GC-Rich Heterochromatin and Ribosomal Genes Mapped in Three Fungus-Farming Ants

Three species of fungus-farming ants collected in Viçosa, in the Brazilian state of Minas Gerais, have yielded new cytogenetic data that help chart how chromosomes have evolved across one of the most distinctive groups of social insects. Using classical staining, C-banding, fluorochrome staining and fluorescence in situ hybridization (FISH), researchers determined the karyotypes of Cyphomyrmex transversus, Mycetomoellerius relictus and Sericomyrmex maravalhas, and mapped the positions of their 18S ribosomal DNA gene clusters. The results, reported for the first time for two of the three genera, add to a growing body of evidence that chromosomal rearrangements have helped shape diversification within the subtribe Attina, the ant lineage that cultivates fungi for food.

The chromosome counts themselves were striking. Cyphomyrmex transversus showed a diploid number of 2n = 18, with a karyotypic formula of 10 metacentric, 2 submetacentric and 6 acrocentric chromosomes. Mycetomoellerius relictus presented 2n = 20, all metacentric, with a haploid complement of n = 10 also confirmed. Sericomyrmex maravalhas carried by far the largest complement of the three, 2n = 48, arranged as 28 metacentric and 20 submetacentric chromosomes. Chromosome morphology was classified according to the ratio of long to short chromosome arms, following the standard scheme in which metacentrics, submetacentrics, subtelocentrics and acrocentrics occupy successive ranges of that ratio.

The most consequential finding concerns heterochromatin. In all three species, most of the heterochromatic regions detected by C-banding, located in centromeric and pericentromeric areas and on the short arms of some chromosomes, were also stained by chromomycin A3, a fluorochrome that preferentially marks GC-rich DNA. This colocalization of heterochromatic blocks and CMA3-positive bands indicates that the constitutive heterochromatin of these ants is rich in guanine and cytosine. The pattern had already been documented in other Attina species, including the paleoattinine Mycocepurus goeldii and the neoattinines Mycetomoellerius urichii and M. holmgreni, but this study reports it for the first time in Sericomyrmex and Cyphomyrmex.

That breadth matters for a long-standing hypothesis. Because GC-rich heterochromatin appears in species belonging to both major clades of the fungus-farming ants, the Paleoattina and the Neoattina, researchers have proposed that this unusual chromatin composition may trace back to a common ancestor of the entire subtribe. The new observations in Sericomyrmex and Cyphomyrmex increase the number of genera showing the trait and lend support to that scenario, although the authors note that the precise nucleotide composition of the heterochromatin has yet to be determined and that further investigation of chromatin composition is needed to corroborate the ancestral-origin hypothesis.

The pattern is also unusual by the standards of ants more broadly. GC-rich heterochromatin is not common in the family Formicidae, with few examples outside Attina, notably in the genus Dolichoderus, which belongs to a different subfamily. In the three newly studied species, heterochromatin was found in the centromeric and pericentromeric regions of all chromosomes, with additional short-arm blocks on the acrocentric chromosomes of Cyphomyrmex transversus and on the short arms of the 7th, 10th and 13th metacentric pairs and all submetacentric pairs of Sericomyrmex maravalhas. Mycetomoellerius relictus showed heterochromatic bands confined to the centromeric regions of its chromosomes.

The ribosomal gene mapping told its own evolutionary story. Each of the three species carried a single chromosome pair bearing 18S rDNA clusters, a pattern consistent with previous findings across fungus-farming ants and considered a plesiomorphic, or ancestral, character state in aculeate Hymenoptera more generally. But the positions differed. In Cyphomyrmex transversus the clusters sat in the pericentromeric region of the short arm of the 2nd metacentric pair; in Mycetomoellerius relictus they occupied an interstitial position on the long arm of the 5th metacentric pair; and in Sericomyrmex maravalhas they lay in the terminal region of the short arm of the 7th metacentric pair.

That terminal placement in S. maravalhas is the derived exception that proves the rule. Most Attina species studied so far carry their rDNA clusters in intrachromosomal, pericentromeric or interstitial positions, from ancient species such as Mycocepurus goeldii and Myrmicocrypta through transition species such as M. holmgreni and M. relictus, to the highly derived leaf-cutting ants. The researchers propose a two-step mechanism to explain the terminal position in S. maravalhas: starting from an ancestor with a low chromosome number and intrachromosomal rDNA clusters, centric fission events would split chromosomes, and a subsequent pericentric inversion would shift the pericentromeric rDNA genes to a terminal position. Terminal rDNA clusters also occur in other fungus-farming ants with derived karyotypes, such as Acromyrmex species and two Mycetophylax species, where inversions have likewise been invoked to explain the shift.

Within Sericomyrmex, the karyotype of S. maravalhas, a member of the basal scrobifer clade, can be compared with that of S. amabilis from the amabilis clade, which has 2n = 50 with only metacentric chromosomes. The authors suggest an increase in chromosome number from 2n = 48 to 2n = 50 along the lineage leading to S. amabilis. The heterochromatic blocks on the short arms of S. maravalhas chromosomes are interpreted as a strong indicator of centric fission events during the genus’s evolution. The absence of subtelocentric and acrocentric chromosomes in both species, they argue, could reflect tandem growth of heterochromatin to stabilize new telomeres after fission, transforming what would have been acrocentric products into submetacentric or metacentric chromosomes. A similar mechanism has been proposed for chromosomal variation in the leaf-cutting ant genus Acromyrmex and in trap-jaw ants of the genus Odontomachus.

The Cyphomyrmex transversus result carries taxonomic weight. The 2n = 18 karyotype found in Minas Gerais is the lowest chromosome number recorded for the genus and differs sharply from previously published counts for the same nominal species: 2n = 24 in French Guiana and 2n = 42 in São Paulo, Brazil, the latter with a notably higher proportion of acrocentric pairs. Such chromosomal divergence among populations of a single described species suggests that C. transversus may in fact be a species complex, and the authors argue that cytogenetic data highlight the need for a taxonomic revision using an integrative approach. Across the genus as a whole, the increase in acrocentric pairs in high-numbered karyotypes has led to the suggestion that centric fissions play a major role in Cyphomyrmex karyotype evolution.

For Mycetomoellerius, the new data fit an emerging picture. Species in one main clade of the genus, defined by molecular phylogeny, carry diploid numbers between 2n = 18 and 2n = 22, with a predominance of metacentric chromosomes, and the authors consider an ancestor in that range likely. The difference in rDNA position between M. relictus, with clusters interstitial on the 5th metacentric pair, and the more basally placed M. holmgreni, with clusters pericentromeric on the 4th metacentric pair, may reflect a paracentric inversion, particularly given how subtle the size difference is between the 4th and 5th pairs in M. relictus, hinting at homeology between the two pairs. Taken together, the study’s authors conclude that chromosomal rearrangements, fissions and inversions alike, have contributed to species diversification in Attina, and that expanding cytogenetic coverage across the roughly 280 described taxa in the subtribe will continue to sharpen understanding of how these agricultural ants evolved.

Subject of Research: Chromosomal evolution and cytogenetics of three fungus-farming ant species (Attina)

Article Title: Distribution of GC-rich heterochromatin and ribosomal genes in three fungus-farming ants ( Myrmicinae , Attini , Attina ): insights on chromosomal evolution

Article References: Amaro Teixeira, G., Antônia Campos Barros, L., Jeferson Alves Cardoso de Aguiar, H., & Meneses Lopes, D. (2021). Distribution of GC-rich heterochromatin and ribosomal genes in three fungus-farming ants (Myrmicinae, Attini, Attina): insights on chromosomal evolution. Comparative Cytogenetics, 15(4), 413-428. https://doi.org/10.3897/compcytogen.v15.i4.73769

Image Credits: AI Generated

DOI: 10.3897/compcytogen.v15.i4.73769

Keywords: fungus-farming ants, Attina, cytogenetics, heterochromatin, GC-rich, 18S rDNA, FISH, karyotype, centric fission, chromosomal inversion, Cyphomyrmex, Sericomyrmex