Acta biol. Colomb., Vol. 18 n.

1, 2013 179 190

POPULATION GENETICS OF Atta sexdens rubropilosa

(HYMENOPTERA: FORMICIDAE)
Gentica de poplaciones de Atta sexdens rubropilosa
(Hymenoptera: Formicidae)
LIRIANA BELIZRIO CANTAGALLI1, Ph. D.; CLAUDETE APARECIDA

MANGOLIN2, Ph. D.; MARIA CLAUDIA COLLA RUVOLOTAKASUSUKI2, Ph. D.

1
Departamento de Biotecnologa, Gentica y Biologa
Celular, Universidad Estatal de Maring.
2

Docente Departamento de Biotecnologa, Gentica y Biologa

Celular, Universidad Estatal de Maring. Av. Colombo, 5790, CEP:
87020-900, Maring, Paran, Brasil. Tel.: 44 3011 4681
Corresponding author: mccrtakasusuki@uem.br

Presentado el 25 de mayo de 2012, aceptado el 27 de septiembre de 2012, correcciones el 7 de febrero de 2013.

ABSTRACT
The genetic variability of Atta sexdens rubropilosa leaf-cutting ants collected from five
brazilian localities was evaluated with PCR-RAPD technique. We used 15 primers
producing 148 fragments of which 123 (83.11 %) contained polymorphisms. The
estimated Shannon index was 0.3836 0.2335 showing that these ants possess high
genetic diversity. The GST value was 0.2372 and PT = 0.184, indicating that the analyzed
populations are moderately differentiated and 82 % of the variation obtained occur within
populations. Although Mantels test had shown correlation between genetic distances
and geographic was observed that Ivatuba and Itamb (33.8 km) have the small
geographical distance and the largest genetic distance. The lower genetic distance was
estimated for Maring and Ivatuba but this localities have a small geographic distance
(42.3 km), indicating that there are no barriers for mating among reproducers in these
populations. The high degree of polymorphism (83.11 %) and the ability to cross among
the populations in the studied regions indicate that this species of leaf-cutting ant is well
adapted to the region; therefore, integrated control programs can be developed.

Keywords: genetic variability, leaf-cutting, PCR-RAPD.

RESUMEN
La variabilidad gentica de las hormigas Atta sexdens rubropilosa colectadas en cinco lugares
distintos de Brasil fueron evaluados por la tcnica PCR-RAPD. Un total de 15 primers
produjeron 148 fragmentos, de los cuales 123 fueron polimrficos, lo que corresponden al

83,11 %. La estimacin de la diversidad gentica por el ndice de Shannon fue 0,3836 y el

desviacin estndar fue de 0,2335. Estos valores demuestran una alta diversidad

180 Artculo de investigacin - Population Genetics of Atta sexdens rubropilosa (Hymenoptera:

Formicidae). Belizrio, et al.

gentica. El valor de GST fue 0,2372 y PT = 0,184 lo que indica que las poblaciones estn
moderadamente diferenciadas y que el 82 % de la variacin obtenida se produce dentro de
las poblaciones. Aunque la prueba de Mantel ha demostrado una correlacin entre la
distancia gentica y geogrfica se observ que Ivatuba e Itamb (33,8 km) tiene una pequea
distancia geogrfica y la mayor distancia gentica. La distancia inferior gentica fue estimada
para Maring e Ivatuba pero estas localidades cuentan con una distancia geogrfica pequea
(42,3 km), lo que indica que no hay barreras para el apareamiento entre los reproductores en
estas poblaciones. El alto valor de polimorfismo (83,11 %) y la capacidad de emparejamiento
entre las poblaciones presentes en las regiones estu-diadas, indican que esta especie de
hormiga cortadora est bien adaptada a la regin, y deben ser desarrollados programas
integrados de control si se convierten en plagas.

Palabras clave: hormigas cortadoras, PCR-RAPD, variabilidad gentica.

INTRODUCTION
Leaf-cutting ants are considered important pests in the neotropics because they
have a wide geographical distribution and are responsible for cutting a wide variety
of native and cultivated plants. In particular, Atta sexdens rubropilosa species (Forel,
1908) harm Eucalyptus and Pinus spp. (Boaretto et al., 1997) by cutting leaves and
tender branches and are capable of completely destroying plants (Oliveira, 1996).

This occurs mainly in homogeneous stands, which favor the development of

pests, such as ants of the Atta and Acromyrmex genera (Della Lucia, 1993).
Forestry companies have used chemicals to control leaf-cutting ants. However,
economic and environmental factors have led companies to improve the operational
efficiency of chemical control techniques. In recent years, there has been an increase in
the number of jobs related to biological and cultural control, especially in relation to plant
resistance, in an attempt to identify alternatives to chemical control strategies or
formulate combinations of different control strategies (Della Lucia, 1993).
Ants also have a variety of ecological functions as seed dispersers, contributing to the
reforestation of many ecosystems and promoting their germination, removed the fruit
pulp (Peternelli et al., 2004). They are responsible for pruning some plants, promoting
their vegetative growth and play an important role in soil aeration (Hlldobler and Wilson,
1990), and incorporate organic matter on earth, making it fertile (Moutinho et al., 2003).
In general, ants provide the unique opportunity to investigate various biological,
evolutionary, and population issues because they form highly complex colonies due to
their diverse social classes. Ants may be used to conduct ecological and population
genetic structure studies, and they also provide rich material for investigating kin
selection and altruism (Brian, 1983; Sudd and Franks, 1987; Hlldobler and Wilson,
1990). Ants are haplodiploid organisms, as the males emerge from parthenogenesis and
females from fertilized eggs. The occurrence of two ploidy levels can be used as an
important tool for genetic analysis (Diehl et al., 2002).
Atta colonies usually consists a queen (mongyne) and thousands or millions of sterile
workers of different sizes and shapes (Hlldobler and Wilson, 2009). The adults, with
rare exceptions, are females and are divided into at least two castes: fertile females or

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181

queens, whose primary function is to egg laying and sterile females, which perform all the
other colony activities, such as water collection and food, feeding the young and queen,
construction and defense of the nest (Wilson, 1976). Males constitute an additional caste and
usually appear only once a year, in mating season, which occurs through the completion of
the nuptial flight (Hlldobler and Wilson, 2009). The queen, after three to eight fertilized by
males during the mating flight, falls to the ground and removes their wings to begin building
the new nest and male die after mating (Forti, 1985).

Some authors have argued that diploid organisms have greater genetic
variability than haplodiploid organisms (Pamilo et al., 1978; Falco and Contel,
1990). Graur (1985), concluded that the level of sociality is more important than
haplodiploidy in determining genetic diversity in hymenoptera.
Since the 1960s, isoenzyme analysis has been the dominant method used for the
evaluation of genetic variation in natural populations. Other techniques are currently
available for the study of genetic diversity, such as random amplification of polymorphic
DNA analysis (RAPD), but all techniques have advantages and disadvantages (Ferreira
and Grattapaglia, 1998). RAPD is based on the identification of polymorphic DNA
randomly amplified using a single primer with an arbitrary sequence, which is capable of
amplifying DNA sequences contained between two annealing sites (Welsh and
Mcclelland, 1990). Although this technique uses random sequences, many studies have
shown that it can be used to effectively identify insect populations (Lopes-da-Silva et al.,
2004) and for genetic variability studies.

Using isozyme polymorphisms (Cantagalli et al., 2010) analyzed the population

structures of Atta capiguara (honeybee-brown ants) in the Tapejara region (PR)
and found low genetic variability for this species. Thus, the RAPD technique
was used in this study to analyze the genetic variability and population structure
of the A. s. rubropilosa leaf-cutter ant to increase the understanding of ant
population genetics and improve the management of this species.
MATERIALS AND METHODS
BIOLOGICAL SAMPLE COLLECTION
Atta sexdens rubropilosa soldiers, which are easily identified by their characteristic lemon
grass smell due to a gland with an alarm function in the head, were collected from five
different regions in the states of Paran and So Paulo, Brazil. In northwestern Paran,
samples were collected from Maring (2326 S, 5156 W), Itamb (233939 S,
515924 W), and Ivatuba (233708 S, 521315 W). In So Paulo, the samples were
collected from Presidente Prudente (220704 S and 512257 W) and Dracena
(212857 S, 513158 W). Collection localities can be visualized in Fig. 1.

In Maring, Itamb, and Ivatuba, four nests were sampled at each location, and
in Presidente Prudente and Dracena, two nests were sampled per site, for a
total of 16 nests. The collected material was stored in plastic bottles at -20 C.
DNA ISOLATION

Total DNA was extracted from five individuals from each nest, with the exception of one
nest from Ivatuba, where it was only possible to extract DNA from four individuals,

182 Artculo de investigacin - Population Genetics of Atta sexdens rubropilosa (Hymenoptera:

Formicidae). Belizrio, et al.

Figure 1. Map of South America, Brazil, States of Paran (PR) and So Paulo (SP) indicating locations

of A. s. rubropilosa collection.

for a total of 79 samples. Only the heads of soldiers were used to prevent
contamination with nucleases that are present in the digestive tract of these insects.
The method used for total DNA extraction was a modified version of that described by
(Yu et al., 1993). The heads were homogenized in 1,5 ml microcentrifuge tubes
containing 300 l of extraction buffer [200 mM Tris-HCl (pH 8,0), 0,5 % SDS, 250 mM
NaCl, 50 mM EDTA, and 100 mg/ml Proteinase K]. After 30 min of incubation at 65 C,
the material was centrifuged for 10 min (10,000 x g) and the supernatant was transferred
to a new tube. An equal volume of chloroform: isoamyl alcohol (24:1) was then added.
After homogenization and centrifugation for phase separation, the upper phase was
transferred to a new tube. The DNA was precipitated with 250 l of cold isopropanol and
incubated at 20 C overnight. The precipitated DNA was separated by centrifugation at
10,300 x g for 10 min. After discarding the supernatant, the precipitate was washed with
1.0 ml of cold 70 % ethanol and left to dry at room temperature. After drying, the DNA
was resuspended for two hours in 50 l of TE buffer, treated with RNAse (10 mg/ml), and
incubated at room temperature for more than 2 hours before storage at -20 C.
The DNA quality was evaluated and quantified on 0.8 % agarose gels with 1X TAE buffer. The
amount of DNA present in each sample was estimated by comparison with known
concentrations of a graded DNA standard ( phage). The gels were stained in ethidium
bromide bath (0.5 mg/mL), and the gel images were visualized under ultraviolet light and
captured with the EDAS system (Kodak 1D Image Analysis 3.5, New York, USA).
AMPLIFICATION OF FRAGMENTS VIA PCR-RAPD, SEPARATION, AND VISUALISATION OF PRODUCTS

To evaluate the genomes of the Atta sexdens rubropilosa populations, 15 primers

were produced by Operon Technologies Inc., Alameda, CA, USA (OPA-01,
OPA-02, OPA-03, OPA-04, OPA-07, OPA-10, OPA-11, OPA-14, OPA-18, OPA19, OPB-11, OPB-12, OPB-17, OPC-20, and OPM-3).
The amplification conditions were based on the methodology described by (Williams et
al., 1990). For a reaction volume of 20 l, it was used 1X Tris-KCl [20 mM Tris-HCl (pH
8.4) and 50 mM KCl], 2.5 mM MgCl2, 0.3 mM of primer, 0.1 mM each dNTP, one

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183

unit of Taq DNA polymerase (Invitrogen), and 20 ng of template DNA. Reactions

were performed in an Eppendorf Mastercycler Gradient thermocycler, as follow 96
C for five minutes followed by 45 cycles of 30 seconds at 94 C, 45 seconds at 35
C, and one minute at 72 C, with a final extension at 72 C for seven minutes. A
negative control reaction (with no DNA template) was used.
The amplified products were separated in a 1.7 % agarose and stained in an ethidium
bromide bath. The DNA fragments were visualized under an ultraviolet light, and the
images were captured with the EDAS system (Kodak 1D Image Analysis 3.5).

POPULATION GENETICS ANALYSIS

The data analysis involved comparison of the molecular weights of the fragments
(bands) obtained from the genomic DNA. Individual bands with identical molecular
weights (located on the same loci) were compared within and between populations
and designated as present (1) or absent (0) for each individual analyzed.
Genetic variability within and between populations was determined by the percentage of
polymorphic loci. The genetic diversity of the populations studied was estimated with the
Shannon index (I) (Lewontin, 1972), GST values were also calculated. Neis (1978)
genetic distance had been used to UPGMA cluster analysis (unweighted pair-group
method using an arithmetic average) and construct a dendrogram. For these analyses,
the Popgene 1.31 program was used (Yeh et al., 1999).

GenALEX 6.5 (Peakall and Smouse, 2012) software was used to estimate
AMOVA (analysis of molecular variance) and phiPT ( PT); to construct genetic
relationships tree among A. s. rubropilosa populations based on Nei and Li/Dice;
to estimate percentage confidence level in the bootstrap analysis (1,000
replicates); spatial analyses involving binary data were performed estimating
principal coordinate analysis (PCA) and Mantel test (1967).
RESULTS
The amount of extracted DNA was ranged between 10 and 40 ng. To verify the genetic
variability of Atta sexdens rubropilosa species collected from different region and the ability to
amplify the extracted DNA via PCR-RAPD, 80 primers were initially tested, of which 15 were
used. Table 1 shows the nucleotide sequences of the selected primers, the numbers of the
total and polymorphic fragments, and the sizes of the amplified fragments.
Primer
OPA- 01

Nucle
5- C

OPA -02

5- G

OPA- 03

5- A

OPA- 04

5- A

OPA- 07

5- G

OPA- 10

5- G

Table 1. Primers, nucleotide sequences, numbers of fragments per primer, and numbers of polymorphic
fragments amplified via RAPD-PCR from DNA extracted from Atta sexdens rubropilosa populations.

184 Artculo de investigacin - Population Genetics of Atta sexdens rubropilosa (Hymenoptera:

Formicidae). Belizrio, et al.

The number of clear and reproducible fragments generated by the primers in all
popu-lations studied ranged from four to 17 fragments, with an average of 9.8
fragments per primer, and the sizes of the amplified products were between 200
and 5000 bp (Fig. 2). The 15 primers used in the RAPD-PCR reactions
produced 148 fragments. It was observed that of out the total number of
fragments analyzed, 123 (83.11 %) displayed polymorphisms.

Figure 2. RAPD electrophoretic profiles of Atta sexdens rubropilosa. Fragments were amplified
with the OPA-03 primer with samples from Maring (01 to 19). M = molecular weight DNA
marker (DNA Ladder, Invitrogen).

The estimated genetic diversity obtained with the Shannon index (0.3836) and the
standard deviation ( 0.2335) indicated that these populations had high genetic diversity.
The high value of GST (0.2372) and PT = 0.184 (significant probability at 0.010) showed
that A. s. rubropilosa populations are moderately differentiated. GST can be considered an
estimate of FST when it is assumed subpopulations are in Hardy-Weinberg equilibrium
(FIS = 0). Therefore, considering Wright (1984) can be considered that G ST values
between 0.15 and 0.25 show that populations are moderately differentiated. AMOVA
analysis showed that 82 % of the variation obtained with RAPD markers occurs within
populations and 18 % among populations.

Principal Component Analysis (PCA) estimated by the covariance matrix showed

that 71.84 % of the variation is explained by the coordinates 1 and 2 (Fig. 3). The
first three coordinates explained 81.30 % of the variation. The coordinates 1, 2 and
3 correspond to 58.51 %, 13.33 % and 9.46 % respectively of variation.
Neis genetic distance values among the five populations analyzed indicated that the
populations from Presidente Prudente and Ivatuba had highest genetic distance (Table
2) but not geographical (215 km, Table 3), whereas the populations from Maring and
Ivatuba had the lowest genetic distance (Table 2) but not geographical (46.5, Table 3).
The correlation between genetic distance and geographical distance was evaluated by
Mantels test. Fig. 4 shows that there is correlation between these variables (R = 0.344,
p = 0.001). A dendrogram based on the arithmetic complement of Neis genetic distance
(1978) (Fig. 5) shows that A. s. rubropilosa analyzed can be separated into two main
groups. The first is formed by Ivatuba-PR, Maring-PR and Presidente Prudente-SP, the
second has populations from Dracena-SP and Itamb-PR (Fig. 5).

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185

Figure 3. Principal component analysis (PCA) shows the coordinates 1 and 2 corresponding
to 71.84 % of the variability.
Pop/ID
Ivatuba
Maring
P. Prudente
Dracena
Itamb
Table 2. Values of genetic identity and Neis genetic distance (1978) obtained with the Popgene
1.31 program for the five Atta sexdens rubropilosa populations. *The genetic identity values are
presented above the diagonal and Neis genetic distance values are below the diagonal.

Km
Ivatuba
Maring
P. Prudente
Dracena
Itamb
Table 3. Geographic distance (Km) between sampling sites of Atta sexdens rubropilosa. SP =
So Paulo state; PR = Paran state.

UPGMA clustering based on the arithmetic complement of the Jaccard

coefficient was not used because the values were not significant at the 70 %
level by bootstrap analysis with 1,000 repetitions.
DISCUSSION

Carvalho and Vieira (2001) reported that the amount of DNA in relation to the primer is
one of the key factors for successful PCR because amplification may not occur if the

186 Artculo de investigacin - Population Genetics of Atta sexdens rubropilosa (Hymenoptera:

Formicidae). Belizrio, et al.

Figure 4. Mantel test showing that there is correlation (p = 0,001) between genetic distance
and geographic distance for A. s. rubropilosa populations analyzed (999 permutations).

Figure 5. Dendrogram based on the arithmetic complement of Neis genetic distance (1978),
which was obtained via RAPD. Individuals in Atta sexdens rubropilosa populations collected
from Ivatuba, Maring, Presidente Prudente, Dracena, and Itamb were grouped with the
UPGMA method using the Popgene 1.31 program.

DNA quantity is too low or high. Carvalho (2000) found that for A. s. rubropilosa,
the optimum amount of DNA for RAPD reactions is between 19 and 30 ng. Our
study shows that it was possible to extract high-quality DNA that ranged
between 10 and 40 ng for amplification.
RAPD-PCR marker proved efficient for genetic populations studies of A. s.
rubropilosa, because 148 fragments reproducible were obtained with 15 primers.
Grutzmacheri et al. (2007) used three primers (UBC 354, UBC 348, and
UBC356) for RAPD to study the genetic variability of the Acromyrmex genus
collected from Rio Grande do Sul and obtained 87 fragments.
Leaf-cutting ants of the Atta genus are indigenous to the Americas. In the Americas,
the distribution of these insects extends from the southern U.S. to northern
Argentina, except for Chile and some of the Antilles islands (Sousa, 1996).
Originally, there were no Atta ants in the forests of Paran. They colonized the state
following the first reforestation event in Paran during the 1940s (Bansho et al., 1994).

Acta biol. Colomb., Vol. 18 n. 1, 2013

187

In the short time since the introduction of A. s. rubropilosa ants in Paran (approximately
70 years), they have obtained a high degree of differentiation (GST = 0.2372), but there is
high variation within populations as shown by AMOVA. Chemical control of A. s.
rubropilosa populations considered pests by farmers, should be careful. The high intrapopulation polymorphism and possible gene flow between populations may promote the
occurrence of cross-resistance to pesticides used to control them.
Despite the short geographical distance (36.5 km) between Ivatuba and Itamb, these
populations have the highest genetic distance (Table 2). It is possible to infer that these
breeding populations do not mate or that there are a small number of hybridizations.
Among the Maring and Ivatuba populations (separated by 42.3 km), the lowest
estimated genetic distance was observed (Table 3). However Mantel test (R = 0.344; p =
0.001) indicate that there is relationship between the genetic and geographic distances
of the studied nests. These results support stepping-stone model proposed by Kimura
(1953). This model assumes that species occupying a wide geographic area, forming
differentiate geographical races (Kimura and Weiss, 1964). Analyzed A. s. rubropilosa
populations do not constitute races, but can be considered subpopulations. The high
genetic differentiation values may be due to the existence of an abundant ancestral
polymorphism that can be maintained by multiple mating of the queen. The high
polymorphism (83.11 %) indicates that this species of leaf-cutter ant is adapted to the
region and integrated programs for the control should be developed.

CONCLUSIONS
Leaf-cutting ants A. s. rubropilosa showed extensive polymorphism and genetic diversity that
were higher within populations than between them. The values of genetic distance and
geographic are not randomized and analyzed populations are moderately differentiated.

ACKNOWLEDGEMENTS
Thanks to Post Graduation in Genetic and Breeding (State University of
Maring/Paran State/Brazil) and to CAPES Program (Coordination
Improvement Higher Education Personnel).
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