Ciência Rural ISSN: 0103-8478 [email protected] Universidade Federal de Santa Maria Brasil

Brayer Pereira, Daniela Isabel; de Avila Botton, Sônia; de Azevedo, Maria Isabel; Urach Monteiro, Danieli; Weiblen, Carla; Schopt Machado, Vanessa; Guimarães Donatti, Michele; Loeck, Alci Enimar Isolation and molecular characterization of symbiotic fungus from ambiguus and Acromyrmex heyeri of Rio Grande do Sul State, Ciência Rural, vol. 45, núm. 7, julio, 2015, pp. 1256-1261 Universidade Federal de Santa Maria Santa Maria, Brasil

Available in: http://www.redalyc.org/articulo.oa?id=33139460017

How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative Ciência1256 Rural, Santa Maria, v.45, n.7, p.1256-1261, jul, 2015 Pereira et al. http://dx.doi.org/10.1590/0103-8478cr20141064 ISSN 0103-8478 MICROBIOLOGY

Isolation and molecular characterization of symbiotic fungus from Acromyrmex ambiguus and Acromyrmex heyeri ants of Rio Grande do Sul State, Brazil

Isolamento e caracterização molecular do fungo simbionte de formigas-cortadeiras (Acromyrmex heyeri e Acromyrmex ambiguus) do Rio Grande do Sul, Brasil

Daniela Isabel Brayer PereiraI Sônia de Avila BottonII Maria Isabel de AzevedoII Danieli Urach MonteiroII Carla WeiblenII Vanessa Schopt MachadoII Michele Guimarães DonattiIII Alci Enimar LoeckIII

ABSTRACT dependentes obrigatórias da simbiose com Leucoagaricus gongylophorus. O objetivo deste estudo foi identificar Leaf-cutting ants of the genera and Acromyrmex morfologicamente e molecularmente, bem como verificar a determine serious agricultural problems and live on symbiosis variabilidade genotípica do fungo simbionte, cultivado por with Leucoagaricus gongylophorus. The aim of this study is to Acromyrmex heyeri e Acromyrmex ambiguus em três regiões identify morphological and molecularly, as well as to verify the do RS, Brasil. Jardins de fungo foram coletados do interior de genotypic variability of the symbiotic fungus cultivated by A. formigueiros e fragmentos de micélio foram cultivados em meio heyeri and A. ambiguus from three different regions of the state de cultura seletivo. O DNA total foi extraído e a amplificação da of Rio Grande do Sul, Brazil. Fungus gardens were collected and região ITS foi realizada por PCR, utilizando primers universais. fragments of mycelia were grown in selective medium. Total DNA Após sequenciamento, os cromatogramas foram montados e as was extracted and amplification of the ITS region was performed análises filogenéticas foram realizadas pelo método de Neighbor- by PCR using universal primers. After DNA sequencing, the Joining. Dos jardins de fungo, obtiveram-se seis isolados de L. chromatograms were assembled and phylogenetic analyzes gongylophorus, confirmados por análise molecular. A análise were performed by the Neighbor-Joining method. A total of six filogenética da região ITS mostrou uma árvore com dois grupos isolates of L. gongylophorus were obtained and their identities distintosem relação aos isolados do fungo oriundos de ninhos de A. were confirmed by molecular analyses. Phylogenetic analysis of heyeri e A. ambiguus. Neste estudo, evidenciou-se que as espécies the ITS region showed a tree with two distinct groups regarding de formigas A. heyeri e A. ambiguus cultivam o mesmo fungo. the fungus isolates from A. heiyeri and A. ambiguous. In this Entretanto, o marcador molecular estudado evidenciou variações study, it was verified that A. heyeri and A. ambiguous, cultivate de L. gongylophorus que permitiram formar duas ramificações the same fungus. Additionally, the molecular marker used in diferentes na árvore filogenética relacionada à espécie de formiga this study showed variations in L. gongylophorus, evidencing que o cultiva. Todavia, para validar as possíveis variações nas two distinct branches in the phylogenetic tree, according to the relações filogenéticas deste fungo simbionte, é necessária a that cultivate L. gongylophorus. However, other inclusão de um maior número de isolados de L. gongylophorus, studies involving the inclusion of a great number of isolates of L. bem como o emprego de outros marcadores moleculares. gongylophorus, as well as the use of other molecular markers to validate the possible variations in the phylogenetic relationship Palavras-chave: Basidiomicetos, Leucoagaricus gongylophorus, of this symbiotic fungus are required. internal transcribed spacer.

Key words: Basidiomycete, Leucoagaricus gongylophorus, internal transcribed spacer. INTRODUCTION RESUMO

Formigas-cortadeiras dos gêneros Atta e Leaf-cutting ants belong to the family Acromyrmex causam elevados prejuízos à agricultura e são Formicidae which are grouped in the Attine tribe

IDepartamento de Microbiologia e Parasitologia, Instituto de Biologia, Universidade Federal de Pelotas (UFPel), 96010-900, Pelotas, RS, Brasil. E-mail: [email protected]. Corresponding author. IIDepartamento de Medicina Veterinária Preventiva, Centro de Ciências Rurais (CCR), Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brasil. IIIDepartamento de Entomologia, Faculdade de Agronomia, UFPel, Pelotas, RS, Brasil. Received 07.17.14 Approved 12.10.14 Returned by the author 04.13.15 CR-2014-1064.R1 Ciência Rural, v.45, n.7, jul, 2015. Isolation and molecular characterization of symbiotic fungus from Acromyrmex ambiguus and Acromyrmex heyeri... 1257 and included in the sub-family . The MATERIAL AND METHOD Attine ants are found only in Neotropical regions and are distributed between southern South America The research was performed with the and the southern United States (Mehdiabadi & species A. heyeri (narrow leaf cutter) and A. Schultz, 2010). The genera Atta and Acromyrmex ambiguus (cutter broadleaf) covering three regions stands out due to the damage they cause, both in of the state of Rio Grande do Sul: South region, in native plants and at cultivated plants, in which they the city of Pelotas (Latitude: 31º46’19”S - Longitude: cut fresh material that serves as a substrate for the 52º20’33”W), campaign region in the city of Bage cultivation of the symbiotic fungus inside their nests (Latitude: 31º19’53” S - Longitude: 54º06’25”W) (CHAPELLA et al., 1994; MUELLER et al., 2001). and Central region, in the municipality of Santa Maria All Attine ants (that includes about 256 described (Latitude: 29º41’03”S-Longitude:53º48’25”W). In species in 15 genera) (SOSA-CALVO et al., 2013) each area, fungi garden of four nests of both ant species are mandatory dependent on this symbiosis, which studied were collected. Fragments of mycelia were involves Lepiotaceaeous fungi (order Agaricales, transferred to Petri plates containing Yeast Nitrogen Basidiomycota division) (Chapela et al., 1994). Base Glucose Chloramphenicol culture media This fungus is considered of monophyletic origin, (YNBGC). The plates were incubated at 25°C for 30 having co-evolved with the ants for 50 million years days. Colonies suspected of having Leucoagaricus through vertical transmission (CHAPELA et al., spp. were micromorphologic evaluated for their 1994; MUELLER et al., 2001). characteristics (presence of gongylidium) and The symbiosis between ants and their picked into Petri dishes containing the same medium fungus has been subject of many studies involving YNBGC without chloramphenicol (YNBG) biochemical (Borba et al., 2006; BACCI JR In order to obtain the total DNA, about et al., 2013), metabolic and molecular aspects 100mg of mycelium grown on YNBG agar was (SILVA-PINHATI et al., 2004).The results of this collected and frozen in liquid nitrogen over night research demonstrate mostly that different species (~16h). Total DNA was extracted according to of leaf-cutting ants cultivate only Leucoagaricus the protocol described by MOLLER et al. (1992) gongylophorus as a single species of symbiotic and KLASSEN et al. (1996) with modifications fungus (Silva-PINHATI et al., 2004; POULSEN (BOTTON et al., 2011), and the frozen isolates were & Boomsma, 2005). However, they could present macerated in a buffer lysis (beta 2-mercaptoethanol, two different strains (Silva-PINHATI et al., 2004). 2% SDS), 10% CTAB and 5N NaCl, followed by Moreover, the behavioral characteristics of fungal phenol extraction and the total DNA was resuspended growth cultivated for different ants species differ in sterile TE. The total DNA concentrations were in different culture media (LOECK et al., 2004; determined using spectrophotometry. Amplification BORBA et al., 2006), and the foraging habits of of the ITS region (intergenic transcribed spacer of different species of leaf cutting ants suggest that the ribosomal RNA), containing partial gene fragments symbiotic fungus can present nutritional differences of the spacer regions 1 and 2 of the internal (Borba et al., 2006). transcribed and the region corresponding to the In Rio Grande do Sul, the main spacing between the 5.8S ribosomal DNA gene was species of leaf-cutting ants belong to the performed by PCR using the universal primers ITS1 Acromyrmex, and plenty of the species (5’-GTAGTCATATGCTTGTCTC-3’) and ITS4 to Acromyrmex heyeri and Acromyrmex (5’CTTCCGTCAATTCCTTTAAG-3’) (WHITE et ambiguus. The first uses monocotyledonous al., 1990). All reactions were performed in a total leaves, while the second uses dicotyledonous volume of 50µl containing: 20 pmol of each primer, leaves for cultivation of their symbiotic fungus 1.25 units Taq DNA polymerase (Invitrogen), 200nM (LOECK & GRUTZMACHER, 2001). Until now, deoxynucleotide (dNTP), 1X of the 10X enzyme molecular studies about the genetic diversity of buffer, 1.5mM MgCl2, and 200ng of DNA samples. these symbiotic fungus species of ants are not Amplifications were performed on thermocycler reported in the literature. The aim of this study PTC-100 (Programmable Thermal Controller, is to identify morphological and molecularly, as MJ Research) under the following amplification well as to verify the genotypic variability of the conditions: 94ºC for 5min, and 30 cycles of 94ºC symbiotic fungus cultivated by A. heyeri and A. for 1min, 55ºC for 1 min and 72ºC for 2 min, ending ambiguus derived from three different regions of with 72ºC for 10 min and 4ºC for 10 min. The PCR the state of Rio Grande do Sul, Brazil. products were separated on 1.2% agarose, stained

Ciência Rural, v.45, n.7, jul, 2015. 1258 Pereira et al.

with ethidium bromide and visualized under UV preliminarily indentified. For each geographical area, light gel. After verification, the amplified products as well as each species of ant it was obtained one were purified by the PureLink kit (Invitrogen) and isolated fungus. the DNA fragments were sequenced in an automatic In the molecular analysis, the DNA sequencer MegaBACE 500 using the DYEnamic ET ITS region derived from the six isolates generated kit (Amersham) employing the same primers used in products of approximately 600pb. The samples were the PCR reactions. amplified, sequenced and compared with the Basic The DNA chromatograms sequences of Local Alignment Search Tool (BLAST), where it was the fungus were mounted and visualized using the possible to confirm the isolates as L. gongylophorus. GAP4 program from the Staden package (STADEN, The DNA sequences obtained from sequencing are 1996). Besides the gene sequences studied, four ITS available in GenBank (Table 1). gene sequences isolates from other Leucoagaricus Phylogenetic analysis of the L. species obtained from GenBank to form the out group this study showed a tree with two distinct groups were used, including: Leucoagaricus barssii (Figure 1). The results showed that the isolates of L. (GQ329062) Leucoagaricus hortensis (AF482843) gongylophorus coming from nests of A. heiyeri and and Tricholoma caligatum (AF319425). Another A. ambiguus collected in three different regions of phylogenetic analysis was carried out using ITS Rio Grande do Sul, were grouped according to the sequences of L. gongylophorus from different regions ant species independently of the geographic region of Brazil, including: AY642809 and AY642810 (Figure 1 and 2). Thus, L. gongylophorus cultivated (Acromyrmex subterraneus molestans); AY642813 by A. heyeri clustered forming the AH group, (Acromyrmex disciger) and AY642814 (Acromyrmex while isolates of L. gongylophorus cultivated by rugosus), previously analyzed by SILVA-PINHATI A. ambiguus constituted the AA group (Figure 1). et al. (2004). Multiple alignment of the data sets was When the phylogenetic analysis was performed, performed by the Clustal W algorithm implemented which included other L. gongylophorus samples in the MEGA5:03 (TAMURA et al., 2011) program. from different regions of Brazil it was observed that Phylogenetic analyzes were performed by analysis isolates from A. ambiguus form a distinct group, of neighbor-joining (NJ) method implemented in whereas isolates derived from L. gongylophorus PAUP4.0b10, using 10,000 bootstrap. from A. heyeri were grouped with the remaining Brazilian isolates analyzed (Figure 2). RESULTS DISCUSSION From 48 collections of fungus gardens it was obtained six isolates with a mycelium growth In this study, which includes Santa Maria, of white cotton wool spots aspect. In the mycelium Bage and Pelotas municipalities (RS/Brazil), it was microscopic it was observed intumescences at the possible to prove that leaf-cutting ants A. heyeri and A. ends of the hyphae, consistent with gongylidium. ambiguus grown L. gongylophorus in their gardens. Based on the macroscopic and microscopic Until now, only BORBA et al. (2006) had reported characteristics obtained L. gongylophorus was the isolation of this symbiotic fungi in the leaf-

Table1 - Isolates of Leucoagaricus gongylophorus derived from Acromyrmex heyeri and Acromyrmex ambiguus from three regions of Rio Grande do Sul, Brazil.

Isolate Ant source City/Region Genbank Access* L. gongylophorusA.h -SM A. heyeri Santa Maria/Central KJ531211 L. gongylophorusA.h - P A. heyeri Pelotas/South KJ531209 L. gongylophorusA.h - B A. heyeri Bagé/Campanha KJ531208 L. gongylophorusA.a – SM A. ambiguus Santa Maria/Central KJ176716 L. gongylophorusA.a – P A. ambiguus Pelotas/South KJ531207 L. gongylophorusA.a - B A. ambiguus Bagé/Campanha KJ531210

*Based on ITS region.

Ciência Rural, v.45, n.7, jul, 2015. Isolation and molecular characterization of symbiotic fungus from Acromyrmex ambiguus and Acromyrmex heyeri... 1259

Figure 1 - Phylogenetic analysis of Brazilian isolates of Leucoagaricus gongylophorus coming from Acromyrmex heyeri (AH) and Acromyrmex ambiguus (AA) based on ITS (Table 1) region using the neighbour-joining method. Bootstrap10,000. cutting ants A. heyeri, A. ambiguus, Acromyrmex gongylophorus come from different genera and species crassipinus and Acromyrmex lundi in the municipal of leaf-cutting ants exhibit different physiological district of Capão do Leão (Brazil/RS), without, behaviors with respect to the culture medium, pH however, including the molecular identification of and temperature (BORBA et al., 2006; BORBA et the fungus. In another study, comprising the southern al., 2007). In addition, POULSEN & BOOMSNA and northern regions of Brazil, SILVA-PINHATI et (2005) showed that agricultural monoculture al. (2004) performed the cultivation and molecular developed by leaf-cutting ants is actively imposed by characterization of L. gongylophorus in the Attine its symbiotic fungus. Previously, SILVA-PINHATI ant tribe including Atta and Acromyrmex species. et al. (2004) evaluated the DNA and the intergenic However, these authors did not include in their study region of L. gongylophorus ants coming from the the A. heyeri and A. ambiguus. Attine tribe in southeastern and northern Brazil and Molecular analysis of the ITS region observed a high similarity of the cultured symbiotic showed that although the species A. ambiguus and fungus, suggesting that leaf-cutting ants cultivate the A. heyeri cultivate the same fungus, fungal intra same fungus species in a wide geographic region of species differences were observed, since it showed South America. In the present study, the results did the formation of two distinct groups shown in the not differ from these authors in relation to the kind phylogenetic tree (Figure 1). Furthermore, it was of symbiotic fungi cultivated by leaf-cutting ants. observed that L. gongylophorus from A. heyeri Moreover, it showed the formation of two distinct showed greater genetic similarity with other isolates groups (AH and AA) determined by the ant species from different regions of Brazil (SILVA-PINHATI independently of the geographical region studied. et al., 2004) that isolates from A. ambiguus, which Agreeing with the results, other studies also claimed formed a distinct group (Figure 2). These differences that different strains of leaf-cutting ants can cultivate may be associated with a co-evolution of the fungus in their nests the same symbiotic fungus (MUELLER, with ants which forage with different leaf types (mono 2001; MIKHEYEV et al., 2007). On the other hand, and dicotyledoneous). In addition to the variation in DOHERTY et al. (2003) using the RAPD (Random the nutritional constitution of sheets, there are also amplified polymorphic DNA) observed genetic differences in the substrate preparation by the ants. variability in isolates originated from symbiotic A. heyeri cultivate the fungus on fragments of entire fungus of Atta cephalotes from Panama and Trinidad. leaves, while A. ambiguus breaks the leaves into Although the ITS region is widely used in tiny pieces. In this sense, studies have shown that L. phylogenetic studies of several fungi, the evolution

Ciência Rural, v.45, n.7, jul, 2015. 1260 Pereira et al.

Figure 2 - Phylogenetic analysis of Brazilian isolates of Leucoagaricus gongylophorus derived from Acromyrmex heyeri (AH), Acromyrmex ambiguus (AA) (Table 1) and Acromyrmex spp.(SILVA-PINHATI et al, 2004) (AB) based in the ITS region using the neighbour-joining method. Bootstrap10,000. of a gene may not represent the evolution of the large number of L. gongylophorus isolates, as well whole genome (SHEN, 2001). The genes encoding as the use of other molecular markers to validate the the structural and metabolic proteins such as possible variations in the phylogenetic relationship of cytochromec oxidase subunit II (COXII) are being this symbiotic fungus. increasingly investigated; since this gene mutations were accumulate throughout evolution, which is Acknowledgements useful to determine phylogenetic relationships (MARTIN, 2000). We thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), grant Whereas L. gongylophorus presents number: 556570/2010-3. metabolic and nutritional changes depending on the kind of the cultivated leaf-cutting ant (BORBA REFERENCES et al., 2006), it can be suggested that future studies evaluating other more sensitive molecular markers BACCI JR., M. et al. A metabolic pathway assembled by enzyme such as COXII, are necessary to evaluate, validate selection may support herbivory of leaf-cutter ants on plant starch. and determine the phylogenetic relationships among Journal of Physiology, v.59, p.525-531, 2013. Available from: . Accessed: Jan. isolates of L. gongylophorus from different strains of 31, 2014. doi: 10.1016/j.jinsphys.2013.02.007. leaf-cutting ants. BORBA, R.S. et al. Crescimento do fungo simbionte de formigas CONCLUSION cortadeiras do gênero Acromyrmex em meios de cultura com diferentes extratos. Ciência Rural, v.36, p.725-730, 2006. Available from: . Accessed: Dec. 10, 2013. was suggested that both species A. heyeri and A. ambiguus, from Rio Grande do Sul, Brazil, cultivated BORBA, R.S. et al. Polimorfismo do fungo simbionte de formigas cortadeiras submetido à luz ultravioleta. Ciência Rural, v.37, the same fungus. However, the molecular marker p.1221-1226, 2007. Available from: . Accessed: Oct. 15, 2014. evidencing two distinct branches in the phylogenetic tree related to the ant species that cultivates the fungus. BOTTON, S.A. et al. Identification of Pythium insidiosum by Nested PCR in cutaneous lesions of Brazilian horses and rabbits. On the other hand, it is required the inclusion of a Current Microbiology, v.62, p.1225-1229, 2011. Available from:

Ciência Rural, v.45, n.7, jul, 2015. Isolation and molecular characterization of symbiotic fungus from Acromyrmex ambiguus and Acromyrmex heyeri... 1261

. Accessed: bodies, and infected plant tissues. Nucleic Acids Research, v.20, Dec. 15, 2013. doi: 10.1007/ s00284-010-9781-4. p.6115-6116, 1992. Available from: . Accessed: Jan. 30, 2013. CHAPELA, I.H. et al. Evolutionary history of the symbiosis between fungus-growing ants and their fungus. Science, v.266, MUELLER, U.G. et al. The origin of the Attine ant-fungus p.1691-1694, 1994. Available from: . Accessed: Nov. 29, 2013. gov/pubmed/11409051>. Accessed: Jan. 31, 2014. doi: PMID:11409051. DOHERTY, K.R. et al. Random amplified polymorphic DNA markers reveal genetic variation in the symbiotic fungus of leaf- POULSEN, M.; BOOMSMA, J.J. Mutualistic fungi control cutting ants. Mycologia, v.95, p.19-23, 2003. Available from: crop diversity in fungus-growing ants. Science, v.4. p.741- . Accessed: 744, 2005. Available from: . Accessed: Nov. 17, 2013. doi: 10.1126/ science.1106688. KLASSEN, G.R. et al. 5S ribosomal RNA gene spacer as species specific probes for eight species of Pythium. Phytopathology, SHEN, Q.S. Molecular phylogenetic analysis of Grifolafrondosa v.86, p.581-587, 1996. Available from: . Accessed: Jan. 30, 2014. dissertation - The Pennsylvania State, University Graduate School.

LOECK, A.E. et al. Growth of symbiont fungi of some higher SILVA-PINHATI, A.C.O. et al. Low variation in ribosomal DNA Attine ants in mineral medium crescimento do fungo simbionte de and internal transcribed spacers of the symbiotic fungi of leaf- alguns Attine superiores em meio mineral. Ciência Rural, v.34, cutting ants (Attini: Formicidae). Brazilian Journal of Medical p.79, 2004. Available from: . Accessed: Dec. 15, 2013. . Accessed: Nov. 27, 2013. doi:10.1590/ LOECK, A.E., GRÜTZMACHER, D.D. Ocorrência de formigas S0100-879X2004001000004. cortadeiras nas principais regiões agropecuárias do Estado do Rio Grande do Sul. Pelotas: UFPEL, 2001. 1v. SOSA-CALVO, J. et al. Cyatta abscondita: , evolution, and natural history of a new fungus-farming ant genus from MARTIN, F.N. Phylogenetic relationships among some Pythium Brazil. PLoS ONE, v.8, p.e80498, 2013. Available from: . Accessed: Oct. 05, 2014. doi: 10.1371/journal. 2000. Available from: . pone.0080498. Accessed: Jan. 29, 2014. doi: 10.2307/3761428. STADEN, R. The Staden sequence analysis package. Molecular MEHDIABADI, N.J.; SCHULTZ, T.R. Natural history and phylogeny Biotechnology, v.5, p.233-241, 1996. Available from:

Ciência Rural, v.45, n.7, jul, 2015.