Two colonies of A. robusta were collected in the city of Aracruz, located in the Brazilian state of Espírito Santo (19°51′S 40°04′W), during January 2012. Specimen collection was authorized by the Brazilian Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio), according to a special collecting permit (SISBio number 31861). This collecting permit was issued to Luísa Antônia Campos Barros. Ant vouchers (workers) were deposited in the reference collection of the Laboratório de Mirmecologia, Centro de Pesquisas do Cacau (CPDC/Brazil), under the record #5727.

Mitotic metaphases were obtained according to Imai et al. [21] by dissecting the cerebral ganglia of the larvae after the meconium elimination. A total of 50 individuals were analyzed. The metaphases were observed and photographed using a BX 60 microscope with a 100× objective, coupled with a Q-Color3 Olympus^® image capture system. A total of 15 metaphases with similar degrees of condensation, without overlapping and evident centromeres, were organized by pairing the chromosomes in order of size, measuring and classifying them according to Levan et al. [22], based on the chromosome arm ratio (r) using the following features: long arm length (L), short arm length (S) and arm ratio between the long and short arms (r = L/S). The chromosomes were classified as: m = metacentric, sm = submetacentric, st = subtelocentric and a = acrocentric. Chromosomes were measured and organized using Image Pro Plus^® and Corel Photopaint X3 software packages, respectively. The C-banding technique was performed according to Sumner [23]. Regions rich in GC and AT base pairs were detected using the fluorochromes Chromomycin A₃ (CMA₃) and 4′,6-diamidin-2-phenylindole (DAPI), respectively [24]. The Hsc-FA technique (secondary constriction heterochromatin-associated bands by fluorescence using Acridine Orange) was applied according to [25]. The NOR banding technique was performed according to Howell and Black [26]. Ribosomal gene clusters were detected by FISH according to Pinkel et al. [27], with the use of a 18S rDNA probe isolated from the bee Melipona quinquefasciata amplified by PCR using the primers 18SF1 (5′-GTCATATGCTTGTCTCAAAGA-3′) and 18SR1.1 (3′-TCTAATTTTTTCAAAGTAAACGC-5′) designed by Pereira [28]. The probes 18S rDNA were labeled by indirect method using digoxigenin-11-dUTP (Roche Applied Science) and the signal was detected with anti-digoxigenin-rhodamine (Roche Applied Science). The metaphases were analyzed with an epifluorescence microscope using WU (330–385 nm) and WG filters (510–550 nm), for DAPI and rhodamine, respectively.

The chromosome number observed was 2n = 22 with the karyotypic formula: 18m + 2sm + 2st (Fig. 1a). A secondary constriction was observed on the fourth metacentric pair (Fig. 1a). The centromeric regions of the chromosomes indicated the presence of weak and little visible blocks of heterochromatin (Fig. 2). The long arm of the fourth pair of metacentric chromosomes showed a region rich in GC base pairs using CMA₃ (Fig. 3a). Regions with differential staining using DAPI were absent, with a uniform pattern along the chromosomes (Fig. 3b). Markings resembling CMA₃ pattern were also observed on the fourth pair of chromosomes when the Hsc-FA technique was applied using Acridine Orange (AO) (Fig. 3c), NOR banding technique (Fig. 3a – inbox) and FISH using 18S rDNA (Fig. 3d).

The chromosome number of A. robusta showed the same general pattern described for other Atta spp.: 2n = 22, with a predominance of metacentric chromosomes. The referred species also presented a conserved morphology of chromosomes when compared with the other species of the genus [17], which was based on the chromosome arm ratio according to Levan et al. [22]. Cytogenetic results of this study confirm the information available for Atta and allow for confirming the conserved chromosome number, morphology and banding pattern within the genus for taxa studied to date, which included species from three of the four groups of Atta indicated by molecular data [29]. It included species in three of the four groups of the genus Atta pertaining to A. laevigata and A. bisphaerica in the Epiatta group; A. colombica in the Atta sensu stricto group; and A. sexdens rubropilosa, A. sexdens piriventris, and A. robusta in the Neoatta group.

The morphological constancy of the chromosomes revealed for Atta spp. differs from that observed for species of the other leafcutter ant genus Acromyrmex, which presented 2n = 38 chromosomes [Barros et al. in prep.]. Morphological differences in the chromosomes may be a consequence of the greater chromosomal number of Acromyrmex spp., which, according to the most robust chromosomal evolution theories of Formicidae proposed by Imai et al. [21], would result from centric fissions that allow for variations in the heterochromatin blocks between different species. The karyotype of A. striatus, however, bears closer similarity to Atta spp. (2n = 22) studied earlier and, associated with the molecular data, it provided new insights into the phylogenetic position of this species among leafcutter ants [30]. According to the authors, it is a sister group of the remainder leafcutter ants that split before the divergence between Acromyrmex.

The heterochromatin distribution pattern of A. robusta is similar to those of other Atta spp., weak centromeric markings, differing from the pattern observed for A. striatus (2n = 22), which is the only Acromyrmex having the same chromosome number as Atta spp. In this ant, cytogenetic studies not only presented weak centromeric markings, but also more evident additional pericentromeric, interstitial, and telomeric heterochromatic GC-rich blocks [27]. Markings observed using the Hsc-FA technique was similar to those observed using the fluorochrome CMA₃ in the present study. Hsc-FA using (AO) marks the NOR-associated heterochromatin (for details, see [31]). The same pattern was also observed for the ants Dinoponera lucida [31] and Wasmannia auropunctata [32]. The only pair of A. robusta chromosomes that showed heterochromatic blocks rich in GC base pairs using the fluorochromes CMA₃ and AO has a pattern similar to that observed in other Atta spp. The negative interstitial region of the fourth chromosome pair was observed with DAPI, indicating complementarity with CMA₃. The same non-specific pattern observed with DAPI along the chromosomes was observed for other fungus-growing ants, e.g., Mycocepurus goeldii [33], A. striatus [30], Trachymyrmex fuscus [34] and Atta spp. [17].

The markings obtained with the FISH technique confirmed the results obtained when employing the fluorochromes CMA₃ and AO, as well as the NOR banding, complementing the data and allowing us to conclude that there is a single NOR for A. robusta. Studies conducted with ants that presented markings in one pair of chromosomes coincided with single NOR e.g., D. lucida [15,31]. It is possible that a single NOR will be found in the genus Atta. Only M. goeldii among fungus-growing ants had its 18S rDNA sites mapped [35], and this study presents the first data for leafcutter ants.

Multiple markings rich in GC base pairs were described in A. striatus [30]. However, they were absent in A. bisphaerica, A. laevigata and A. sexdens rubropilosa [17] and also in the chromosomes of A. robusta stained with CMA₃. A single NOR was confirmed for A. robusta; however, the characteristics of the heterochromatin blocks rich in GC base pairs remain unknown for A. striatus and only further molecular cytogenetic techniques (FISH) could confirm the presence or absence of rDNA in the chromosomes. The fungus-growing ant M. goeldii showed single NOR [35], although it presented multiple GC-rich regions [33].

At the long arm of the fourth chromosome pair markings were observed in its interstitial region using the NOR banding technique, indicating the presence of active ribosomal genes in the last interphase in this pair. The repeatability obtained in the metaphases subjected to this technique is low. However, the clear secondary constriction associated with the presence of heterochromatic blocks rich in GC base pairs, using the CMA₃ and AO, in addition to 18S rDNA-FISH, confirm that this chromosome pair is the NOR bearer. Imai et al. [36] raised the possibility that, somehow, the chromatin of the ribosomal genes was little accessible, which hinders the argentophilic proteins from associating with the chromatin and that silver nitrate later binds to them.

The endemic status of A. robusta suggests peculiarities in its biology if compared to other leafcutter ants. Moreover, strong anthropic pressure on the biome of this species may greatly reduce the variability of its populations by subjecting them to a bottleneck effect capable of reducing its frequency of occurrence. This effect could be expected in function of its cytogenetic configuration; however, the results of this study, which represent the first cytogenetic information on an endemic species of Atta with a restricted distribution, showed no differences in the karyotype compared to other Atta spp. Although samples were not collected in other populations of A. robusta, the constancy of the chromosomal characters found in Atta suggests that variations in the karyotype of A. robusta are unlikely.

Populations of A. robusta have been management target for conservation purposes since 2008 due to the intense process of plant cover reduction in restinga areas. The use of cytogenetic data in animal relocation and breeding programs is of great significance to prevent the reduction of fitness or infertility, because chromosomally divergent groups need to be avoided [14,37]. Therefore, it is important to ensure, especially when dealing with threatened species, that the animals used as founders do not carry chromosomal rearrangements [28]. Considering the constant karyotype (number, morphology and banding patterns) of the threatened A. robusta and other Atta spp., cytogenetic data do not indicate restrictions in relocation or reintroduction in areas where populations were extinct due to the conserved karyotype. However, other data need to be considered, including molecular biology that indicates haplogroups for the species [13,17]. It differs from the giant ant Dinoponera lucida that presents high chromosome number variation in some populations of the state of Bahia (Brazil). In this case, cytogenetic data need to be considered for relocation or reintroduction in areas where the populations were extinct in association with other fields of knowledge. Considering the status of the threatened A. robusta, all information is important for conservation and chromosome data is not a restriction. Furthermore, efforts for preserving restinga environments are needed to preserve A. robusta populations along with many other organisms.

The authors declare that they have no conflicts of interest concerning this article.