5–5.5 μm, with a septum 0–6.5 μm from the foot cell. Conidiophore branches 16.5–97.5 μm long, formed in in one or two levels, usually at right angles, alternate. Stipes on branches 1–17.5 μm long, with a septum 1–9.5 μm from conidiophore axis. Vesicles cylindrical, 20.5–84.5 × 4–8 μm, predominantly aseptate, less frequently septate (one septum), formed on conidiophore axis or on the axis of branches. Vesicle stipe1–17 μm long, aseptate. Phialides formed on vesicles, 5.5–10 μm long, lageniform, 0.5–

1.5 × 0.5–1.5 μm at the base, 2–3.5 × 1.5–2.5 μm at the swollen section, 1.5–5.5 × 0.5 μm at the neck. Conidia formed in chains, subglobose, 1–4 × 1–3 μm, Olive-Ochre (XXX21′′), with smooth and thick wall. Chlamydospores very common, 10–17 × 8–16 μm, formed in chains on aerial mycelium, intercalary.

Culture characteristics: Colonies growing at 20, 25, and 30 °C on CMD, PDA, and MEA. Growth starts on the first day at all temperatures, on all media. Colony radius, after 4 d at 20 °C: 9–14 mm on CMD, 16–20 mm on MEA and 30–40 mm on PDA; at 25 °C: 13–15 mm on CMD, 32–40 mm on MEA and 40 mm on PDA; at 30 °C: 10–15 mm on CMD, 36–20 mm on MEA and 30–40 mm on PDA. Colony morphology — CMD 25 °C, 7 d: Colonies with scattered aerial mycelium, spread by stolons, White (LIII73(10)) to Margerite Yellow (XXX23′′f). MEA 25 °C, 7 d: Colonies with cottony aerial mycelium, White (LIII73(10)) to Margerite Yellow (XXX23′′f). PDA 25 °C, 7 d: Colonies with abundant cottony aerial mycelium, spread by stolons, White (LIII73(10)) to Colonial Buff (XXX21′′d). White (LIII73(10)) to Colonial Buff (XXX21′′d) pustule-like formations only on PDA. Soluble pigments absent.

Ecology: Unknown.

Distribution: This species is found in Novo Airão, Amazonas, Brazil in fungus gardens of the attine ant genera Acromyrmex, Apterostigma, and Trachymyrmex.

Additional materials examined: Brazil, Amazonas, Novo Airão, Parque Nacional de Anavilhanas, 2°16’15.7’’S, 61°01’8.46’’W, fungus garden of Acromyrmex sp., 24 Jan. 2017, Q.V. Montoya, LESF 961, ibid., LESF 963; Amazonas, Novo Airão, Parque Nacional de Anavilhanas, 2°31’26.04’’S, 60°49’31.62’’W, fungus garden of Trachymyrmex sp., 20 Jan. 2017, Q.V. Montoya, LESF 991; Amazonas, Novo Airão, Parque Nacional de Anavilhanas, 2°26’52.56”S, 59°45’53.4”W, fungus garden of Trachymyrmex sp., 9 Mar. 2017, Q.V. Montoya, LESF 1026.

Notes: Escovopsis chlamydosporosa is closely related to E. rectangula. Unlike strains of E. rectangula, E. chlamydosporosa does not grow at 10 °C. Furthermore, its conidiophores are usually longer, more branched, and irregularly shaped, compared to of E. rectangula, which are shorter and slightly rectangular.

Escovopsis clavata Q.V. Montoya et al., Myco*keys 46: 102. 2019. MycoBank MB 828328. Fig. 7.

Diagnosis: Escovopsis clavata usually forms conidiophores with swollen cells and with terminal sterile hypha (conidiophore apex does not end in a vesicle).

Typus: Brazil, Santa Catarina, Florianópolis, 27°44’39.6’’S, 48°31’10.14’’W, elev. 46 m, fungus garden of Apterostigma sp., Aug. 2015, A. Rodrigues, LESF 853 (holotype CBS H-23845 dried culture, ex-type culture CBS 145326).

Description: Conidiophores forming 2–8 vesicles, sometimes with swollen cells, hyaline, irregular shape, smooth-walled, alternate or opposite, formed on aerial hyphae. Mono-vesiculate conidiophores 10–50 μm, polyvesiculate up to 780 μm long. Conidiophore stipes 10–40 μm × 5–8 μm, with a septum 2–9 μm from the foot cell. Conidiophore axis usually ends in a vesicle, sometimes in an infertile hypha and less frequently in a terminal swollen cell 10–18 × 7–9 μm. Conidiophore branches 16–138 μm long (usually shorter, sometimes as long as conidiophore axis), formed in one or two levels, usually at right angles and sometimes slightly curved up or down, alternate or opposite. Swollen cells form 2–4 branches, 28–35 μm long, mostly curved upward or less frequently at right angles. Stipes on branches 9–38 μm long, with a septum 2–6 μm from conidiophore axis. Vesicles of various shapes, i.e., globose, sub-globose, capitate, obovoid, prolate, spatulate, predominantly clavate, cymbiform, and cylindric, 9–27 × 7–20 μm, aseptate, formed on the tips of conidiophore and branches. Vesicle stipe 10–30 μm long, with two or six septa. Phialides formed on vesicles, 5–8 μm long, lageniform 0.5–1.5 × 0.5–1 μm at the base, 1.5–2.5 × 1–3 μm at the swollen section, 1.5–4 × 0.5 μm at the neck. Conidia formed in chains, ellipsoidal to oblong, 1.5–2.5 × 0.5–1.5 μm, Olive-Ochre (XXX21′′), with smooth and slightly thick walls. Chlamydospores absent.

Culture characteristics: Colonies growing at 20 and 25 °C on CMD, PDA, and MEA. At both temperatures, growth starts on the third day on all media. Colony radius, after 4 d at 20 °C: 0–3 mm on CMD, 1–4 mm on MEA and 3–5 mm on PDA; at 25 °C: 2–8 mm on CMD, 4–6 mm on MEA and 6–11 mm on PDA. Colony morphology — CMD 25 °C, 7 d: Colonies with diffuse aerial mycelium, Margerite Yellow (XXX23′′f) to Colonial Buff (XXX21′′d). MEA and PDA 25 °C, 7 d: Colonies with dense floccose aerial mycelium forming concentric rings, Margerite Yellow (XXX23′′f) to Colonial Buff (XXX21′′d) at centre and Margerite Yellow (XXX23′′f) at margin. Rarely forming stolons. Pustule-like formations and soluble pigments absent.

Ecology: Unknown.

Distribution: This species is found in different regions of Brazil in fungus gardens of the attine ant genus Apterostigma.

Additional materials examined: Brazil, Santa Catarina, Florianópolis, 27°44’38.94’’S, 48°31’9.3’’W, elev. 32 m, fungus garden of Apterostigma sp., Aug. 2015, A. Rodrigues, LESF 854; Santa Catarina, Florianópolis, 27°44’39.49’’S, 48°31’9.72’’W, elev. 38 m, fungus garden of Apterostigma sp., Aug. 2015, A. Rodrigues, LESF 855.

Notes: Escovopsis clavata is closely related to E. multiformis. Unlike strains of E. multiformis, which grow at 10, 20, 25 and 30 °C, E. clavata only grows at 20 and 25 °C. Conidiophores of E. clavata are usually larger and more branched than those of E. multiformis and end in a sterile elongation not observed in E. multiformis. Compared to the latter species, swollen cells are less frequent and shorter in E. clavata.

Escovopsis diminuta Q.V. Montoya, M.J.S. Martiarena & A. Rodrigues, sp. nov. MycoBank MB 847814. Fig. 8.

Etymology: “diminuta” (diminuta = reduced in size) in reference to the reduced size of the conidiophores.

Diagnosis: Escovopsis diminuta forms short and rarely branched conidiophores with globose vesicles.

Typus: Brazil, Amazonas, Novo Airão, Parque Nacional de Anavilhanas, 2°31’23.4’’S, 60°49’31.9’’W, fungus garden of Trachymyrmex sp., 20 Jan. 2017, Q.V. Montoya, LESF 969 (holotype CBS 149747 preserved as metabolically inactive culture, ex-type culture CBS 149747).

Description: Conidiophores forming 2–8 vesicles, hyaline, irregularly shaped, smooth-walled, alternate, formed on aerial hyphae. Mono-vesiculate conidiophores 22–80 μm, polyvesiculate 52–130 μm long. Conidiophore stipe 8–73 × 3.5–8.5 μm, with a septum 0–5 μm from the foot cell. Conidiophore branches 21–61 μm long, formed in one level, in almost right angles, alternate or opposite. Stipes on branches 3–30 μm long, with a septum 0.5–10 μm from conidiophore axis. Vesicles globose 17–30 × 15–30 μm, aseptate, formed on the tips of conidiophore and branches. Vesicle stipe 0.5–10 μm long, with two septa. Phialides formed on vesicles, 6–9 μm long, lageniform, 0–1 × 1–2 μm at the base, 3–5 × 2–4 μm at the swollen section and 2–4 × 0.5–1 μm at the neck. Conidia formed in chains, oblong, 2–3 × 1.5–2.5 μm, Olive-Ochre (XXX21′′, smooth and thick wall. Chlamydospores absent.

Culture characteristics: Colonies growing at 20, and 25 °C on CMD, PDA, and MEA. At 20 °C growth starts on second day, and at 25 °C on the first day. Colony radius, after 4 d at 20 °C: 4–10 mm on CMD, 3–15 mm on MEA and 12–15 mm on PDA; at 25 °C: 10–16 mm on CMD, 15–18 mm on MEA and 14–21 mm on PDA. Colony morphology — CMD 25 °C, 7 d: colonies with diffuse aerial mycelia, with pustule-like formations, White (LIII73(10)) to Colonial Buff (XXX21′′d) (Colonial buff (XXX21′′d) at centre, White (LIII73(10)) at margin). MEA 25 °C, 7 d: colonies with dense cottony aerial mycelium, few stolons, White (LIII73(10)) to Margerite Yellow (XXX23′′f), *Olive-Yellow (XXX23′′) to Olive-Ochre (XXX21′′) (*Olive-Yellow (XXX23′′) to Olive-Ochre (XXX21′′) at centre, White (LIII73(10)) to Margerite Yellow (XXX23′′f) at margin). PDA 25 °C, 7 d: colonies with dense aerial mycelium, few stolons, few pustule-like formations, White (LIII73(10)), Colonial buff (XXX21′′d), Olive-Ochre (XXX21′′) (Olive-Ochre (XXX21′′) to Colonial Buff (XXX21′′d) at centre, White (LIII73(10)) at margin). Light Yellow-Green (VI31d) soluble pigments only on MEA.

Ecology: Unknown.

Distribution: This species was found in the Amazon regions of Brazil in fungus gardens of the attine ant genera Apterostigma and Trachymyrmex.

Additional materials examined: Brazil, Amazonas, Novo Airão, Parque Nacional de Anavilhanas, 2°32’02.7’’S, 60°50’11.7’’W, fungus garden of Apterostigma sp., 19 Jan. 2017, Q.V. Montoya, LESF 996; Amazonas, Novo Airão, Parque Nacional de Anavilhanas, 2°32’1.4’’S, 60°50’0.4’’W, fungus garden of Trachymyrmex sp., 21 Jan. 2017, Q.V. Montoya, LESF 1003.

Notes: Escovopsis diminuta is closely related to E. rosisimilis and E. lentecrescens. Escovopsis diminuta grows faster than E. rosisimilis but slower than E. lentecrescens. Furthermore, E. diminuta has shorter conidiophores than E. rosisimilis and E. lentecrescens.

Escovopsis elongatistipitata Q.V. Montoya, M.J.S. Martiarena & A. Rodrigues, sp. nov. MycoBank MB 847810. Fig. 9.

Etymology: “elongatistipitata” (elongati = Latin feminine for elongate, stipitata = stipe) in reference to the elongate stipes of both the conidiophores and the conidiophore branches.

Diagnosis: Escovopsis elongatistipitata forms conidiophores and conidiophore branches with long stipes.

Typus: Brazil, Amazonas, Novo Airão, Parque Nacional de Anavilhanas, 2°31’23.4’’S, 60°49’31.9’’W, fungus garden of Trachymyrmex sp., 20 Jan. 2017, Q.V. Montoya, LESF 999, (holotype CBS 149750 preserved as metabolically inactive culture, ex-type culture LESF 999 = CBS 149750).

Description: Conidiophores forming 2–13 vesicles, hyaline, irregular shaped, smooth-walled, alternate or opposite, formed on aerial hyphae. Mono-vesiculate conidiophores rare, 54–120 μm, polyvesiculate 56.5–380 μm long. Conidiophore stipes 10.5–190 × 2–6.5 μm, with a septum 0.5–9 μm from the foot cell. Conidiophore branches 20–230 μm long, formed in one or two levels, usually at right angles, less frequently at angles less than 90°, alternate or opposite. Stipes on branches 3–220 μm long, with a septum commonly 0–2 μm and rarely 6–20 μm from conidiophore axis. Vesicles mainly cylindric, less frequently clavate, 14–74.5 × 2–7.5 μm, aseptate, formed on conidiophore axis or on the axis of branches. Vesicle stipe 0–76 μm long, with one or two septa. Phialides formed on vesicles, 4–13 μm long, lageniform, 0–1 × 1–2 μm at the base, 2–7 × 1.5–3 μm at the swollen section and 1–3.5 × 0.5–1.5 μm at the neck. Conidia formed in chains, oblong, 3–5 × 2–3.5 μm, Olive-Ochre (XXX21′′), with ornamented and thick wall. Chlamydospores absent.

Culture characteristics: Colonies growing at 20, and 25 °C on CMD, MEA, PDA. Growth starts on second day at both temperatures, on all media. Colony radius, after 4 d at 20 °C: 5–9 mm on CMD, 10–15 mm on MEA and 12–15 mm on PDA; at 25 °C: 8–12 mm on CMD, 25–30 mm on MEA and 20–23 mm on PDA. Colony morphology — CMD 25 °C, 7 d: colonies with diffuse cottony aerial mycelium, White (LIII73(10)) to Margerite Yellow (XXX23′′f). MEA 25 °C, 7 d: colonies with cottony aerial mycelium, sometimes spread by stolons, forming concentric rings, White (LIII73(10)) to Margerite Yellow (XXX23′′f) and Colonial Buff (XXX21′′d) (Colonial buff (XXX21′′d) at centre White (LIII73(10)) to Margerite Yellow (XXX23′′f) at margin). PDA 25 °C, 7 d: colonies with cottony aerial mycelium, spread by stolons, White (LIII73(10)) to Margerite Yellow (XXX23′′f). Pustule-like formations and soluble pigments absent.

Ecology: Unknown.

Distribution: This species was found in the amazon regions of Brazil in fungus gardens of the attine ant Trachymyrmex.

Additional materials examined: Brazil, Amazonas, Novo Airão, Parque Naciona de Anavilhanas, 2°16’8.7’’S, 59°27’32.32’’W, fungus garden of Trachymyrmex sp., 20 Jan. 2017, Q.V. Montoya, LESF 1021; Amazonas, Novo Airão, Parque Nacional de Anavilhanas, 2°18’52.09’’S, 60°27’29.41.02’’W, fungus garden of Trachymyrmex sp., 20 Jan. 2017, Q.V. Montoya, LESF 985.

Notes: Escovopsis elongatistipitata is closely related to E. phialicopiosa. Unlike strains of the latter species, which can grow at 30 °C on MEA and PDA and do not form concentric rings on any media, E. elongatistipitata does not grow at 30 °C and eventually forms concentric rings on MEA and PDA.

Escovopsis gracilis Q.V. Montoya, M.J.S. Martiarena & A. Rodrigues, sp. nov. MycoBank MB 847807. Fig. 10.

Etymology: “gracilis” (gracilis = thin) in reference to the narrow conidiophores and vesicles.

Diagnosis: Escovopsis gracilis forms cottony colonies with disperse narrow conidiophores and vesicles.

Typus: Brazil, Bahia, Camacan, Serra Bonita, 14°47’56.8’’S, 39°10’16.4’’W, fungus garden of Atta cephalotes, 3 Jul. 2012, A. Rodrigues, LESF 325 (holotype CBS 149743 preserved as metabolically inactive culture, extype culture CBS 149743).

Description: Conidiophores forming 2–10 vesicles, scarce, thin, hyaline, irregular shaped, smooth-walled, alternate, formed on aerial hyphae. Mono-vesiculate conidiophores 27–150 μm, polyvesiculate 78–680 μm long. Conidiophore stipes 7.5–550 μm × 3–5.5 μm, with a septum 2–58.5 μm from the foot cell. Conidiophore branches 32.5–120 μm long, formed in one level, rarely in two levels, usually at angles less than 90°, less frequently at right angles, alternate. Stipes on branches 2–90 μm long, with a septum 0–22.5 μm from conidiophore axis. Vesicles thin, cylindrical, 19.5–81 × 2.5–5.5 μm, predominantly aseptate, less frequently septate (1–2 septa), formed on conidiophore axis or on the axis of branches. Vesicle stipe 0.5–7.5 μm long, septate (1–2 septa). Phialides formed on vesicles, 5–13 μm long, lageniform, 1–2 × 0.5–2 μm at the base (sometimes base absent), 3–7.5 × 2–4 μm at the swollen cell and 0.5–7 × 0.5–1 μm at the neck. Conidia formed in chains, subglobose, 3–7 × 2.5–4.5 μm, Olive-Ochre (XXX21′′), with smooth and thick wall. Chlamydospores absent.

Culture characteristics: Colonies growing at 20, and 25 °C on CMD, PDA, and MEA. Growth starts on the first day at both temperatures, on all media. Colony radius, after 4 d at 20 °C: 32–33 mm on CMD, 33–37 mm on MEA and 40 mm on PDA; at 25 °C: 12–19 mm on CMD and 40 mm on MEA and PDA. Colony morphology — CMD 25 °C, 7 d: Colonies with scattered aerial mycelium, spread by stolons, few short White (LIII73(10)) pustule-like formations, White (LIII73(10)) to Margerite Yellow (XXX23′′f). MEA 25 °C, 7 d: Colonies with abundant cottony aerial mycelium (sometimes forming concentric rings), spread by stolons, without pustule-like formations, White (LIII73(10)) to Margerite Yellow (XXX23′′f). PDA 25 °C, 7 d: Colonies with abundant dense aerial mycelium (sometimes forming concentric rings), spread by stolons, abundant White (LIII73(10)) to Colonial Buff (XXX21′′d) pustule-like formations, White (LIII73(10)) to Colonial buff (XXX21′′d). Soluble pigments absent.

Ecology: Unknown.

Distribution: This species is found in Bahia-Brazil in fungus gardens of the attine ant Atta cephalotes.

Additional materials examined: Brazil, Bahia, Camacan, Fazenda Paris, 14°47’51.18’’S, 39°10’17.4’’W, fungus garden of Atta cephalotes, 15 Mar. 2013, A. Rodrigues, LESF 843; Bahia, Camacan, Serra Bonita, 15°23’14.82’’S, 39°33’28.38’’W, fungus garden of Atta cephalotes, 21 Feb. 2013, A. Rodrigues, LESF 844.

Notes: Escovopsis gracilis is closely related to E. breviramosa. Unlike strains of the latter species, E. gracilis does not grow at 10 or 30 °C. Conidiophores of E. gracilis are usually thinner, longer and more branched than those of E. breviramosa.

Escovopsis lentecrescens H.C. Evans & J.O. Augustin, PLoS ONE 8 (12): e82265, 5. 2013. MycoBank MB 800441. Fig. 11.

Diagnosis: Escovopsis lentecrescens has the slowest growth rate in culture among the known species of the genus.

Typus: Brazil, Minas Gerais, Viçosa, Mata do Paraíso, elev. 700 m, fungal garden of Acromyrmex subterraneus subterraneus, Apr. 2010, J.O. Augustin & H.C. Evans, AUJ9 (holotype IMI 501179, isotypes CBS 135750, DOA628 and VIC 31755).

Description: Conidiophores forming 1–10 vesicles, hyaline, of irregular shape, smooth-walled, alternate or opposite, formed on aerial hyphae. Mono-vesiculate conidiophores 36–150 μm, polyvesiculate 57–200 μm long. Conidiophore stipes 28–49 μm × 5–7 μm, with a septum 2–6 μm from the foot cell. Conidiophore branches 20–80 μm long, formed in one to three levels, in almost right angles, alternate. The second branching level is usually much longer than other branching levels. Stipes on branches 7–31 μm long, with a septum up to 9 μm from conidiophore axis. Vesicles of various shapes, i.e., predominantly globose, subglobose, spathulate, oblanceolate and cylindric, 14–27 μm × 13–27 μm, aseptate, formed on the tips of conidiophore and branches. Vesicle stipe 10–94 μm long with 1–6 septa. Phialides formed on vesicles, 6–8.5 μm long, ampulliform, 0.5–1 × 1–1.5 μm at the base, 4–5 × 2.5–3 μm at the swollen section and 2–3 × 0.5–0.6 μm at the neck. Conidia formed in chains, globose to oblong, 2–3.5 × 1.5–2 μm, Olive-Ochre (XXX21′′), with smooth and slightly thick walls. Rarely chlamydospores intercalary, hyaline, 9.5–23 × 8.5–16 μm.

Culture characteristics: Colonies growing at 20, and 25 °C on CMD, PDA, and MEA. At 20 °C, growth starts on the third day, and at 25 °C, growth starts on the second day, on all media. Colony radius, after 4 d at 20 °C: 0–2 mm on CMD, 0–1 mm on MEA and 0–1 mm on PDA; at 25 °C: 2–3 mm on CMD, 2–5 mm on MEA and 2–3 mm on PDA. Colony morphology — CMD 25 °C, 7 d: Colonies with diffuse aerial mycelium, spread by stolons, *Vinaceous-Cinnamon (XXIX13′′b) to Colonial Buff (XXX21′′d). MEA 25 °C, 7 d: Colonies with dense cottony aerial mycelium, forming concentric rings, White (LIII73(10)) to Margerite Yellow (XXX23′′f). PDA 25 °C, 7 d: Colonies with dense cottony aerial mycelium, forming concentric rings, White (LIII73(10)) to Light Brownish olive (XXX19′′k) (Colonial buff (XXX21′′d) to Light Brownish Olive (XXX19′′k) at centre and White (LIII73(10)) at margin). Rarely forming stolons. Pustule-like formations and soluble pigments absent.

Ecology: Unknown.

Distribution: This species has been found only in one region in Brazil in fungus garden of Acromyrmex subterraneus.

Notes: Escovopsis lentecrescens is closely related to E. diminuta. Unlike strains of E. diminuta, which form yellowish-brown to brown colonies, E. lentecrescens usually has white to light-brown or beige colonies. In addition, conidiophores formed by E. lentecrescens are slightly longer and more branched than those of E. diminuta.

Escovopsis maculosa Q.V. Montoya, M.J.S. Martiarena & A. Rodrigues, sp. nov. MycoBank MB 847815. Fig. 12.

Etymology: “maculosa” (maculosa = mottled, full of spots) in reference to the mottled aspect of the mycelial growth displayed by strains of this species on PDA.

Diagnosis: Escovopsis maculosa displays a mottled aspect on the base of plates containing PDA. This is caused by a dense pattern of stolons that give the appearance of spots on the reverse of the colonies.

Typus: Brazil, Amazonas, Novo Airão, Parque Nacional de Anavilhanas, 2°16’15.7’’S, 61°01’8.5’’W, fungus garden of Acromyrmex sp., 24 Jan. 2017, Q.V. Montoya, LESF 962 (holotype CBS 149746 preserved as metabolically inactive culture, ex-type culture CBS 149746).

Description: Conidiophores forming 2–14 vesicles, hyaline, irregularly shaped, smooth-walled, alternate or opposite, formed on aerial hyphae. Mono-vesiculate conidiophores 44–82 μm (less frequent), polyvesiculate 57–180 μm long. Conidiophore stipes 10–83 × 4–8 μm, with a septum 0.5–15 μm from the foot cell. Conidiophore branches 26–100 μm long, formed in one level, in almost right angles, alternate or opposite. Stipes on branches 2–75 μm long, with a septum 1–8 μm from conidiophore axis. Vesicles globose, 13–24 × 12–22 μm, aseptate, formed on the tips of conidiophore and branches. Vesicle stipe 5–70 μm long, usually with two to three septa. Phialides formed on vesicles, 5–8 μm long, lageniform, 0.5–1.5 × 0.5–2 μm at the base, 2–4.5 × 2–3.5 μm at the swollen cell and 1–3.5 × 0.5–1 μm at the neck. Conidia formed in chains, oblong, 2–4 × 1.5–2.5 μm, Olive-Ochre (XXX21′′, smooth and thick walls. Chlamydospores absent.

Culture characteristics: Colonies growing at 20, and 25 °C on CMD, PDA, and MEA. At 20 °C, growth starts on third day, on all media. At 25 °C growth starts on third day, on CMA and MEA and on first day on PDA. Colony radius, after 4 d at 20 °C: 2–4 mm on CMD, 2–5 mm on MEA and 2–7 mm on PDA; at 25 °C: 3–5 mm on CMD, 5–7 mm on MEA and 19–30 mm on PDA. Colony morphology — CMD 25 °C, 7 d: colonies with diffuse aerial mycelium, usually spread by stolons, with abundant conidia, White (LIII73(10)) to Olive-Ochre (XXX21′′). MEA 25 °C, 7 d: colonies with diffuse aerial mycelium, spread by stolons, Margerite Yellow (XXX23′′f) to Light Yellow-Green (VI31d). PDA 25 °C, 7 d: colonies with cottony aerial mycelium, abundant stolons, Margerite Yellow (XXX23′′f) to Light Yellow-Green (VI31d). colonies with a mottled aspect on the reverse of the plate on all media, but more visible on PDA. Pustule-like formations and soluble pigments absent.

Ecology: Unknown.

Distribution: This species was found in the amazon regions of Brazil in fungus garden of the attine ant genus Acromyrmex.

Notes: Escovopsis maculosa is closely related to E. aspergilloides. However, E. maculosa grows faster and forms shorter and less branched conidiophores than E. aspergilloides. Unlike E. aspergilloides, which forms vesicles of various shapes i.e., globose, sub-globose, capitate, obovoid, prolate and spatulate, E. maculosa forms mainly globose vesicles.

Escovopsis moelleri H.C. Evans & J.O. Augustin, PLoS ONE 8 (12): e82265, 4. 2013. MycoBank MB 800440. Fig. 13.

Diagnosis: Escovopsis moelleri forms mostly subulate vesicles and conidia with thickened cell walls and ornamentations.

Typus: Brazil, Minas Gerais, Viçosa, Mata do Paraíso, elev. 700 m, fungus garden of Acromyrmex subterraneus molestans Forel, Mar. 2010, J.O. Augustin & H.C. Evans, AUJ5 (holotype IMI 501176, ex-type culture CBS 135748 = DOA626 = VIC 31753). GenBank: JQ815077 (ITS); JQ855715 (28S); MT305413 (rpb1); MT305538(rpb2); JQ855712 (tef1).

Description: Conidiophores forming 2–9 vesicles, hyaline, usually pyramidal, less frequently of irregular shape, smooth-walled, mostly alternate, less frequently opposite, formed on aerial hyphae. Mono-vesiculate conidiophores, rare, 34–54 μm long, and polyvesiculate 70–230 μm long. Conidiophores stipes 2–52 μm × 6–10 μm, with 1–5 septa, first septum 2–7 μm from the foot cell. Conidiophore branches 30–89 μm long, formed in one or two levels, in almost right angles and sometimes slightly curved upward, mostly alternated, less frequent opposite. Stipes on branches 2–20 μm long, with a septum at 2–8.5 μm from conidiophore axis. Vesicles of various shapes, i.e., subulate, oblanceolate, and clavate, 22–60 μm × 5–10 μm, predominantly aseptate and rarely with one septum (clavate-septate), formed on the tips of conidiophore and branches. Vesicle stipe 1.5–17 μm long, with one or three septa. Phialides formed on vesicles, 5–7 μm long, ampulliform, 1.7–3 × 0.5–1 μm at the base, 4.4–5.6 × 3.6–4.8 μm at the swollen section and 1–1.7 × 1–2 μm at the neck. Conidia formed on phialides, predominantly solitary, less frequent in short chains, oblong-ornamented, 6–7 × 3–3.8 μm, Olive-Ochre (XXX21′′), with ornamentation and thick walls. Chlamydospores absent.

Culture characteristics: Colonies growing at 10, 20, and 25 °C on CMD, PDA and MEA. At 10 °C, growth starts between second and third day. At 20 °C, growth starts between first and second day and at 25 °C, growth starts on the first day, on all media. Colony radius, after 4 d at 10 °C: Inconspicuous growth (the colony barely grows on the inoculum); at 20 °C: 23–38 mm on CMD, 35–40 mm on MEA and 31–40 mm on PDA; at 25 °C: >40 mm on CMD, MEA and PDA (colonies reach the plate edge between the third and fourth day). Colony morphology — CMD 25 °C, 7 d: Colonies with thin aerial mycelium, spread by stolons and submerged mycelium, Margerite Yellow (XXX23′′f) to Colonial Buff (XXX21′′d). MEA 25 °C, 7 d: colonies with short aerial mycelium, mostly spread by submerged mycelium, White (LIII73(10)) to Margerite Yellow (XXX23′′f). PDA 25 °C, 7 d: Colonies with cottony aerial mycelium, spread predominantly by submerged mycelium and less by stolons, Margerite Yellow (XXX23′′f) to Olive-Ochre (XXX21′′) (Olive-Ochre (XXX21′′) at centre and Margerite Yellow (XXX23′′f) at margin). Pustule-like formations and soluble pigments absent.

Ecology: Unknown.

Distribution: This species has been found only in one region in Brazil in fungus garden of Acromyrmex subterraneus molestans.

Notes: Escovopsis moelleri is closely related to E. spicaticlavata. Unlike species of its sister clade, which form clavate vesicles, those of E. moelleri are mostly subulate. In addition, E. moelleri does not grow at 30 °C and at 25 °C its colonies grow faster than those of E. spicaticlavata.

Escovopsis multiformis Q.V. Montoya et al., Myco*keys 46: 106. 2019. MycoBank MB 828329. Fig. 14.

Diagnosis: Escovopsis multiformis is characterized by forming vesicles of multiple shapes. Swollen cells are commonly present on the conidiophores.

Typus: Brazil, Santa Catarina, Florianópolis, 27°28’11.28’’S, 48°22’39.48’’W, elev. 119 m, fungus garden of Apterostigma sp., Aug. 2015, A. Rodrigues, LESF 847 (holotype CBS H-23846, ex-type culture CBS 145327). GenBank: MH715091 (ITS); MH715105 (28S); MT305420 (rpb1); MT305545 (rpb2); MH724265 (tef1).

Description: Conidiophores forming 2–9 vesicles, sometimes with swollen cells, hyaline, usually of irregular shape, smooth-walled, alternate or opposite, formed on aerial hyphae. Mono-vesiculate conidiophores 66–130 μm, and polyvesiculate 82–290 μm long. Conidiophore stipes 16–56 μm × 7–9 μm, with 1–3 septa, first septum 1–2 μm from the foot cell. Conidiophore axis usually ends in a vesicle, and sometimes in a swollen cell 16–34 μm × 9–20 μm. Conidiophore branches 32–84 μm long (usually short, sometimes as long as the conidiophore axis), formed in one or three branching levels, usually at right angles and sometimes slightly curved upward, alternate. Swollen cells form 2–6 branches, 28–35 μm long, mostly curved upward, less frequently at right angles. Swollen-cell branch usually ends in a vesicle but sometimes forms an additional swollen cell with 2–4 new branches. Stipes on branches 22–70 μm long, with a septum at 1–2 μm from conidiophore axis. Vesicles of various shapes, i.e., globose, predominantly subglobose, capitate, obovoid, prolate, spatulate, cymbiform, and cylindric, 12–27 × 9–17 μm, aseptate, formed on the tips of conidiophore and branches. Vesicle stipe 22–70 μm long, with one or four septa. Phialides formed on vesicles, 6–10 μm long, lageniform, 1–2.5 × 0.5–1μm at the base, 2.5–4.5 × 2–3.5 μm at the swollen section, 1–4.5 × 0.5–1 μm at the neck. Conidia formed in chains, globose to oblong, 2.5–3.5 μm × 1.5–2.5 μm, Olive-Ochre (XXX21′′), with smooth and slightly thick wall. Chlamydospores absent.

Culture characteristics: Colonies growing at 10 °C on PDA and MEA, and at 20, 25 and 30 °C on CMD, PDA and MEA. At 10 °C growth starts between the second and third day on PDA and MEA and after fourth day on CMD. At 20 °C and 30 °C, growth starts on second day on CMD and on third day on MEA and PDA. At 25 °C, growth starts on second day on all media. Colony radius, after 4 d at 10 °C: Inconspicuous growth (the colony barely grows on the inoculum); at 20 °C: 4–10 mm on CMD, 3–6 mm on MEA and 2–5 mm on PDA; at 25 °C: 6–10 mm on CMD, 4–7 mm on MEA and 4–7 mm on PDA; at 30 °C: 4–7 mm on CMD, mm on 0–3 MEA and 0–2 mm on PDA. Colony morphology — CMD 25 °C, 7 d: Colonies with diffuse aerial mycelium, spread by stolons, submerged mycelium forming dense circular zones, pustule-like formations, Margerite Yellow (XXX23′′f) to Colonial buff (XXX21′′d). MEA 25 °C, 7 d: Colonies with dense cottony mycelium, White (LIII73(10)) to Margerite Yellow (XXX23′′f), forming Margerite Yellow (XXX23′′f) exudates. PDA 25 °C, 7 d: Colonies with raised cottony mycelium, White (LIII73(10)) to Olive-Ochre (XXX21′′) colours (Olive-Ochre (XXX21′′) at centre and White (LIII73(10)) at margin). Rarely forming stolons on MEA and PDA. Soluble pigments absent.

Ecology: Unknown.

Distribution: This species is found in different regions in Brazil and Panama in fungus gardens of the attine genus Apterostigma.

Additional material examined: Brazil, Mato Grosso, Cotriguaçu, 09°49’22.74’’S, 58°15’32.04’’W, elev. 252 m, fungus garden of Apterostigma sp., Oct. 2017. Q.V. Montoya, LESF 1136.

Notes: Escovopsis multiformis is closely related to E. clavata. Unlike strains of E. clavata, which grow only at 20 and 25 °C, E. multiformis also grows at 10 and 30 °C. Conidiophores of E. multiformis are usually shorter and less branched than those of E. clavata, and frequently with swollen cells more frequently, which are larger than those of E. clavata.

Escovopsis papillata Q.V. Montoya, M.J.S. Martiarena & A. Rodrigues, sp. nov. MycoBank MB 847816. Fig. 15.

Etymology: “papillata” (papillata = shaped like a nipple) in reference to the nippled aspect of some immature vesicles before they form phialides.

Diagnosis: Escovopsis papillata usually has some vesicles that have a papilla at the terminal part (nipple-shaped). This is more common on immature vesicles when these start to form the phialides.

Typus: Brazil, Amazonas, Novo Airão, Parque Nacional de Anavilhanas, 2°31’25.8’’S, 60°49’28.62’’W, fungus garden of Apterostigma sp., 23 Jan. 2017, Q.V. Montoya, LESF 960 (holotype CBS 149745 preserved as metabolically inactive culture, ex-type culture CBS 149745). GenBank: OQ589840 (ITS); OQ589790 (28S); OQ596413 (rpb1); OQ603883 (rpb2); OQ603933 (tef1).

Description: Conidiophores forming 2–7 vesicles, hyaline, irregularly shaped, smooth-walled, mostly alternate and less opposite, formed on aerial hyphae. Mono-vesiculate conidiophores 6.5–116 μm, polyvesiculate 45–170 μm long. Conidiophore stipes 7–76 × 3–7 μm, with a septum 0.5–13 μm from the foot cell. Conidiophore branches 25–72 μm long, formed in one level, at right and less than 90° angles, commonly opposite and less frequently alternate. Stipes on branches 9–84 μm long, with a septum 0–4 μm from conidiophore axis. Vesicles mostly obovoid, 18–59 × 15–31 μm, aseptate, formed on the tips of conidiophore and branches. Vesicle stipe 6.5–115 μm long with two to three septa. Phialides formed on vesicles, 5–8 μm long, lageniform, 0.5–2 × 1–2 μm at the base, 2.5–5 × 1.5–3 μm at the swollen cell and 2–3 × 0.5–1 μm at the neck. Conidia formed in chains, oblong, 2–5 × 1.5–2.5 μm, Olive-Ochre (XXX21′′, smooth and thick wall. Chlamydospores absent.

Culture characteristics: Colonies growing at 20, and 25 °C on CMD, PDA, and MEA. At 20 °C, growth starts on the third day, and between the second and third day at 25 °C. Colony radius, after 4 d at 20 °C: 2–5 mm on CMD, 1–2 mm on MEA and 5–10 mm on PDA; at 25 °C: 1–4 mm on CMD, 2–4 mm on MEA and 5–10 mm on PDA. Colony morphology — CMD 25 °C, 7 d: colonies with diffuse aerial mycelium, White (LIII73(10)) to Margerite Yellow (XXX23′′f). MEA 25 °C, 7 d: colonies with dense aerial mycelium, few stolons, Margerite Yellow (XXX23′′f) to Light Yellow-Green (VI31d). PDA 25 °C, 7 d: colonies with diffuse fluffy aerial mycelium, few stolons, White (LIII73(10)) and Margerite Yellow (XXX23′′f) to Colonial Buff (XXX21′′d) (Colonial buff (XXX21′′d) at centre, White (LIII73(10)) and Margerite Yellow (XXX23′′f) at margin). Pustule-like formations and pigments absent.

Ecology: Unknown.

Distribution: This species was found in the Amazon regions of Brazil in fungus gardens of the attine ant genus Apterostigma.

Additional material examined: Brazil, Amazonas, Novo Airão, Parque Nacional de Anavilhanas, 2°31’25.8’’S, 60°49’28.62’’W, fungus garden of Apterostigma sp., 20 Jan. 2017, Q.V. Montoya, LESF 959.

Notes: Escovopsis papillata is closely related to E. clavata and E. multiformis. Escovopsis papillata grows slower than E. clavata and E. multiformis and does not grow at 30 °C, as is also the case for some strains of E. multiformis. In addition, unlike strains of E. clavata and E. multiformis, which form conidiophores with swollen cells, E. papillata lacks such structures on its conidiophores.

Escovopsis peniculiformis Q.V. Montoya, M.J.S. Martiarena & A. Rodrigues, sp. nov. MycoBank MB 847804. Fig. 16.

Etymology: “peniculiformis” (peniculus = duster, formis = shape) in reference to the duster shape of the conidiophores.

Diagnosis: Escovopsis peniculiformis is characterized by forming conidiophores with short branches and very long and thin vesicles at the apex of their conidiophores.

Typus: Panama, Gamboa, fungus garden of Atta colombica, 19 Jan. 2001, N.M. Gerardo, LESF 876 (holotype CBS 149744 preserved as metabolically inactive culture, ex-type culture CBS 149744). GenBank: KM817101 (ITS); OQ589724 (28S); OQ596347 (rpb1); OQ603817 (rpb2); KM817162 (tef1).

Description: Conidiophores forming 2–25 vesicles, hyaline, usually pyramidal, smooth-walled, alternate or less frequent opposite, formed on aerial hyphae. Mono-vesiculate conidiophores 9–77 μm, polyvesiculate 40–1 380 μm long. Conidiophore stipes 12– 570 × 3.5–7 μm, with a septum 1.5–29.5 μm from the foot cell. Conidiophore branches 11–86 μm long, mostly formed by long vesicles, rarely in one or two levels, in almost right angles, alternate and opposite. Stipes on branches 1–24 μm long, with a septum 1–9 μm from conidiophore axis. Vesicles cylindrical, 12–280 × 4.5–7 μm, predominantly aseptate and less frequently septate, predominantly formed on conidiophore axis, less frequently on the axis of branches. Vesicle stipe 0.5–49 μm long, with one or two septa. The terminal vesicle is usually the longest and thinnest and appear to be an extension of the conidiophore apex rather than a vesicle. Phialides formed mainly on vesicles and less frequently on the aerial mycelium, 4.5–10 μm long, lageniform, 0.5–1 × 0.5–1.5 μm at the base, 2–4 × 1.5–3 μm at the swollen section and 1–6 × 0.5–1 μm at the neck. Conidia formed in chains, ellipsoidal, 1.5–3.5 × 1–2.5 μm, Olive-Ochre (XXX21′′), with smooth and thick wall. Chlamydospores absent.

Culture characteristics: Colonies growing at 20, 25, and 30 °C on CMD, PDA, and MEA. Growth starts between the first and second day at all temperatures and on all media. Colony radius, after 4 d at 20 °C: 10–12 mm on CMD, 14–20 mm on MEA and 18–22 mm on PDA; at 25 °C: 18–30 mm on CMD, 39–40 mm on MEA and > 40 mm on PDA (colonies reach plate edge on third day); at 30 °C: 19–20 mm on CMD, 20–25 mm on MEA and 40 mm on PDA. Colony morphology — CMD 25 °C, 7 d: Colonies with submerged and diffuse aerial mycelium, spread by stolons, White (LIII73(10)) to Olive-Ochre (XXX21′′). MEA 25 °C, 7 d: Colonies with dense aerial mycelium, spread by stolons, Colonial buff (XXX21′′d) to Light Brownish olive (XXX19′′k) at centre and Light Yellow-Green (VI31d) to White (LIII73(10)) at margin. On this medium, colonies sometimes Deep Colonial Buff (XXX21′′b) and *Vinaceous-Cinnamon (XXIX13′′b), with submerged mycelium forming dense circular zones. PDA 25 °C, 7 d: Colonies forming abundant aerial mycelium, spread by stolons, White (LIII73(10)) and *Olive-Yellow (XXX23′′) to Ecru-Olive (XXX21′′i) at the centre and White (LIII73(10)) to Margerite Yellow (XXX23′′f) at margin. Commonly forming pustule-like formations. Rarely forming soluble pigments.

Ecology: Unknown.

Distribution: This species is found in Panama and Austin (Texas, USA) in fungus gardens of the attine ant genera Atta, Acromyrmex, and Apterostigma.

Additional materials examined: Panama, fungus garden of Apterostigma sp., 6 Jan. 2003, U.G. Mueller, LESF878. USA, Texas, Austin, 30°22’9.9”N; 97°47’49.8”W, elev. 157.8 m, fungus garden of fungus-growing ant, 19 Nov. 2005, U.G. Mueller, LESF 297.

Notes: Escovopsis peniculiformis is closely related to E. weberi. However, unlike strains of E. weberi, E. peniculiformis does not grow at 10 °C. Conidiophores of E. peniculiformis are usually longer and less branched than those of E. weberi and unlike strains of E. weberi (which form phialides only on vesicles), E. peniculiformis forms phialides on both the vesicles and aerial mycelium (less frequently).

Escovopsis phialicopiosa Q.V. Montoya, M.J.S. Martiarena & A. Rodrigues, sp. nov. MycoBank MB 847809. Fig. 17.

Etymology: “phialicopiosa” (phiali = phialide, copiosa = abundant) in reference to the abundant number of phialides formed by strains of this species on their vesicles.

Diagnosis: Escovopsis phialicopiosa forms conidiophores with vesicles covered with phialides to such an extent that they are difficult to observe individually.

Typus: Brazil, Minas Gerais, Uberlândia, Panga Ecological Station, 19°17’17.5”S, 48°39’40.2”W, fungus garden of Trachymyrmex sp., 22 Sep. 2008, A. Rodrigues, LESF 048 (holotype CBS 149738 preserved as metabolically inactive culture, ex-type culture CBS 149738). GenBank: KM817088 (ITS); OQ589739 (28S); OQ596362 (rpb1); OQ603832 (rpb2); KF240731 (tef1).

Description: Conidiophores forming 2–11 vesicles, hyaline, pyramidal, smooth-walled, alternate, formed on aerial hyphae. Mono-vesiculate conidiophores 14–41 μm, polyvesiculate 12–150 μm long. Conidiophore stipes 0–79 μm × 3–7.5 μm, with a septum 0–14 μm from the foot cell. Conidiophore branches 11–62 μm long, formed mostly by a vesicle, rarely with two levels, usually at right angles, alternate or opposite. Stipes on branches 0.5–6 μm long, with a septum 0–1.5 μm from conidiophore axis. Vesicles mostly prolate, 13–42 × 6–12.5 μm, aseptate, formed on conidiophore axis or on the axis of branches. Vesicle stipe 0–1 μm long, aseptate. Phialides formed on vesicles, 4.5–8 μm long, lageniform, 0.5–1 × 0.5–1.5 μm at the base, 2–3 × 1.5–2.5 μm at the swollen section and 2–4 × 0.5–1 μm at the neck. Conidia formed in chains, ellipsoidal, 1.5–4 × 1–2.5 μm, Olive-Ochre (XXX21′′), with smooth and thick wall. Chlamydospores absent.

Culture characteristics: Colonies growing at 20, 25 °C, on CMD, PDA, and MEA and only on PDA and MEA at 30 °C. Growth starts on the third day at 20, and 25 °C on CMD, and between the first and second day at all temperatures, on PDA, and MEA. Colony radius, after 4 d at 20 °C: 1–2 mm on CMD, 4–10 mm on MEA and 12–33 mm on PDA; at 25 °C: 4–7 mm on CMD, 11–25 mm on MEA and 10–25 mm on PDA; at 30 °C: 10–23 mm on MEA and 10–24 mm on PDA. Colony morphology — CMD 25 °C, 7 d: colonies with scatter aerial mycelium, few conidia, without pustule-like formation, White (LIII73(10)) to Margerite Yellow (XXX23′′f). MEA 25 °C, 7 d: colonies with dense cottony aerial mycelium, spread by stolons, few pustule-like formations, White (LIII73(10)) to Margerite Yellow (XXX23′′f) PDA 25 °C, 7 d: colonies with wispy cottony aerial mycelium, spread by stolons, pustule-like formations, White (LIII73(10)) to Olive-Ochre (XXX21′′). Soluble pigments absent.

Ecology: Unknown.

Distribution: This species is distributed in different regions in Brazil in fungus gardens of the attine ants Atta sexdens, Mycetomoellerius dichrous, and Trachymyrmex sp. sensu lato.

Additional materials examined: Brazil, Goiás, Fazenda Pau, fungus garden of Trachymyrmex sp., 8 Apr. 2008, A. Rodrigues, LESF 047; Minas Gerais, Uberlândia, Panga Ecological Station, 19°17’17.5”S, 48°39’40.2”W, fungus garden of Mycetomoellerius dichrous, 22 Sep. 2008, A. Rodrigues, LESF 106; São Paulo, Rio Claro, São Paulo State University (UNESP), fungus garden of Atta sexdens, unknown date, A. Rodrigues, LESF 021.

Notes: Escovopsis phialicopiosa is closely related to E. elongatistipitata. Unlike strains of the latter species, which do not grow at 30 °C and eventually form concentric rings on MEA and PDA, colonies of E. phialicopiosa grow at 30 °C on MEA and PDA and does not produce concentric rings on any media. Escovopsis phialicopiosa forms conidiophores with shorter stipes than those of E. elongatistipitata.

Escovopsis pseudocylindrica Q.V. Montoya, M.J.S. Martiarena & A. Rodrigues, sp. nov. MycoBank MB 847811. Fig. 18.

Etymology: “pseudocylindrica” (pseudo = false, cylindrica = Latin feminine of cylindrical) in reference to the “cylindrical” collapsed vesicles observed in old colonies of this species.

Diagnosis: Escovopsis pseudocylindrica forms conidiophores with oblong vesicles that start collapsing after 7 d, as they form phialides and conidia.

Typus: Brazil, Amazonas, Novo Airão, Parque Nacional de Anavilhanas, 2°31’29.64’’S, 60°49’28.92’’W, fungus garden of Trachymyrmex sp., 20 Jan. 2017, Q.V. Montoya, LESF 993 (holotype CBS 149749 preserved as metabolically inactive culture, ex-type culture CBS 149749). GenBank: OQ589819 (ITS); OQ589769 (28S); OQ596392 (rpb1); OQ603862 (rpb2); OQ603912 (tef1).

Description: Conidiophores forming 2–14 vesicles, hyaline, irregularly shaped, smooth-walled, alternate, less frequent opposite, formed on aerial hyphae. Mono-vesiculate conidiophores 20–36 μm, polyvesiculate 46–270 μm long. Conidiophore stipes 2–112 × 4–8 μm, with a septum 2–14 μm from the foot cell. Conidiophore branches 21–148 μm long, formed in one or two levels, at angles less than 90°, alternate or opposite. Stipes on branches 1.5–38.5 μm long, with a septum 0.5–4 μm from conidiophore axis. Vesicles mainly prolate, 9–45 × 4–12 μm, aseptate, formed on the tips of conidiophore and branches. Vesicle stipe 1–26 μm long, with one or three septa. Phialides formed on vesicles, 4–7 μm long, lageniform, 0–1.5 × 1–2 μm at the base, 2–4 × 2–3 μm at the swollen section and 1–2.5 × 0.5–1 μm at the neck. Conidia formed in chains, oblong, 2–6 × 3–4 μm, Olive-Ochre (XXX21′′), with ornamented and thick wall. Chlamydospores absent.

Culture characteristics: Colonies growing at 20, 25 °C on CMD, PDA, and MEA, and only on PDA at 30 °C. At 20 °C growth starts on the second day, at 25 °C on the first day, and at 30 °C on third day. Colony radius, after 4 d at 20 °C: 3–9 mm on CMD, 9–14 mm on MEA and 10–15 mm on PDA; at 25 °C: 10–15 mm on CMD, 16–20 mm on MEA and 26–35 mm on PDA; at 30 °C: 2–6 mm on PDA. Colony morphology — CMD 25 °C, 7 d: colonies with diffuse aerial mycelium, Margerite Yellow (XXX23′′f) and White (LIII73(10)) to Colonial Buff (XXX21′′d). MEA and PDA 25 °C, 7 d: colonies with cottony aerial mycelium, spread by stolons; White (LIII73(10)), Colonial buff (XXX21′′d), Olive-Ochre (XXX21′′) and Ecru-Olive (XXX21′′i) (Ecru-Olive (XXX21′′i) at centre, White (LIII73(10)) at margin); sometimes Light Yellow-Green (VI31d), *Olive-Yellow (XXX23′′). Pustule-like formations and soluble pigments absent.

Ecology: Unknown.

Distribution: This species was found in the amazon regions of Brazil in fungus garden of the attine ant Trachymyrmex.

Additional material examined: Brazil, Amazonas, Novo Airão, Parque Nacional de Anavilhanas, S2°16’9.3’’S, 59° 27’32.54’’W, fungus garden of Trachymyrmex sp., 24 Jan. 2017, Q.V. Montoya, QVM157, LESF 1029.

Notes: Escovopsis pseudocylindrica is closely related to E. spicaticlavata. Unlike strains of the latter species, which grow only on PDA at 30 °C, E. pseudocylindrica can grow on all media at this temperature. In addition, conidiophores of E. pseudocylindrica are more branched than those of E. spicaticlavata.

Escovopsis rectangula Q.V. Montoya, M.J.S. Martiarena & A. Rodrigues, sp. nov. MycoBank MB 847808. Fig. 19.

Etymology: “rectangula” (rectangula = right angled) in reference to the slightly rectangular shape of the conidiophores formed by strains of this species.

Diagnosis: Escovopsis rectangula forms slightly rectangular conidiophores, usually with branches formed by a long cylindrical vesicle.

Typus: Brazil, Rondônia, Fazenda São Sebastião, fungus garden of Acromyrmex sp., 7 Oct. 2018, A. Rodrigues, LESF 050 (holotype CBS 149739 preserved as metabolically inactive culture, ex-type culture CBS 149739). GenBank: KM817091 (ITS); OQ589729 (28S); OQ596352 (rpb1); OQ603822 (rpb2); KM817152 (tef1).

Description: Conidiophores forming 2–34 vesicles, hyaline, slightly rectangular shape, smooth-walled, alternately, formed on aerial hypha. Mono-vesiculate conidiophores 24–56 μm, polyvesiculate 47–250 μm long. Conidiophore stipe 2.5–72.5 μm × 4–6.5 μm, with a septum 0–12.5 μm from the foot cell. Conidiophore branches 16.5–220 μm long, formed in one or two levels, usually at right angles, alternate. Stipes on branches 1–87 μm long, with a septum 1–28 μm from conidiophore axis. Vesicles cylindrical, 20–58 × 4–9 μm, predominantly non-septate, less frequently septate (1 septum), formed on conidiophore axis or on the axis of branches. Vesicle stipe 1–12 μm long, septate (1 septum). Phialides formed on vesicles, 5–8 μm long, lageniform, 0.5–1 × 0.5–2 μm at the base, 2–3.5 × 1–3 μm at the swollen section and 1–5 × 0.5 μm at the neck. Conidia formed in chains, subglobose, 2–4 × 1.5–3 μm, Olive-Ochre (XXX21′′), with smooth and thick wall. Chlamydospores absent.

Culture characteristics: Colonies growing at 10, 20, 25, and 30 °C on CMD, PDA, and MEA. At 10 °C growth starts between second and third day, and at 20, 25, and 30 °C growth starts on the first day, on all media. Colony radius, after 4 d at 10 °C: Inconspicuous growth (the colony barely grows on the inoculum) but with few conidia production; at 20 °C: 8–11 mm on CMD, 6–24 mm on MEA and 13–35 mm on PDA; at 25 and 30 °C: 10–20 mm on CMD, 27–40 mm on MEA and 40 mm on PDA. Colony morphology — CMD 25 °C, 7 d: colonies with scattered aerial mycelium, abundant short pustule-like formations, White (LIII73(10)) to Olive-Ochre (XXX21′′) (Olive-Ochre (XXX21′′) at centre, White (LIII73(10)) at margin). MEA 25 °C, 7 d: colonies with dense cottony aerial mycelium, without pustule-like formations, White (LIII73(10)) to Margerite Yellow (XXX23′′f). PDA 25 °C, 7 d: colonies with dense cottony aerial mycelium, spread by stolons, abundant pustule-like formations, White (LIII73(10)) to Light Brownish olive (XXX19′′k) (White (LIII73(10)) at centre, Light Brownish olive (XXX19′′k) at margin forming a ring). Soluble pigments absent.

Ecology: Unknown.

Distribution: This species is found in Brazil, Mexico and Panama in fungus gardens of the attine ant genera Acromyrmex, Apterostigma, and Trachymyrmex.

Additional materials examined: Brazil, Pernambuco, Frei Caneca, fungus garden of Atta cephalotes, 21 Jan. 2004, A. Rodrigues, LESF 022; Bahia, Camacan, Santa Cruz State University (UESC), 14°47’56.8’’S, 39°10’16.4’’W, fungus garden of Atta cephalotes, 15 Mar. 2013, A. Rodrigues, LESF 326. Mexico, Guadeloupe island, fungus garden of Acromyrmex octospinosus, 24 Dec. 2003, N.M. Gerardo, LESF 865. Panama, fungus garden of Apterostigma dentigerum, 9 Jul. 2002, N.M. Gerardo, LESF 863.

Notes: Escovopsis rectangula is closely related to E. chlamydosporosa. Unlike strains of the latter species, which do not grow at 10 °C and usually form chlamydospores, E. rectangula grows at 10 °C and rarely forms these structures. Conidiophores of E. rectangula are short, less branched and have a slightly rectangular shape, while conidiophores of E. chlamydosporosa are longer, more branched, and irregularly shaped.

Escovopsis rosisimilis Q.V. Montoya, M.J.S. Martiarena & A. Rodrigues, sp. nov. MycoBank MB 847813. Fig. 20.

Etymology: “rosisimilis” (rosi = roses, similis = like) in reference to the shape of blooming roses displayed by the conidiophore aggregations on the aerial mycelium.

Diagnosis: Escovopsis rosisimilis forms clusters of short, tangled conidiophores on the aerial mycelium that resemble blooming roses.

Typus: Brazil, Minas Gerais, Uberlândia, Panga Ecological Station, 19°17’17.5”S, 48°39’40.2”W, fungus garden of Trachymyrmex sp., 20 Aug. 2008, A. Rodrigues, LESF 135 (holotype CBS 149742 preserved as metabolically inactive culture, ex-type culture CBS 149742). GenBank: KM817086 (ITS); OQ589740 (28S); OQ596363 (rpb1); OQ603833 (rpb2); KM817148 (tef1).

Description: Conidiophores forming 2–16 vesicles, hyaline, irregularly shaped, smooth-walled, alternate or opposite, formed on aerial hyphae. Mono-vesiculate conidiophores 21–80 μm, polyvesiculate 47–210 μm long. Conidiophore stipes 5–65 × 5–15 μm, with a septum 1–7 μm from the foot cell. Conidiophore branches 21–110 μm long, formed in up to two levels, in almost right angles, alternate or opposite. Stipes on branches 5–38 μm long, with a septum 1–9 μm from conidiophore axis. Vesicles globose, 13–62 × 14–58 μm, aseptate, formed on the tips of conidiophore and branches. Vesicle stipe 2–34 μm long, with two septa. Phialides formed on vesicles, 5–8 μm long, lageniform, 0.5–1 × 0.5–2 μm at the base, 2–3 × 1–3 μm at the swollen section and 1.5–3 × 0.5–1 μm at the neck. Conidia formed in chains, oblong, 1.5–3 × 1–2 μm, Olive-Ochre (XXX21′′), smooth and thick wall. Chlamydospores absent.

Culture characteristics: Colonies growing at 20, and 25 °C on CMD, PDA, and MEA. At 20 and 25 °C growth starts on the third day on CMD and MEA, and on the second day on PDA, on all media. Colony radius, after 4 d at 20 °C: 2–18 mm on CMD, 4–7 mm on MEA and 15–22 mm on PDA; at 25 °C: 5–7 mm on CMD, 5–10 mm on MEA and 20–32 mm on PDA. Colony morphology — CMD 25 °C, 7 d: colonies with diffuse aerial mycelium, Margerite Yellow (XXX23′′f) to Light Yellow-Green (VI31d). MEA 25 °C, 7 d: colonies with dense aerial mycelium, Margerite Yellow (XXX23′′f) to Picnic Yellow (IV23d) and *Olive-Yellow (XXX23′′). PDA 25 °C, 7 d: colonies with dense cottony aerial mycelium, spread by stolons, mostly White (LIII73(10)) and less frequently Light Yellow-Green (VI31d) (Light Yellow-Green (VI31d) at centre, White (LIII73(10)) at margin). Pustule-like formations and soluble pigments absent.

Ecology: Unknown.

Distribution: This species was found in Minas Gerais and in the Amazon regions of Brazil in fungus gardens of the attine ant Trachymyrmex.

Notes: Escovopsis rosisimilis is closely related to E. diminuta and E. lentecrescens. Escovopsis rosisimilis grows faster than

E. lentecrescens, but slower than E. diminuta. Furthermore, E. rosisimilis forms slightly longer and more entangled conidiophores on the aerial mycelium than E. lentecrescens and E. diminuta.

Escovopsis spicaticlavata Q.V. Montoya, M.J.S. Martiarena & A. Rodrigues, sp. nov. MycoBank MB 847812. Fig. 21.

Etymology: “spicaticlavata” (spicati = spikes, clava = club) in reference to the spiked club shape of the vesicles formed by this species.

Diagnosis: Escovopsis spicaticlavata forms irregularly shaped conidiophores with mostly prolate vesicles that resemble a spiked club because of the phialides jutting out from it.

Typus: Brazil, Amazonas, Manaus, Biological Dynamics of Forest Fragments Project (PDBFF–Camp 41), 2°26’54.84’’S, 59°46’10.02’’W, fungus garden of Paratrachymyrmex diversus, 9 Jan. 2009, A. Rodrigues, LESF 052 (holotype CBS 149740 preserved as metabolically inactive culture, ex-type culture CBS 149740). GenBank: KM817093 (ITS); MH715124 (28S); MT305437 (rpb1); MT305562 (rpb2); KM817154 (tef1).

Description: Conidiophores forming 2–15 vesicles, sometimes with swollen cells, hyaline, irregular shaped, smooth-walled, alternate or opposite, formed on aerial hyphae. Mono-vesiculate conidiophores rarely, 22–110 μm long, polyvesiculate 63.5–400 μm long. Conidiophore stipe 18.5–150 × 3–10 μm, with a septum mostly 0–5 μm and rarely 8–15 μm from the foot cell. Conidiophore axis usually ends in a vesicle, less frequently in a terminal swollen cell. Conidiophore branches 28–120 μm long, formed on conidiophore axis or on swollen cells, in one level, almost at right angles, sometimes curved upward or down, alternate or opposite. Conidiophore branches sometimes ends in a swollen cell. Swollen cells 11–25 × 9–17 μm, form up to three branches. Stipes on branches 5–38 μm long, with a septum 0–7 μm from conidiophore axis. Vesicles mainly prolate, 14.5–39 × 7–15 μm, aseptate, formed on conidiophore axis or on swollen cells. Vesicle stipe 2–69 μm long, with one or four septa. Phialides formed on vesicles, 5–9.5 μm long, lageniform, 0–2.5 × 1–2 μm at the base, 2–5.5 × 2–3 μm at the swollen section and 1–3 × 0.5–1.5 μm at the neck. Conidia formed in chains, oblong, 2–5 × 2–3.5 μm, Olive-Ochre (XXX21′′), with ornamented thick wall. Chlamydospores absent.

Culture characteristics: Colonies growing at 20, 25, and 30 °C on CMD, PDA, and MEA. At 20 °C, growth starts on the second day and at 25 and 30 °C on the first day. Colony radius, after 4 d at 20 °C: 9–14 mm on CMD, 10–30 mm on MEA and 15–28 mm on PDA; at 25 °C: 15–20 mm on CMD, 25–35 mm on MEA and 20–30 mm on PDA; at 30 °C: 15–20 mm on CMD, 25–35 mm on MEA and 20–28 mm on PDA. Colony morphology — CMD 25 °C, 7 d: colonies with diffuse aerial mycelium, White (LIII73(10)) or Margerite Yellow (XXX23′′f) to Colonial buff (XXX21′′d). MEA 25 °C, 7 d: colonies with diffuse cottony aerial mycelium, spread by stolons, White (LIII73(10)) or Margerite Yellow (XXX23′′f) to *Olive-Yellow (XXX23′′) (*Olive-Yellow (XXX23′′) at centre, White (LIII73(10)) to Margerite Yellow (XXX23′′f) at margin). PDA 25 °C, 7 d: Colonies with cottony aerial mycelium, spread by stolons, White (LIII73(10)) and Colonial Buff (XXX21′′d) (Colonial Buff (XXX21′′d) at centre, White (LIII73(10)) at margin). Pustule-like formations and soluble pigments absent.

Ecology: Unknown.

Distribution: This species was found in the Amazon regions of Brazil in fungus garden of the attine ant Trachymyrmex.

Additional materials examined: Brazil, Amazonas, Novo Airão, Parque Nacional de Anavilhanas, 2°31’25.3’’S, 60°49’33.1’’W, fungus garden of Trachymyrmex sp., 20 Jan. 2017, Q.V. Montoya, LESF 975; Amazonas, Novo Airão, Parque Nacional de Anavilhanas, fungus garden of Trachymyrmex sp., 24 Jan. 2017, Q.V. Montoya, LESF 979.

Notes: Escovopsis spicaticlavata is closely related to E. pseudocylindrica. Unlike strains of the latter species, which grow at 30 °C on all media, E. spicaticlavata grows only on PDA at this temperature. Conidiophores of E. spicaticlavata are less branched than those of E. pseudocylindrica.

Escovopsis weberi J.J. Muchovej & Della Lucia, Mycotaxon 37: 192. 1990. MycoBank MB 127786. Fig. 22.

Synonym: Escovopsis microspora H.C. Evans & J.O. Augustin, PLoS ONE 8: e82265, 4. 2013. MycoBank MB 800442. Diagnostic characters: Escovopsis weberi grows faster than other known Escovopsis species and exhibits the most variable colony colours in the genus.

Typus: Brazil, Minas Gerais, Viçosa, ant colony, 25 Mar. 1987, Della Lucia (Herbário, UFV–Universidade Federal de Viçosa) (holotype ATCC 64542 preserved as metabolically inactive culture). GenBank: KF293285 (ITS); KF293281 (28S); MT305412 (rpb1); MT305537 (rpb2); MZ170961 (tef1).

Description: Conidiophores forming 1–48 vesicles, hyaline, usually pyramidal, less frequently of irregular shape, smooth-walled, alternate or less frequent opposite, formed on aerial hyphae. Mono-vesiculate conidiophores 20–60 μm, polyvesiculate 30–570 μm long. Conidiophore stipes 45–90 × 5–7 μm, with a septum 2–5 μm from the foot cell. Conidiophore branches 34–120 μm long, formed in one or two levels, in almost right angles and sometimes slightly curved upward, mostly alternate and occasionally opposite. Stipes on branches 10–36 μm long, with a septum 1.5–3 μm from conidiophore axis. Vesicles of various shapes, i.e., cylindrical, clavate, or filiform, 21–63 × 6–12 μm, predominantly aseptate, less frequently septate (1–2 septa), formed directly on conidiophore axis or on the axis of branches. Vesicle stipe 1–12 μm long, without septa. Phialides formed on vesicles, 7–9 μm long, lageniform, 0.5– 1 × 1–1.5 μm at the base, 2.5–3 × 2.5–3 μm at the swollen section and 3–4 × 0.5–0.8 μm at the neck. Conidia formed in chains, ellipsoidal to oblong, 2.5–3 × 1.5–2.5 μm, Olive-Ochre (XXX21′′), with smooth and thick wall. Chlamydospores absent.

Culture characteristics: Colony growing at 10, 20, 25, and 30 °C on CMD, PDA, and MEA. At 10 °C, growth starts on the third day, and at 20, 25, and 30 °C, growth starts on the first day on all media. Colony radius, after 4 d at 10 °C: Inconspicuous growth (the colony barely grows on the inoculum); at 20 °C: 20–24 mm on CMD, 30– 40 mm on MEA and 40 mm on PDA; at 25 °C: 17–36 mm on CMD, 24–40 mm on MEA and > 40 mm on PDA (in this case colonies reach plate edge on third day); at 30 °C: 37–40 mm on CMD, 12–15 mm on MEA and 37–40 mm on PDA. Colony morphology — CMD 25 °C, 7 d: Colonies with diffuse aerial mycelium, spread by stolons, forming small pustule-like structures, White (LIII73(10)) to Olive-Ochre (XXX21′′). MEA 25 °C, 7 d: colonies with abundant aerial mycelium; varying in colour, usually Colonial buff (XXX21′′d) to Olive-Ochre (XXX21′′) at centre (often Picnic Yellow (IV23d) and Deep Colonial Buff (XXX21′′b) are also observed), surrounded by *Vinaceous-Cinnamon (XXIX13′′b) to Colonial buff (XXX21′′d) and Margerite Yellow (XXX23′′f) to Ecru-Olive (XXX21′′i) regions, White (LIII73(10)) to Light Yellow-Green (VI31d) at margin; submerged mycelium forming dense circular zones, with White (LIII73(10)) to Olive-Ochre (XXX21′′) pustules. PDA 25 °C, 7 d: colonies with abundant aerial mycelium spread by stolons; White (LIII73(10)) to Olive-Ochre (XXX21′′), sometimes Picnic Yellow (IV23d) to Ecru-Olive (XXX21′′i) and *Vinaceous-Cinnamon (XXIX13′′b) to Deep Colonial Buff (XXX21′′b); eventually with pustule-like formations. Occasionally forming soluble pigments.

Ecology: Some representatives of this species are opportunists in the fungus gardens of leaf-cutting ants and a few strains were reported as mycoparasites of Leucoagaricus gongylophorus, the fungal cultivar of Atta.

Distribution: Across Brazil.

Additional materials examined: Brazil, Bahia, Ilhéus, Santa Cruz State University (UESC), 14°47’56.8’’S, 39°10’16.4’’W, fungus garden of Acromryrmex balzanii, unknown collection date, A. Rodrigues, LESF 054; Mato Grosso, Alta Floresta, fungus garden of Atta cephalotes, unknown collection date, A. Rodrigues, LESF 023; Rio Grande do Sul, Chuvisca, grassland, 30°50’10.2”S, 51°55’10.4”W, fungus garden of Acromyrmex lundii, unknown collection date, A. Rodrigues, LESF 042; Rio Grande do Sul, Chuvisca, grassland, 30°50’10.2”S, 51°55’10.4”W, fungus garden of Acromyrmex heyeri, unknown collection date, A. Rodrigues, LESF 043; São Paulo, Botucatu, Fazenda Santana, 22°50’45.8’’S, 48°26’09.4’’W, fungus garden of Atta sexdens rubropilosa, unknown collection date, A. Rodrigues, LESF 019; São Paulo, Botucatu, Fazenda Santana, 22°50’46.4”S, 48°26’09.6”W, fungus garden of Atta capiguara, unknown collection date, A. Rodrigues, LESF 292; São Paulo, Corumbataí, Fazenda Corumbataí, 22°17’22’’S, 47°39’23’’W, fungus garden of Atta sexdens, unknown collection date, A. Rodrigues, LESF 031; São Paulo, Corumbataí, Fazenda Corumbataí, 22°17’21.7’’S, 47°39’22.8’’W, fungus garden of Atta sexdens rubropilosa, unknown collection date, A. Rodrigues, LESF 156; São Paulo, Thermas de Santa Bárbara, 22°49’10.6”S, 49°16’06.2”W, fungus garden of Atta laevigata, unknown collection date, A. Rodrigues, LESF 324.

Notes: Escovopsis weberi is closely related to E. peniculiformis. The conidiophores of E. weberi are usually shorter but more branched than those of E. peniculiformis. Furthermore, the vesicles of E. weberi are shorter and wider than those of E. peniculiformis. The morphological characters of E. weberi do not differentiate it from E. microspora. In addition, the ex-type strains have only one nucleotide difference in the ITS, rpb1 and rpb2 sequences, no difference in the 28S and tef1, and they form together with other isolates of E. weberi a well-supported clade. Although E. microspora was described based on the supposition that the conidial sizes differ from E. weberi, the measurements of Augustin et al. (2013) are within the range observed in the broader sampling of E. weberi examined here.

Dichotomous key to known Escovopsis species

1a. Colony growth < 20 mm at 25 °C on PDA ................................................................................................................................................. 2

1b. Colony growth > 20 mm at 25 °C on PDA ................................................................................................................................................. 8

2a. Aerial mycelium Margerite Yellow (XXX23′′f), Light Yellow-Green (VI31d) to Picnic Yellow (IV23d) on CMD ........................................... 3

2b. Aerial mycelium White (LIII73(10)) on CMD .............................................................................................................................................. 6

3a. Colonies growing at 10 °C on CMD, PDA, and MEA ............................................................................................................ E. multiformis

3b. Colonies not growing at 10 °C ................................................................................................................................................................... 4

4a. Aerial mycelium Light Yellow-Green (VI31d) to Picnic Yellow (IV23d) on CMD ................................................................ E. aspergilloides

4b. Aerial mycelium Light Brownish olive (XXX19′′k) on CMD ........................................................................................................................ 5

5a. Aerial mycelium Margerite Yellow (XXX23′′f) on CMD ................................................................................................................. E. clavata

5b. Aerial mycelium Light Yellow-Green (VI31d) on CMD ...................................................................................................... E. lentecrescens

6a. Aerial mycelium Margerite Yellow (XXX23′′f) on CMD ............................................................................................................................... 7

6b. Aerial mycelium *Olive-Yellow (XXX23′′) on CMD ..................................................................................................................... E. diminuta

7a. Aerial mycelium White (LIII73(10)) on MEA ........................................................................................................................ E. phialicopiosa

7b. Aerial mycelium Light Yellow-Green (VI31d) on MEA ................................................................................................................ E. papillata

8a. Aerial mycelium Margerite Yellow (XXX23′′f) on MEA ............................................................................................................................... 9

8b. Aerial mycelium Colonial buff (XXX21′′d) on MEA ................................................................................................................................... 16

9a. Colonies with clusters of short, tangled conidiophores on the aerial mycelium ....................................................................... E. rosisimilis

9b. Colonies without clusters of short, tangled conidiophores on the aerial mycelium .................................................................................. 10

10a. Colonies not growing at 10 °C ............................................................................................................................................................... 11

10b. Colonies growing at 10 °C on CMD, PDA, and MEA ............................................................................................................................ 14

11a. Colonies growing at 30 °C on CMD, PDA, and MEA ............................................................................................................................. 12

11b. Colonies do not grow at 30 °C on CMD, PDA, and MEA ....................................................................................................................... 13

12a. Colonies with a mottled aspect on the reverse of the plate ................................................................................................... E. maculosa

12b. Colonies with a uniform aspect on the reverse of the plate ....................................................................................................... E. gracilis

13a. Colonies forming abundant chlamydospores ............................................................................................................ E. chamydosporosa

13b. Colonies rarely forming chlamydospores ........................................................................................................................ E. spicaticlavata

14a. Aerial mycelium forming slightly rectangular conidiophores ................................................................................................. E. rectangula

14b. Aerial mycelium forming pyramidal or irregularly shaped conidiophores ............................................................................................... 15

15a. Colonies growing at 30 °C on CMD, PDA, and MEA, conidia ornamented .............................................................................. E. moelleri

15b. Colonies not growing at 30 °C on any media, conidia without ornamentations ......................................................................... E. weberi

16a. Colonies growing at 10 °C on CMD, PDA, and MEA ........................................................................................................ E. breviramosa

16b. Colonies not growing at 10 °C on CMD, PDA, and MEA ...................................................................................................................... 17

17a. Aerial mycelium white on CMD, Light Yellow-Green (VI31d) on PDA and *Olive-Yellow (XXX23′′) and Ecru-Olive (XXX21′′i) on MEA ............................................................................................................................................................................................. E. pseudocylindrica

17b. Aerial mycelium, Colonial buff (XXX21′′d) or Margerite Yellow (XXX23′′f) on CMD, Margerite Yellow (XXX23′′f) on PDA and without *Olive-Yellow (XXX23′′) and Ecru-Olive (XXX21′′i) colours on MEA .............................................................................................................. 18

18a. Colonies growing at 30 °C .............................................................................................................................................. E. peniculiformis

18b. Colonies not growing at 30 °C ..................................................................................................................................... E. elongatistipitata