Genetic relationship among Camponotus rufipes Fabricius ( Hymenoptera : Formicidae ) nests by RAPD molecular markers

Random amplified polymorphic DNA (RAPD) markers were used to investigate the genetic relationship among nests of the carpenter ant, Camponotus rufipes, located in the same area. Five random oligodecamers were used to amplify DNA from 108 ant workers collected from six nests. A total of 47 RAPD markers were identified, which revealed low levels of genetic differentiation among nests (Φst = 0.00218) and a low average Shannon index (0.3727) among workers within nests. These results together suggest that the C. rufipes nest may be formed by a single, once-mated queen and that nests produced by queens that are genetically related tend to keep their nests in close proximity to one other.


Introduction
The number of queens in an ant colony is an important characteristic to understand the genetic structure of this social insect (ROSS, 2001).Camponotus species have been associated with monogyny, i.e., nests headed by only one queen where the nestmates are usually full or half siblings (HEINZE et al., 1994;HÖLLDOBLER;WILSON, 1990).However, other studies have revealed cases of polygyny or polyandry in this genus (AKRE et al., 1994;FRASER et al., 2000;GADAU et al., 1996;GERTSCH et al., 1995).
Camponotus rufipes is a wood-harvester ant that inhabits areas ranging from sea level up to 3,000 m altitude in the neotropical region (FERNÁNDEZ, 2003).Different studies of C. rufipes have been published regarding the nestmate-recognition system (JAFFÉ; SANCHEZ, 1984), morphometric patterns of their castes (DINIZ et al., 1994), karyotypes (MARIANO et al., 2001), foraging activities (TAVARES et al., 2008), neural plasticity and behavior (SOARES et al., 2008), sucrose solution intake (SCHILMAN; ROCES, 2008), as biological indicators of environmental conditions (SILVA et al., 2006) or even protection against ectoparasites in which tufted capuchins (Cebus apella) rub their fur with carpenter ants (VERDERANE et al., 2007).However, as far as we know, no study has examined the genetic relationship of C. rufipes nests built in the same geographical area.Elucidating the genetic relationship among nests is important for understanding other aspects of ant biology such as kin discrimination, division of labor, reproductive success of individual queens in polygynous societies, and the organization of founder groups (HEINZE et al., 1994;HÖLLDOBLER;WILSON, 1990).Thus, the present study aims to present a preliminary investigation of the genetic variability within and among C. rufipes nests built near each other, and verify genetic evidence of monogyny, since this species displays worker polymorphism and soldier caste aggression typical of monogyny ants (BUENO; CAMPOS-FARINHA, 1999;LAMON;TOPOFF, 1981).

Material and methods
Camponotus rufipes specimens were collected from six nests found along a cross-section in the Tietê Ecological Park (Figure 1), which is located on the east side of the city of São Paulo (23° 25' S; 46º 28' W).The distances between the nests were the following: 23 m between nests 1 and 2, 0.56 m between 2 and 3, 57 m between 3 and 4, 37 m between 4 and 5, 85 m between 5 and 6, and 202 m between 6 and 1. Eighteen ants were collected alive from each of the six nests and to the laboratory for DNA isolation.Total DNA was extracted using the modified protocol of Taggart et al. (1992), in which an STE buffer was used (0.1 M NaCl, 0.05 M Tris-HCl and 0.001 M EDTA, pH 8.0).In the RAPD reactions, 2 ng μL -1 of DNA template, 10 pmol of the primer, 3.0 mM of MgCl 2 , 1U of Taq DNA Polymerase, 2.5 mM of dNTP and ultrapure water were used, for a total of 10 μL.The following five RAPD primers were used for genetic evaluation: OPB-10, OPB-12, OPA-2, OPA-3 and OPA-5, from Operon Technologies, USA.The RAPD reactions were performed in a thermocycler (PTC-100 MJ Research) under the following conditions: 94 for 5, 92 for 1, 31 -35 for 1, and 72° for 2 minutes, and the final extension at 72° for 5 minutes.The samples were subjected to electrophoresis in 2% agarose gel.All RAPD reactions were carried out twice to ensure the repeatability of the bands.The bands selected to be used in the analysis were based on their resolution, signal strength and reliability.The presence (1) and absence (0) matrices were obtained through analysis of the agarose gels as performed with the following programs: ARLEQUIN version 2.0 (SCHNEIDER et al., 2000) to quantify the genetic differentiation between nests, TFPGA 1.3 (MILLER, 1997) to estimate Nei's genetic distance (NEY, 1978) and POPGENE 1.31 (YEH et al., 1999) to produce the Shannon index to calculate intra-nest genetic diversity.

Results and discussion
The UPGMA dendrogram (Figure 2) based on Nei's genetic distance showed a pattern of genetic differentiation among the six nests.AMOVA (Table 1) analysis was implemented considering all nests as one group.A total of 47 bands using five RAPD primers were identified.The genetic differences between nests was low (Φst = 0.00218) which may be a result of the low number of polymorphic bands produced by the RAPD marker.Heinze et al. (1994), using multilocus DNA fingerprinting for C. floridanus (Buckley) found a considerable amount of genetic variation (Fst = 0.19), although using a more polymorphic molecular marker.The RAPD evaluation of the six nest workers showed a high degree of genetic similarity among them.This finding suggests that the C. rufipes queens of the nests analyzed herein were derived from the same genetic origin and were possibly females from the same colony.Another hypothesis for this low level of genetic differentiation is the presence of satellite nests, which are usually found in this species (BUENO; CAMPOS-FARINHA, 1999).
Average intra-nest genetic variability based on the Shannon index was low (0.3727).This indicates  , 1996).Nests produced by queens that are genetically related can keep their nests close to each other, even if the workers recognize conspecifics from other nests as alien (JAFFÉ; SANCHEZ, 1984).This situation seems to be happening with the C. rufipes nests analyzed herein because of the proximity in which they were built.
Further investigations using other more polymorphic markers such as microsatellites (BOOTH et al., 2009;CROZIER et al., 1999;SEPPÄ;GERTSCH, 1996) should be carried out to verify the sociogenetic structure found herein and develop a better understanding of the social genetic structure of C. rufipes.

Conclusion
Genetic differentiation was low between ant workers from different nests and among workers within the same nest.This suggests that the queens of the colonies analyzed herein have the same genetic origin, or that they came from satellite nests.

Figure 1 .
Figure 1.Distance between each nest C. rufipes, in the Tietê Ecological Park.

Figure 2 .
Figure 2. Dendrogram generated using UPGMA Nei's genetic distance from samples of C. rufipes workers inferred from RAPD.Bootstrap values based on 1000 replications.

Table 1 .
AMOVA analysis from the RAPD data of Camponotus rufipes workers collected from six nests built in the same geographical area.