The phylogenetic analyses confirmed the taxonomic

The phylogenetic analyses confirmed the taxonomic identity of the Leucoagaricus isolate from A. pubescens nest from the North of Argentina as L. gongylophorus. The isolate obtained in this study is phylogenetically indistinguishable from L. gongylophorus isolated from other XL184 of leaf-cutting ants belonging to the genera Atta and Acromyrmex, at least using this particular molecular marker.
Also according with this molecular marker, all isolates of L. gongylophorus from leaf-cutting ants had little genetic distance among them, but were clearly distinguished from other strains of L. gongylophorus grown by lower fungus-growing ants (CLADE 3). Moreover, isolates of L. gongylophorus from leaf-cutting ants were genetically distinguished from other species of free living forms of Leucoagaricus.

Gongylidia are typical structures of the symbiotic fungi of higher attine ants (Chapela et al., 1994; Silva et al., 2004). These structures were recognized in the isolated fungus in our study. Miyashira et al. (2010) and Lugo et al. (2013) completed the morphologic identification of the symbiont fungus of Atta and Acromyrmex leaf-cutting ants using macro- and microscopic characters. In our study, the morphologic identification of the fungus symbiont isolate from one A. pubescens nest was consistent with current taxonomic identification of L. gongylophorus.
Molecular methods represent a more sensitive and rapid strategy than morphological techniques to identify species, also molecular identification strategies eliminate diverse technical limitations (Chakraborty et al., 2011). Molecular methods do not replace morphological characterization, conversely they complement morphological methods. ITS region is typically useful for molecular systematics of species (Baldwin, 1992; Schmidt and Moreth, 2002; Schoch et al., 2012), because ITS sequences can accumulate mutations at a faster rate than the 5.8S, 18S, and 28S rRNA genes.
The phylogeny of Leucoagaricus genus was reconstructed by employing the ITS sequence from our isolate and from data of the GenBank. The phylogeny is depicted in Fig. 3.
With respect to their taxonomic and systematic location, the genus Leucoagaricus is controversial; Lugo et al. (2013) analyzed the phylogeny of L. gongylophorus with ITS sequences from diverse species of Atta and Acromyrmex and demonstrated that the fungus grown in ant nests is a unique species. The latter findings also support the results found in our study.
Pereira et al. (2015) made a Phylogenetic analysis of the ITS region where the tree showed two distinct groups (branches in tree) regarding the symbiont fungus isolates from Acromyrmex heyeri and Acromyrmex ambiguous evidencing differences in the strain. So in that study the authors proposed that each of the latter leaf-cutting ant species cultivate the same L. gongylophorus species but their own strain. Those findings were in contrast with a part of our results, because our Phylogenetic tree using more taxa (L. gongylophorus from more leaf-cutting ants and from more regions) made a unique clade.
Otherwise, based on phylogenetic evidence the strains of L. gongylophorus that occur in the phylogenetically basal genera of ants (i.e. Trachymyrmex and Sericomyrmex) formed a distinct clade from those that occur in the phylogenetically derived leaf-cutting ants (i.e. Acromyrmex and Atta). This clade could be explained by the hypothesis that those strains of L. gongylophorus were recently domesticated from free-living populations of Agaricaceae (Mueller et al., 1998). Vo et al. (2009) also found that fungi of lower attine ants have close free-living relatives.
Chapela et al. (1994) worked with the phylogeny of the nuclear 28S ribosomal DNA of the fungal symbiont and found a similar phylogenetic tree, with a clade of fungi from derived higher attine ants and another from phylogenetically basal ants.
All strains of L. gongylophorus from Atta and Acromyrmex from diverse regions of South America were grouped together with low genetic variability. Interestingly the phylogenetic tree couldn’t separate L. gongylophorus strains from Atta from those L. gongylophorus strains isolated from Acromyrmex. The fact that strains of L. gongylophorus of the higher Attini ants made a sole clade could indicate the conservation of the phylogenetic relationship of these strains of fungal symbiont. Generally it had been proposed that these fungal strains are clonally propagated by their ant hosts (Chapela et al., 1994). One noteworthy example of the evolutionary cost to cultivar clonality is a slower evolutionary response to parasites (Currie et al., 1999).