Research Article |
Corresponding author: Luísa Antônia Campos Barros ( luufv@yahoo.com.br ) Academic editor: Natalia Golub
© 2016 Luísa Antônia Campos Barros, Hiton Jeferson Alves Cardoso de Aguiar, Cléa dos Santos Ferreira Mariano, Vanderly Andrade-Souza, Jacques Hubert Delabie, M. A. Costa, Silvia das Graças Pompolo.
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Citation:
Barros LAC, de Aguiar HJAC, Mariano CSF, Andrade-Souza V, Costa MA, Delabie JHC, Pompolo SG (2016) Cytogenetic data on six leafcutter ants of the genus Acromyrmex Mayr, 1865 (Hymenoptera, Formicidae, Myrmicinae): insights into chromosome evolution and taxonomic implications. Comparative Cytogenetics 10(2): 229-243. https://doi.org/10.3897/CompCytogen.v10i2.7612
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Cytogenetic data for the genus Acromyrmex Mayr, 1865 are available, to date, for a few species from Brazil and Uruguay, which have uniform chromosome numbers (2n = 38). The recent cytogenetic data of Acromyrmex striatus (Roger, 1863), including its banding patterns, showed a distinct karyotype (2n = 22), similar to earlier studied Atta Fabricius, 1804 species. Karyological data are still scarce for the leafcutter ants and many gaps are still present for a proper understanding of this group. Therefore, this study aimed at increasing cytogenetic knowledge of the genus through the characterization of other six species: Acromyrmex balzani (Emery, 1890), A. coronatus Fabricius, 1804, A. disciger (Mayr, 1887), A. echinatior (Forel, 1899), A. niger (Smith, 1858) and A. rugosus (Smith, 1858), all of which were collected in Minas Gerais – Brazil, except for A. echinatior which was collected in Barro Colorado – Panama. The number and morphology of the chromosomes were studied and the following banding techniques were applied: C-banding, fluorochromes CMA3 and DAPI, as well as the detection of 45S rDNA using FISH technique. All the six species had the same chromosome number observed for already studied species, i.e. 2n = 38. A. balzani had a different karyotype compared with other species mainly due to the first metacentric pair. The heterochromatin distribution also showed interspecific variation. Nevertheless, all the studied species had a pair of bands in the short arm of the first subtelocentric pair. The fluorochrome CMA3 visualized bands in the short arm of the first subtelocentric pair for all the six species, while A. rugosus and A. niger also demonstrated in the other chromosomes. The AT-rich regions with differential staining using DAPI were not observed. 45S ribosomal genes were identified by FISH in the short arm of the first subtelocentric pair in A. coronatus, A. disciger and A. niger. The uniform chromosome number in the genus Acromyrmex (2n = 38) suggests that A. striatus (2n = 22) should be transferred to a new genus. Other aspects of the chromosome evolution in ants are also discussed.
Chromosome evolution, karyotype, fungus-growing ants, biodiversity, heterochromatin, FISH
Fungus-growing ants belong to the Atta-genus group (
The genus Acromyrmex contains 33 described species (or more than 60 taxa if all subspecies and variations are included) (
The genus Acromyrmex has been subdivided into two subgenera, Acromyrmex and Moellerius Forel, 1893 (
Leafcutter ants are one of the most studied groups of fungus-growing ants (
Cytogenetic data on the leafcutter ants are scarce. Namely, these data are available for five Atta species (
Six cytogenetically studied Acromyrmex species were collected between August 2008 and March 2010 in the state of Minas Gerais – Brazil, except for Acromyrmex echinatior (Forel, 1899) which was collected in Panama (Table
Acromyrmex spp. cytogenetically studied in this paper. Locality, sample size (number of colonies/individuals stained with Giemsa), diploid (2n) and haploid (n) chromosome number and karyotypic formula.
Acromyrmex species | Locality (coordinates) | Colony – Individuals | 2n (n) | Karyotypic formula |
---|---|---|---|---|
A. (Moellerius) balzani (Emery, 1890) | Viçosa – MG – Brasil (20°45'S; 42°51'W) | 3 – 12 | 38 | 2n = 12m + 10sm + 14st + 2a |
A. (Moellerius) balzani (Emery, 1890) | Paraopeba – MG – Brazil (19° 17'S; 44° 29'W) | 2 – 15 | 38 | 2n = 12m + 10sm + 14st + 2a |
A. (A.) coronatus Fabricius, 1804 | São Tiago – MG – Brazil (20°54'S; 44°30'W) | 1 – 10 | 38 (19) | 2n = 12m + 8sm + 16st + 2a |
A. (A.) coronatus Fabricius, 1804 | Paraopeba – MG – Brazil (19°17'S; 44°29'W) | 5 – 20 | 38 | 2n = 12m + 8sm + 16st + 2a |
A. (A.) disciger (Mayr, 1887) | Santos Dumont – MG – Brazil (21°27'S; 43°32'W) | 2 – 15 | 38 | 2n = 10m + 12sm + 14st + 2a |
A. (A.) niger (Smith, F. 1858) | Viçosa – MG – Brazil (20°45'S; 42°51'W) | 3 – 21 | 38 | 2n = 12m + 14sm + 10st + 2a |
A. (A.) rugosus (Smith, F. 1858) | Florestal – MG – Brazil (19°52'S; 44°24'W) | 1 – 6 | 38 | 2n = 16m + 12sm + 8st + 2a |
A. (A.) rugosus (Smith, F. 1858) | Paraopeba – MG – Brazil (19° 17'S; 44° 29'W) | 5 – 22 | 38 | 2n = 16m + 12sm + 8st + 2a |
A. (A.) echinatior (Forel, 1899) | Barro Colorado – Panama (9°9'N; 79°50'W) | 2 – 10 | 38 | 2n = 8m + 6sm + 14st + 10a |
Adult ant specimens were identified by J.H.C. Delabie and deposited in the ant collection at the Laboratório de Mirmecologia do Centro de Pesquisas do Cacau (
All studied species had the same diploid chromosome number, 2n = 38 (Table
Karyotype of Acromyrmex species. a A. balzani b A. coronatus c A. disciger d A. rugosus e A. niger f A. echinatior. All species have 2n = 38. Bar = 5 µm.
Chromosome measurements revealed morphological differences between similar karyotypes (Table
The C-banding results of A. balzani, A. coronatus, A. disciger (Mayr, 1887) and A. rugosus (Smith, 1858) indicated bands in some chromosomes: in the short arms of the submetacentric and subtelocentric and also in the centromeric regions of the metacentric chromosomes (Fig.
C-banded metaphases of Acromyrmex species. a A. balzani b A. coronatus c A. disciger d A. rugosus e A. niger f A. echinatior. Arrows indicate C-bands in the largest subtelocentric chromosome pair. Bar = 5 µm.
The short arms of the ST1 pair revealed differences in the banding patterns among the species with fluorochrome CMA3. A. disciger (Fig.
Metaphases of Acromyrmex species stained with CMA3 and DAPI, respectively. a A. balzani b A. coronatus c A. disciger d A. rugosus e A. niger f A. echinatior. Arrows indicate CMA3-positive bands in the largest subtelocentric pair. Arrowheads indicate additional CMA3-positive bands in A. niger and A. rugosus. Bar = 5 µm.
DAPI-positive bright bands which could correspond to AT-rich regions were not revealed (Fig.
Karyotypes of Acromyrmex species observed in this study can be distinguished only on the basis of chromosomal measurements. Differential heterochromatin growth is therefore responsible for small but robust differences in chromosomal morphology, and these differences could not be observed using classification proposed by
The largest metacentric pair of A. balzani strongly differs in size from that of other species probably due to complex chromosomal rearrangements that need to be further investigated. This can be explained by the fact that A. balzani forms a separate clade together with A. landolti according to the molecular phylogeny presented by
The six studied species showed heterochromatic segments on the short arms of ST1 pair. It was observed that these GC-rich heterochromatic regions correspond to NOR which is, in turn, confirmed by FISH with the 45S rDNA probe in the chromosomes of A. coronatus, A. disciger and A. niger. In the latter species, this technique revealed a single NOR, although additional multiple CMA3-positive bands also were observed. This means that these additional bands are not related to the ribosomal genes. NORs are generally GC-rich and CMA3-positive in different organisms (
The nonspecific banding pattern of DAPI staining revealed in the present work is similar to those observed for other fungus-growing ants such as M. goeldii (
Up to now, 12 Acromyrmex species (plus the only subspecies) are cytogenetically studied. All of them show 2n = 38, including both subgenera Acromyrmex and Moellerius (
Patterns of heterochromatin distribution on short arms of some submetacentric and subtelocentric chromosomes of Acromyrmex species suggest that centric fissions which contributed to the origin of the derived karyotype with 2n = 38, probably occurred in the karyotype of the most recent common ancestor of this group. Moreover, recent molecular phylogenetic reconstruction by
Besides data on the chromosome numbers, multiple GC-rich segments were observed in the fungus-growing ants M. goeldii (
Cytogenetic data permitted the differentiation among four of the six Acromyrmex species studied. A. balzani, included in the Moellerius subgenus, showed the largest metacentric chromosome pair with lower size compared with the other species. A. echinatior, besides the higher quantity of acrocentric chromosomes, also had interstitial bands in the ST1 pair for the fluorochrome CMA3, differing from the other species, which suggest the possibility of inversion. A. niger showed multiple CMA3-positive bands: in the telomeric regions of the short arms of the ST1 pair, in the pericentromeric regions of the long arm of the ST1 pair and in the second largest subtelocentric pair. A. rugosus had a greater proportion of metacentric chromosomes compared with the other Acromyrmex and also showed small bands in the telomeric regions of at least three other chromosomes. A. coronatus and A. disciger could only be cytogenetically differentiated from the other species by slight differences in the morphology that are probably due to the differential growth of heterochromatin on the short arms of the chromosomes.
Five of the six Acromyrmex species studied in this paper were collected in a particular area in the South East of South America. However, A. echinatior was collected in Central America, which is more than 5,000 km from the main study area. Moreover, another three species analyzed by
Conserved chromosome numbers were found in certain ant genera, as in Pogonomyrmex Mayr, 1868 in which 13 of 15 studied species had the same chromosome number. The two other species were transferred to another subgenus Ephebomyrmex Wheeler, 1902 (
Our data confirmed uniformity of the chromosome number (2n = 38) in the studied Acromyrmex species. However, chromosomal rearrangements such as heterochromatin growth are likely to be responsible for karyotypic differentiation in this ant group. Location of rDNA clusters of other leafcutter ants (especially A. striatus) also needs to be determined using molecular cytogenetic techniques (FISH). Moreover, cytogenetic studies of other members of fungus-growing ants, e.g. of the genus Trachymyrmex Forel, 1893 which represents the sister group to leafcutter ants, will be important for better understanding of chromosomal evolution of this group and Neotropical Formicidae in general.
We are grateful to Terezinha M.C. Della Lucia and Danival de Souza for providing larvae of some species and for helpful suggestions on a previous version of the manuscript; to Pedro Lorite and Vladimir Gokhman for valuable suggestions, to Manoel José de Souza for assistance in the field work, to V & M Florestal for their support in the field work at Paraopeba, and to Global Edico for language reviewing. We are grateful to CAPES and CNPq for the scholarships granted to LACB, as well as to CNPq for the research grant to JHCD and MAC. This research was supported by FAPEMIG and FAPESB.