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.

Isolates and their associated metadata used in the phylogenetic analysis of Escovopsis based on five gene markers (Figs 3, S1). In addition to 138 isolates of Escovopsis spp., Sympodiorosea kreiselii CBS 139320 was used as the outgroup.

^T Holotype; ^ET Ex-type cultures; $: strains used to assess the morphological characters of Escovopsis species; +: Inactive strains; LESF: Laboratory of Fungal Ecology and Systematics (UNESP, Rio Claro, Brazil); QVM: Quimi Vidaurre Montoya.

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).