Abstracts
Leaf-cutting ants of the genera Atta and Acromyrmex determine serious agricultural problems and live on symbiosis with Leucoagaricus gongylophorus. The aim of this study is to identify morphological and molecularly, as well as to verify the genotypic variability of the symbiotic fungus cultivated by A. heyeri and A. ambiguus from three different regions of the state of Rio Grande do Sul, Brazil. Fungus gardens were collected and fragments of mycelia were grown in selective medium. Total DNA was extracted and amplification of the ITS region was performed by PCR using universal primers. After DNA sequencing, the chromatograms were assembled and phylogenetic analyzes were performed by the Neighbor-Joining method. A total of six isolates of L. gongylophorus were obtained and their identities were confirmed by molecular analyses. Phylogenetic analysis of the ITS region showed a tree with two distinct groups regarding the fungus isolates from A. heiyeri and A. ambiguous. In this study, it was verified that A. heyeri and A. ambiguous, cultivate the same fungus. Additionally, the molecular marker used in this study showed variations in L. gongylophorus, evidencing two distinct branches in the phylogenetic tree, according to the ant species that cultivate L. gongylophorus. However, other studies involving the inclusion of a great number of isolates of L. gongylophorus, as well as the use of other molecular markers to validate the possible variations in the phylogenetic relationship of this symbiotic fungus are required.
Basidiomycete; Leucoagaricus gongylophorus; internal transcribed spacer.
Formigas-cortadeiras dos gêneros Atta e Acromyrmex causam elevados prejuízos à agricultura e são dependentes obrigatórias da simbiose com Leucoagaricus gongylophorus. O objetivo deste estudo foi identificar morfologicamente e molecularmente, bem como verificar a variabilidade genotípica do fungo simbionte,cultivado por Acromyrmex heyeri e Acromyrmex ambiguus em três regiões do RS, Brasil. Jardins de fungo foram coletados do interior de formigueiros e fragmentos de micélio foram cultivados em meio de cultura seletivo. O DNA total foi extraído e a amplificação da região ITS foi realizada por PCR, utilizando primers universais. Após sequenciamento, os cromatogramas foram montados e as análises filogenéticas foram realizadas pelo método de Neighbor-Joining. Dos jardins de fungo, obtiveram-se seis isolados de L. gongylophorus, confirmados por análise molecular. A análise filogenética da região ITS mostrou uma árvore com dois grupos distintosem relação aos isolados do fungo oriundos de ninhos de A. heyeri e A. ambiguus. Neste estudo, evidenciou-se que as espécies de formigas A. heyeri e A. ambiguus cultivam o mesmo fungo. Entretanto, o marcador molecular estudado evidenciou variações de L. gongylophorus que permitiram formar duas ramificações diferentes na árvore filogenética relacionada à espécie de formiga que o cultiva. Todavia, para validar as possíveis variações nas relações filogenéticas deste fungo simbionte, é necessária a inclusão de um maior número de isolados de L. gongylophorus, bem como o emprego de outros marcadores moleculares.
Basidiomicetos; Leucoagaricus gongylophorus; internal transcribed spacer.
INTRODUCTION
Leaf-cutting ants belong to the family Formicidae which are grouped in
the Attine tribe and included in the sub-family Myrmicinae. The Attine
ants are found only in Neotropical regions and are distributed between southern South
America and the southern United States (MEHDIABADI &
SCHULTZ, 2010MARTIN, F.N. Phylogenetic relationships among some Pythium species
inferred from sequence analysis of the mitochondrially encoded cytochrome oxidase II
gene. Mycologia, v.92, p.711-727, 2000. Available from:
<http://www.jstor.org/stable/3761428>.Accessed: Jan. 29, 2014. doi:
10.2307/3761428.
http://www.jstor.org/stable/3761428...
). The genera Atta and Acromyrmex
stands out due to the damage they cause, both in native plants and at
cultivated plants, in which they cut fresh material that serves as a substrate for the
cultivation of the symbiotic fungus inside their nests (CHAPELLA et al., 1994CHAPELA, I.H. et al. Evolutionary history of the symbiosis between
fungus-growing ants and their fungus. Science, v.266, p.1691-1694, 1994. Available
from:
<http://entomology.si.edu/staffpages/schultz%20et%20al/Pubs/1994_Science266-EvolHist.pdf>.
Accessed: Nov. 29, 2013.
http://entomology.si.edu/staffpages/schu...
; MUELLER et al.,
2001MUELLER, U.G. et al. The origin of the Attine ant-fungus mutualism.
Quarterly Review of Biology, v.76, p.169-197, 2001. Available from:
<http://www.ncbi.nlm.nih.gov/pubmed/11409051>. Accessed: Jan. 31, 2014.doi:
PMID:11409051.
http://www.ncbi.nlm.nih.gov/pubmed/11409...
). All Attine ants (that includes about 256 described species in 15
genera) (SOSA-CALVO et al., 2013SOSA-CALVO, J. et al. Cyatta abscondita: taxonomy, evolution, and
natural history of a new fungus-farming ant genus from Brazil. PLoS ONE, v.8,
p.e80498, 2013. Available from:
<http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0080498>.
Accessed: Oct. 05, 2014. doi: 10.1371/journal.pone.0080498.
http://www.plosone.org/article/info%3Ado...
) are mandatory
dependent on this symbiosis, which involves Lepiotaceaeous fungi (order
Agaricales, Basidiomycota division) (CHAPELA et al., 1994CHAPELA, I.H. et al. Evolutionary history of the symbiosis between
fungus-growing ants and their fungus. Science, v.266, p.1691-1694, 1994. Available
from:
<http://entomology.si.edu/staffpages/schultz%20et%20al/Pubs/1994_Science266-EvolHist.pdf>.
Accessed: Nov. 29, 2013.
http://entomology.si.edu/staffpages/schu...
). This fungus is considered of
monophyletic origin, having co-evolved with the ants for 50 million years through
vertical transmission (CHAPELA et al., 1994CHAPELA, I.H. et al. Evolutionary history of the symbiosis between
fungus-growing ants and their fungus. Science, v.266, p.1691-1694, 1994. Available
from:
<http://entomology.si.edu/staffpages/schultz%20et%20al/Pubs/1994_Science266-EvolHist.pdf>.
Accessed: Nov. 29, 2013.
http://entomology.si.edu/staffpages/schu...
; MUELLER et al., 2001MUELLER, U.G. et al. The origin of the Attine ant-fungus mutualism.
Quarterly Review of Biology, v.76, p.169-197, 2001. Available from:
<http://www.ncbi.nlm.nih.gov/pubmed/11409051>. Accessed: Jan. 31, 2014.doi:
PMID:11409051.
http://www.ncbi.nlm.nih.gov/pubmed/11409...
).
The symbiosis between ants and their fungus has been subject of many studies involving
biochemical (BORBA et al., 2006BORBA, R.S. et al. Crescimento do fungo simbionte de formigas
cortadeiras do gênero Acromymex em meios de cultura com diferentes extratos Ciência
Rural, v.36, p.725-730, 2006. Available from:
<http://www.scielo.br/pdf/cr/v36n3/a02v36n3.pdf>. Accessed: Dec. 10,
2013.
http://www.scielo.br/pdf/cr/v36n3/a02v36...
; BACCI JR et al., 2013BACCI JR., M. et al. A metabolic pathway assembled by enzyme selection
may support herbivory of leaf-cutter ants on plant starch. Journal of Insect
Physiology, v.59, p.525-531, 2013. Available from:
<http://www.ncbi.nlm.nih.gov/pubmed/23500892>. Accessed: Jan. 31, 2014. doi:
10.1016/j.jinsphys.2013.02.007.
http://www.ncbi.nlm.nih.gov/pubmed/23500...
), metabolic and molecular
aspects (SILVA-PINHATI et al., 2004SILVA-PINHATI, A.C.O. et al. Low variation in ribosomal DNA and internal
transcribed spacers of the symbiotic fungi of leaf-cutting ants (Attini: Formicidae).
Brazilian Journal of Medical and Biological Research, v.37, p.1463-1472, 2004.
Available from:
<http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2004001000004>.
Accessed: Nov. 27, 2013. doi:10.1590/S0100-879X2004001000004.
http://www.scielo.br/scielo.php?script=s...
).The results
of this research demonstrate mostly that different species of leaf-cutting ants
cultivate only Leucoagaricus gongylophorus as a single species of
symbiotic fungus (SILVA-PINHATI et al., 2004SILVA-PINHATI, A.C.O. et al. Low variation in ribosomal DNA and internal
transcribed spacers of the symbiotic fungi of leaf-cutting ants (Attini: Formicidae).
Brazilian Journal of Medical and Biological Research, v.37, p.1463-1472, 2004.
Available from:
<http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2004001000004>.
Accessed: Nov. 27, 2013. doi:10.1590/S0100-879X2004001000004.
http://www.scielo.br/scielo.php?script=s...
;
POULSEN & BOOMSMA, 2005MUELLER, U.G. et al. The origin of the Attine ant-fungus mutualism.
Quarterly Review of Biology, v.76, p.169-197, 2001. Available from:
<http://www.ncbi.nlm.nih.gov/pubmed/11409051>. Accessed: Jan. 31, 2014.doi:
PMID:11409051.
http://www.ncbi.nlm.nih.gov/pubmed/11409...
). However, they
could present two different strains (SILVA-PINHATI et
al., 2004SILVA-PINHATI, A.C.O. et al. Low variation in ribosomal DNA and internal
transcribed spacers of the symbiotic fungi of leaf-cutting ants (Attini: Formicidae).
Brazilian Journal of Medical and Biological Research, v.37, p.1463-1472, 2004.
Available from:
<http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2004001000004>.
Accessed: Nov. 27, 2013. doi:10.1590/S0100-879X2004001000004.
http://www.scielo.br/scielo.php?script=s...
). Moreover, the behavioral characteristics of fungal growth
cultivated for different ants species differ in different culture media (LOECK et al., 2004LOECK, A.E. et al. Growth of symbiont fungi of some higher Attine ants
in mineral medium crescimento do fungo simbionte de alguns Attine superiores em meio
mineral. Ciência Rural, v.34, p.79, 2004. Available from:
<http://www.scielo.br/pdf/cr/v34n1/a12v34n1.pdf>. Accessed: Dec. 15,
2013.
http://www.scielo.br/pdf/cr/v34n1/a12v34...
; BORBA et al., 2006BORBA, R.S. et al. Crescimento do fungo simbionte de formigas
cortadeiras do gênero Acromymex em meios de cultura com diferentes extratos Ciência
Rural, v.36, p.725-730, 2006. Available from:
<http://www.scielo.br/pdf/cr/v36n3/a02v36n3.pdf>. Accessed: Dec. 10,
2013.
http://www.scielo.br/pdf/cr/v36n3/a02v36...
), and the foraging habits of different species of leaf
cutting ants suggest that the symbiotic fungus can present nutritional differences
(BORBA et al., 2006BORBA, R.S. et al. Crescimento do fungo simbionte de formigas
cortadeiras do gênero Acromymex em meios de cultura com diferentes extratos Ciência
Rural, v.36, p.725-730, 2006. Available from:
<http://www.scielo.br/pdf/cr/v36n3/a02v36n3.pdf>. Accessed: Dec. 10,
2013.
http://www.scielo.br/pdf/cr/v36n3/a02v36...
).
In Rio Grande do Sul, the main species of leaf-cutting ants belong to the genus
Acromyrmex, and plenty of the species to Acromyrmex
heyeri and Acromyrmex ambiguus. The first uses
monocotyledonous leaves, while the second uses dicotyledonous leaves for cultivation of
their symbiotic fungus (LOECK & GRUTZMACHER,
2001LOECK, A.E. et al. Growth of symbiont fungi of some higher Attine ants
in mineral medium crescimento do fungo simbionte de alguns Attine superiores em meio
mineral. Ciência Rural, v.34, p.79, 2004. Available from:
<http://www.scielo.br/pdf/cr/v34n1/a12v34n1.pdf>. Accessed: Dec. 15,
2013.
http://www.scielo.br/pdf/cr/v34n1/a12v34...
). Until now, molecular studies about the genetic diversity of these
symbiotic fungus species of ants are not reported in the literature. The aim of this
study is to identify morphological and molecularly, as well as to verify the genotypic
variability of the symbiotic fungus cultivated by A. heyeri and
A. ambiguus derived from three different regions of the state of Rio
Grande do Sul, Brazil.
MATERIAL AND METHOD
The research was performed with the species A. heyeri (narrow leaf cutter) and A. ambiguus (cutter broadleaf) covering three regions of the state of Rio Grande do Sul: South region, in the city of Pelotas (Latitude: 31º46'19"S - Longitude: 52º20'33"W), campaign region in the city of Bage (Latitude: 31º19'53" S - Longitude: 54º06'25"W) and Central region, in the municipality of Santa Maria (Latitude: 29º41'03"S-Longitude:53º48'25"W). In each area, fungi garden of four nests of both ant species studied were collected. Fragments of mycelia were transferred to Petri plates containing Yeast Nitrogen Base Glucose Chloramphenicol culture media (YNBGC). The plates were incubated at 25°C for 30 days. Colonies suspected of having Leucoagaricus spp. were micromorphologic evaluated for their characteristics (presence of gongylidium) and picked into Petri dishes containing the same medium YNBGC without chloramphenicol (YNBG)
In order to obtain the total DNA, about 100mg of mycelium grown on YNBG agar was
collected and frozen in liquid nitrogen over night (~16h). Total DNA was extracted
according to the protocol described by MOLLER et al.
(1992MOLLER, E.M. et al. A simple and efficient protocol for isolation of
high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant
tissues. Nucleic Acids Research, v.20, p.6115-6116, 1992. Available from:
<http://www.ncbi.nlm.nih.gov/pmc/articles/PMC334490/>. Accessed: Jan. 30,
2013.
http://www.ncbi.nlm.nih.gov/pmc/articles...
) and KLASSEN et al.(1996KLASSEN, G.R. et al. 5S ribosomal RNA gene spacer as species specific
probes for eight species of Pythium. Phytopathology, v.86, p.581-587, 1996. Available
from:
<https://www.apsnet.org/publications/phytopathology/backissues/Documents/1996Articles/Phyto86n06_581.pdf>.
Accessed: Jan. 30, 2014.
https://www.apsnet.org/publications/phyt...
) with
modifications (BOTTON et al., 2011BOTTON, S.A. et al. Identification of Pythium insidiosum by Nested PCR
in cutaneous lesions of Brazilian horses and rabbits. Current Microbiology, v.62,
p.1225-1229, 2011. Available from:
<http://www.ncbi.nlm.nih.gov/pubmed/21188592>. Accessed: Dec. 15, 2013. doi:
10.1007/ s00284-010-9781-4.
http://www.ncbi.nlm.nih.gov/pubmed/21188...
), and the
frozen isolates were macerated in a buffer lysis (beta 2-mercaptoethanol, 2% SDS), 10%
CTAB and 5N NaCl, followed by phenol extraction and the total DNA was resuspended in
sterile TE. The total DNA concentrations were determined using spectrophotometry.
Amplification of the ITS region (intergenic transcribed spacer of ribosomal RNA),
containing partial gene fragments of the spacer regions 1 and 2 of the internal
transcribed and the region corresponding to the spacing between the 5.8S ribosomal DNA
gene was performed by PCR using the universal primers ITS1 (5'-GTAGTCATATGCTTGTCTC-3')
and ITS4 (5'CTTCCGTCAATTCCTTTAAG-3') (WHITE et al.,
1990WHITE, T. et al. Amplification and direct sequencing of fungal ribosomal
RNA genes for phylogenetics. In: INNIS, M. PCR protocols: a guide to methods and
applications. New York: Academic, 1990. p.315-322.). All reactions were performed in a total volume of 50µl containing: 20
pmol of each primer, 1.25 units Taq DNA polymerase (Invitrogen), 200nM deoxynucleotide
(dNTP), 1X of the 10X enzyme buffer, 1.5mM MgCl2, and 200ng of DNA samples.
Amplifications were performed on thermocycler PTC-100 (Programmable Thermal Controller,
MJ Research) under the following amplification conditions: 94ºC for 5min, and 30 cycles
of 94ºC for 1min, 55ºC for 1 min and 72ºC for 2 min, ending with 72ºC for 10 min and 4ºC
for 10 min. The PCR products were separated on 1.2% agarose, stained with ethidium
bromide and visualized under UV light gel. After verification, the amplified products
were purified by the PureLink kit (Invitrogen) and the DNA fragments were sequenced in
an automatic sequencer MegaBACE 500 using the DYEnamic ET kit (Amersham) employing the
same primers used in the PCR reactions
The DNA chromatograms sequences of the fungus were mounted and visualized using the GAP4
program from the Staden package (STADEN, 1996STADEN, R.The Staden sequence analysis package. Molecular Biotechnology,
v.5, p.233-241, 1996. Available from:
<http://link.springer.com/article/10.1007/BF02900361#page-1>. Accessed: Dec.
03, 2013.doi: 10.1007/BF02900361.
http://link.springer.com/article/10.1007...
).
Besides the gene sequences studied, four ITS gene sequences isolates from other
Leucoagaricus species obtained from GenBank <www.ncbi.nlm.nih.gov/GenBank> to form the out group were used,
including: Leucoagaricus barssii (GQ329062) Leucoagaricus
hortensis (AF482843) and Tricholoma caligatum (AF319425).
Another phylogenetic analysis was carried out using ITS sequences of L.
gongylophorus from different regions of Brazil, including: AY642809 and
AY642810 (Acromyrmex subterraneus molestans); AY642813
(Acromyrmex disciger) and AY642814 (Acromyrmex
rugosus), previously analyzed by SILVA-PINHATI et al. (2004SILVA-PINHATI, A.C.O. et al. Low variation in ribosomal DNA and internal
transcribed spacers of the symbiotic fungi of leaf-cutting ants (Attini: Formicidae).
Brazilian Journal of Medical and Biological Research, v.37, p.1463-1472, 2004.
Available from:
<http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2004001000004>.
Accessed: Nov. 27, 2013. doi:10.1590/S0100-879X2004001000004.
http://www.scielo.br/scielo.php?script=s...
). Multiple alignment of the data sets was
performed by the Clustal W algorithm implemented in the MEGA5:03 (TAMURA et al., 2011TAMURA, K. et al. MEGA5: molecular evolutionary genetics analysis using
maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular
Biology and Evolution, v.28, p.2731-2739, 2011. Available from:
<http://mbe.oxfordjournals.org/content/28/10/2731.full.pdf+html>. Accessed:
Dec. 03 2013.doi: 10.1093/molbev/msr121.
http://mbe.oxfordjournals.org/content/28...
) program. Phylogenetic analyzes were performed
by analysis of neighbor-joining (NJ) method implemented in PAUP4.0b10, using 10,000
bootstrap.
RESULTS
From 48 collections of fungus gardens it was obtained six isolates with a mycelium growth of white cotton wool spots aspect. In the mycelium microscopic it was observed intumescences at the ends of the hyphae, consistent with gongylidium. Based on the macroscopic and microscopic characteristics obtained L. gongylophorus was preliminarily indentified. For each geographical area, as well as each species of ant it was obtained one isolated fungus.
In the molecular analysis, the DNA ITS region derived from the six isolates generated products of approximately 600pb. The samples were amplified, sequenced and compared with the Basic Local Alignment Search Tool (BLAST), where it was possible to confirm the isolates as L. gongylophorus. The DNA sequences obtained from sequencing are available in GenBank (Table 1).
Phylogenetic analysis of the L. gongylophorus ITS region isolates obtained in this study showed a tree with two distinct groups (Figure 1). The results showed that the isolates of L. gongylophorus coming from nests of A. heiyeri and A. ambiguus collected in three different regions of Rio Grande do Sul, were grouped according to the ant species independently of the geographic region (Figure 1 and 2). Thus, L. gongylophorus cultivated by A. heyeri clustered forming the AH group, while isolates of L. gongylophorus cultivated by A. ambiguus constituted the AA group (Figure 1). When the phylogenetic analysis was performed, which included other L. gongylophorus samples from different regions of Brazil it was observed that isolates from A. ambiguus form a distinct group, whereas isolates derived from L. gongylophorus from A. heyeri were grouped with the remaining Brazilian isolates analyzed (Figure 2).
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.
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, 2004SILVA-PINHATI, A.C.O. et al. Low variation in ribosomal DNA and internal transcribed spacers of the symbiotic fungi of leaf-cutting ants (Attini: Formicidae). Brazilian Journal of Medical and Biological Research, v.37, p.1463-1472, 2004. Available from: <http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2004001000004>. Accessed: Nov. 27, 2013. doi:10.1590/S0100-879X2004001000004.
http://www.scielo.br/scielo.php?script=s... ) (AB) based in the ITS region using the neighbour-joining method. Bootstrap10,000.
DISCUSSION
In this study, which includes Santa Maria, Bage and Pelotas municipalities (RS/Brazil),
it was possible to prove that leaf-cutting ants A. heyeri and
A. ambiguus grown L. gongylophorus in their
gardens. Until now, only BORBA et al. (2006BORBA, R.S. et al. Crescimento do fungo simbionte de formigas
cortadeiras do gênero Acromymex em meios de cultura com diferentes extratos Ciência
Rural, v.36, p.725-730, 2006. Available from:
<http://www.scielo.br/pdf/cr/v36n3/a02v36n3.pdf>. Accessed: Dec. 10,
2013.
http://www.scielo.br/pdf/cr/v36n3/a02v36...
) had
reported the isolation of this symbiotic fungi in the leaf-cutting ants A.
heyeri, A. ambiguus, Acromyrmex
crassipinus and Acromyrmex lundi in the municipal district
of Capão do Leão (Brazil/RS), without, however, including the molecular identification
of the fungus. In another study, comprising the southern and northern regions of Brazil,
SILVA-PINHATI et al. (2004SILVA-PINHATI, A.C.O. et al. Low variation in ribosomal DNA and internal
transcribed spacers of the symbiotic fungi of leaf-cutting ants (Attini: Formicidae).
Brazilian Journal of Medical and Biological Research, v.37, p.1463-1472, 2004.
Available from:
<http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2004001000004>.
Accessed: Nov. 27, 2013. doi:10.1590/S0100-879X2004001000004.
http://www.scielo.br/scielo.php?script=s...
) performed the
cultivation and molecular characterization of L.gongylophorus in the Attine
ant tribe including Atta and Acromyrmex species.
However, these authors did not include in their study the A. heyeri and
A. ambiguus.
Molecular analysis of the ITS region showed that although the species A.
ambiguus and A. heyeri cultivate the same fungus, fungal
intra species differences were observed, since it showed the formation of two distinct
groups shown in the phylogenetic tree (Figure 1).
Furthermore, it was observed that L. gongylophorus from A.
heyeri showed greater genetic similarity with other isolates from different
regions of Brazil (SILVA-PINHATI et al., 2004SILVA-PINHATI, A.C.O. et al. Low variation in ribosomal DNA and internal
transcribed spacers of the symbiotic fungi of leaf-cutting ants (Attini: Formicidae).
Brazilian Journal of Medical and Biological Research, v.37, p.1463-1472, 2004.
Available from:
<http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2004001000004>.
Accessed: Nov. 27, 2013. doi:10.1590/S0100-879X2004001000004.
http://www.scielo.br/scielo.php?script=s...
)
that isolates from A.ambiguus, which formed a distinct group (Figure 2). These differences may be associated with a
co-evolution of the fungus with ants which forage with different leaf types (mono and
dicotyledoneous). In addition to the variation in the nutritional constitution of
sheets, there are also differences in the substrate preparation by the ants.
A.heyeri cultivate the fungus on fragments of entire leaves, while
A. ambiguus breaks the leaves into tiny pieces. In this sense,
studies have shown that L. gongylophorus come from different genera and
species of leaf-cutting ants exhibit different physiological behaviors with respect to
the culture medium, pH and temperature (BORBA et al.,
2006BORBA, R.S. et al. Crescimento do fungo simbionte de formigas
cortadeiras do gênero Acromymex em meios de cultura com diferentes extratos Ciência
Rural, v.36, p.725-730, 2006. Available from:
<http://www.scielo.br/pdf/cr/v36n3/a02v36n3.pdf>. Accessed: Dec. 10,
2013.
http://www.scielo.br/pdf/cr/v36n3/a02v36...
; BORBA et al., 2007). In addition, POULSEN & BOOMSNA (2005MUELLER, U.G. et al. The origin of the Attine ant-fungus mutualism.
Quarterly Review of Biology, v.76, p.169-197, 2001. Available from:
<http://www.ncbi.nlm.nih.gov/pubmed/11409051>. Accessed: Jan. 31, 2014.doi:
PMID:11409051.
http://www.ncbi.nlm.nih.gov/pubmed/11409...
) showed that agricultural monoculture developed
by leaf-cutting ants is actively imposed by its symbiotic fungus. Previously, SILVA-PINHATI et al. (2004SHEN, Q.S. Molecular phylogenetic analysis of Grifolafrondosa (Maitake)
and related species and the influence of selected nutrient supplements on mushroom
yield. 2001. 141f. Doctoral dissertation - The Pennsylvania State, University
Graduate School.) evaluated the DNA and
the intergenic region of L. gongylophorus ants coming from the Attine
tribe in southeastern and northern Brazil and observed a high similarity of the cultured
symbiotic fungus, suggesting that leaf-cutting ants cultivate the same fungus species in
a wide geographic region of South America. In the present study, the results did not
differ from these authors in relation to the kind of symbiotic fungi cultivated by
leaf-cutting ants. Moreover, it showed the formation of two distinct groups (AH and AA)
determined by the ant species independently of the geographical region studied. Agreeing
with the results, other studies also claimed that different strains of leaf-cutting ants
can cultivate in their nests the same symbiotic fungus (MUELLER, 2001MOLLER, E.M. et al. A simple and efficient protocol for isolation of
high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant
tissues. Nucleic Acids Research, v.20, p.6115-6116, 1992. Available from:
<http://www.ncbi.nlm.nih.gov/pmc/articles/PMC334490/>. Accessed: Jan. 30,
2013.
http://www.ncbi.nlm.nih.gov/pmc/articles...
; MIKHEYEV et al., 2007MEHDIABADI, N.J.; SCHULTZ, T.R. Natural history and phylogeny of the
fungus-farming ants (Hymenoptera: Formicidae: Myrmicinae: Attini). Myrmecological
News, v.13, p.37-55, 2010. Available from:
<http://myrmecologicalnews.org/cms/index.php?option=com_content&view=category&id=399:myrmecol-news-13-37-55-online-earlier&Itemid=64&layout=default>.
Accessed: Nov. 09, 2013.
http://myrmecologicalnews.org/cms/index....
).
On the other hand, DOHERTY et al. (2003DOHERTY, K.R. et al. Random amplified polymorphic DNA markers reveal
genetic variation in the symbiotic fungus of leaf-cutting ants. Mycologia, v.95,
p.19-23, 2003. Available from: <http://www.ncbi.nlm.nih.gov/pubmed/21156584>.
Accessed: Nov. 30, 2013.
http://www.ncbi.nlm.nih.gov/pubmed/21156...
) using the
RAPD (Random amplified polymorphic DNA) observed genetic variability in isolates
originated from symbiotic fungus of Atta cephalotes from Panama and
Trinidad.
Although the ITS region is widely used in phylogenetic studies of several fungi, the
evolution of a gene may not represent the evolution of the whole genome (SHEN, 2001POULSEN, M.; BOOMSMA, J.J. Mutualistic fungi control crop diversity in
fungus-growing ants. Science, v.4. p.741-744, 2005. Available from:
<http://www.ncbi.nlm.nih.gov/pubmed/15692054>. Accessed: Nov. 17,
2013.doi:10.1126/science.1106688.
http://www.ncbi.nlm.nih.gov/pubmed/15692...
). The genes encoding the structural and
metabolic proteins such as cytochromec oxidase subunit II (COXII) are being increasingly
investigated; since this gene mutations were accumulate throughout evolution, which is
useful to determine phylogenetic relationships (MARTIN,
2000LOECK, A.E., GRÜTZMACHER, D.D. Ocorrência de formigas cortadeiras nas
principais regiões agropecuárias do Estado do Rio Grande do Sul. Pelotas: UFPEL,
2001. 1v.).
Whereas L. gongylophorus presents metabolic and nutritional changes
depending on the kind of the cultivated leaf-cutting ant (BORBA et al., 2006BORBA, R.S. et al. Crescimento do fungo simbionte de formigas
cortadeiras do gênero Acromymex em meios de cultura com diferentes extratos Ciência
Rural, v.36, p.725-730, 2006. Available from:
<http://www.scielo.br/pdf/cr/v36n3/a02v36n3.pdf>. Accessed: Dec. 10,
2013.
http://www.scielo.br/pdf/cr/v36n3/a02v36...
), it can be suggested that future studies evaluating other
more sensitive molecular markers such as COXII, are necessary to evaluate, validate and
determine the phylogenetic relationships among isolates of L. gongylophorus
from different strains of leaf-cutting ants.
CONCLUSION
Based on the results of this study, it was suggested that both species A. heyeri and A. ambiguus, from Rio Grande do Sul, Brazil, cultivated the same fungus. However, the molecular marker studied showed variations of L. gongylophorus evidencing two distinct branches in the phylogenetic tree related to the ant species that cultivates the fungus. On the other hand, it is required the inclusion of a large number of L. gongylophorus isolates, as well as the use of other molecular markers to validate the possible variations in the phylogenetic relationship of this symbiotic fungus.
ACKNOWLEDGEMENTS
We thank the Brazilian Institute Nacional Council for Scientific and Techonology Development of Brazil (CNPq), grant number: 556570/2010-3.
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Erratum
Article “Isolation and molecular characterization of symbiotic fungus from Acromyrmex ambiguus and Acromyrmex heyeri ants of Rio Grande do Sul State, Brazil” published in volume 45 number 7 July 2015 in the journal Ciência Rural pages 1256-1261 on the affiliation of the authors where we read:“Vanessa Schopt Machado”should read:“Vanessa Schopf Machado”
Publication Dates
-
Publication in this collection
22 May 2015 -
Date of issue
July 2015
History
-
Received
17 July 2014 -
Accepted
10 Dec 2014