Saudi Journal of Biological Sciences (2016) xxx, xxx–xxx King Saud University Saudi Journal of Biological Sciences www.ksu.edu.sa www.sciencedirect.com ORIGINAL ARTICLE Isolation of the symbiotic fungus of Acromyrmex pubescens and phylogeny of Leucoagaricus gongylophorus from leaf-cutting ants G.A. Bich a,b,*, M.L. Castrillo a,b, L.L. Villalba b, P.D. Zapata b a Microbiology and Immunology Laboratory, Misiones National University, 1375, Mariano Moreno Ave., 3300 Posadas, Misiones, Argentina b Biotechnology Institute of Misiones ‘‘Marı´a Ebe Reca”, 12 Road, km 7, 5, 3300 Misiones, Argentina Received 7 August 2015; revised 21 April 2016; accepted 10 May 2016 KEYWORDS Abstract Leaf-cutting ants live in an obligate symbiosis with a Leucoagaricus species, a basid- ITS; iomycete that serves as a food source to the larvae and queen. The aim of this work was to isolate, Leaf-cutting ants; identify and complete the phylogenetic study of Leucoagaricus gongylophorus species of Acromyr- Leucoagaricus; mex pubescens. Macroscopic and microscopic features were used to identify the fungal symbiont Phylogeny of the ants. The ITS1-5.8S-ITS2 region was used as molecular marker for the molecular identifica- tion and to evaluate the phylogeny within the Leucoagaricus genus. One fungal symbiont associated with A. pubescens was isolated and identified as L. gongylophorus. The phylogeny of Leucoagaricus obtained using the ITS molecular marker revealed three well established monophyletic groups. It was possible to recognize one clade of Leucoagaricus associated with phylogenetically derived leaf-cutting ants (Acromyrmex and Atta). A second clade of free living forms of Leucoagaricus (non-cultivated), and a third clade of Leucoagaricus associated with phylogenetically basal genera of ants were also recognized. The clades corresponded to traditional taxonomic groups, and were differentiated by ecological habitats of different species. Ó 2016 The Authors. Published by Elsevier B.V. on behalf of King Saud University This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction * Corresponding author at: Misiones National University, 1375, There is an ancient, highly evolved, mutualism between Mariano Moreno Ave., 3300 Posadas, Misiones, Argentina. Tel.: +54 fungus-growing ants (Hymenoptera: Formicidae: Attini) and (0376) 4435118; fax: +54 (0376) 4425414. their fungi (Agaricales) (Currie, 2001; Wetterer et al., 1998; E-mail address: [email protected] (G.A. Bich). Wilson, 1971). Peer review under responsibility of King Saud University. The ability to cultivate fungi occurs just in a few groups of ants, and all these ants belong to the Attini tribe which is com- posed of 12 genera and approximately 210 species (Schultz and Meier, 1995). Most of these genera of ants use dead matter to Production and hosting by Elsevier http://dx.doi.org/10.1016/j.sjbs.2016.05.010 1319-562X Ó 2016 The Authors. Published by Elsevier B.V. on behalf of King Saud University This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Bich, G.A. et al., Isolation of the symbiotic fungus of Acromyrmex pubescens and phylogeny of Leucoagaricus gongylophorus from leaf-cutting ants. Saudi Journal of Biological Sciences (2016), http://dx.doi.org/10.1016/j.sjbs.2016.05.010 2 G.A. Bich et al. cultivate their fungal symbiont except the leaf-cutting ants. of ants (Chapela et al., 1994; Lugo et al., 2013; Ortiz et al., The latter ants use fresh plant materials for manuring their 2008; Silva et al., 2004). gardens and belong to the Atta and Acromyrmex genera In Argentina there is only one initial molecular study of the (Currie, 2001). Leucoagaricus fungus (Lugo et al., 2013). These authors iso- Atta and Acromyrmex genera of ants are the most serious lated and identified both, morphologically and genetically agricultural pest of tropical and subtropical America, causing the fungi cultured by two populations of Acromyrmex lobicor- enormous economic damage to the neotropic agricultural nis from the center of Argentina. But studies on morphological industry (Wirth et al., 2003). The possibility of controlling and molecular characterization of leaf-cutting ant symbionts leaf-cutting ants with biological methods has generated great isolated from other regions of Argentina still remain to be interest in diverse studies involving the fungi cultivated by done. Therefore, the contribution of species inhabiting in Mis- ants. iones (located in the North of Argentina) will allow further Ants of the Attini tribe live in obligate symbiosis with a understanding on new populations. basidiomycete fungus of the Agaricaceae family which is the Currently there are several hypotheses about the phyloge- essential food source for the larvae and queen (Currie, 2001; netic origin of fungi associated with Attini ants. Accordingly, Fisher et al., 1994; Lange and Grell, 2014; Miyashira et al., nowadays phylogenetic relationship studies about ant’s fungal 2010). On the other hand, the ants provide the fungal symbiont symbiont are under extensive discussion (Bot et al., 2001; with a variety of substrates and also stimulate the fungal Chapela et al., 1994; Silva et al., 2004). growth (Currie, 2001; Lugo et al., 2013; North et al., 1997). The objectives of our study were: (1) to determine the tax- The Agaricales order is one of the most diverse and largest onomic identity of one Acromyrmex pubescens fungal sym- orders of the Basidiomycota Phylum (Misra et al., 2012). The biont strain using morphological and molecular family Agaricaceae is a widely distributed group of sapro- characterization; and (2) to study the phylogenetic relation- trophic fungi and many taxa exist in this family which makes ships among the fungal symbiont strain and related species the taxonomical identification quite complex (Misra et al., from the Leucoagaricus genus. 2012; Moncalvo et al., 2000; Vellinga et al., 2003, 2010). To date, according to Kirk et al. (2008), this family comprises 2. Materials and methods 85 genera and 1340 species. Several authors reported the fungal symbiont of the fungus 2.1. Fungal isolate and culture conditions growing ants as Leucoagaricus gongylophorus species (Fisher et al., 1994; Miyashira et al., 2010; Silva et al., 2004). Accord- Portions of fungal garden were collected from nineteen nests of ing to the literature Leucoagaricus species belong to the family Acromyrmex ants from Misiones province, in the North of Agaricaceae and Leucoagaricus genus has approximately 75 Argentina. Dextrose Potato Agar and Malt Extract Agar LP species (Ortiz et al., 2008). Media were used for the isolation of the mycelial form of the An understanding of mutualism between the fungal sym- fungus (Miyashira et al., 2010) (Collection date: September biont and the Attini ants is greatly hindered by the lack of 2014. Collectors/isolators: Castrillo and Bich). The fungal gar- information regarding the taxonomic placement and evolu- den portions were subcultured periodically until pure isolates tionary history of the fungal cultivars (Currie, 2001). The tax- were attained. The fungus garden material was incubated in onomy and hence the diversity of the symbiotic fungi of ants the dark at 28 °C and 80% humidity for at least 20 days. have not yet been completely elucidated (Silva et al., 2004). The isolate was deposited in culture collection of the Universi- The traditional methods for fungal taxonomy and system- dad Nacional de Misiones with accession number LBM 190. atics depend on the morphology of the fruiting body. Ferreira and Cortez (2012) described some macromycetes 2.2. Morphological identification of Leucoagaricus isolates belonging to the Leucoagaricus genus in the region. Since the fungi cultivated by Attini ants do not actually produce these sexual structures in association with the ants or in pure culture, After a 5–10-day period, the macroscopic characteristics of their identification becomes quite complex (Fisher et al., 1994; each fungal colony developed from the fungus garden material Hervey et al., 1977; Lugo et al., 2013; North et al., 1997). were described through the observation of the following Other methods for fungal taxonomy and systematics use parameters: growth rate, aspect and colonial color upside molecular markers. Nowadays, the systematic relationships and reverse. of fungi have been greatly assisted by new evidence from Microscopic features like presence/absence of septum, molecular systematics, mostly using ribosomal DNA sequence mycelia size, basidia or other structures, etc. were observed data (Schoch et al., 2012; White et al., 1990). for morphological identification. Leucoagaricus genus typically Moncalvo et al. (2002) conducted a molecular phylogenetic presents a special mycelial structure called gongylidia and the study of Euagarics with nuclear large ribosomal subunit gene absence of fibula (Currie, 2001; Miyashira et al., 2010). sequences (nLSU) and although they demonstrated the exis- tence of several clades composed of members of diverse tradi- 2.3. DNA extraction tional groups, they placed most Leucoagaricus species and fungal symbionts of leaf-cutting ants in one single clade. Approximately 200 mg mycelium was placed in a 1.5-mL In some studies, genetic markers such as ribosomal gene microcentrifuge tube. The mycelium was disrupted with a glass (rDNA), internal transcribed spacer (ITS), and part of elonga- pestle that fitted inside the tube with DNA extraction Buffer tion factor 1-a gene (EF 1-a), have been used for the molecular (Tris EDTA buffer pH 8, containing 1.5 M sodium chloride, characterization and phylogenetic studies of fungal symbionts Proteinase K 0.1 mg/mL, b-mercaptoethanol 10 mM and Please cite this article in press as: Bich, G.A. et al., Isolation of the symbiotic fungus of Acromyrmex pubescens and phylogeny of Leucoagaricus gongylophorus from leaf-cutting ants. Saudi Journal of Biological Sciences (2016), http://dx.doi.org/10.1016/j.sjbs.2016.05.010 Isolation of the symbiotic fungus 3 SDS 2% (w/v).