Ribosomal DNA has long been a workhorse of comparative cytogenetics. The 45S rDNA cluster, which contains the genes for the 18S, 5.8S and 28S ribosomal RNA subunits, exists in long tandem arrays that mark the nucleolus organizer regions, the chromosomal sites where ribosomes are assembled. Because these arrays are repetitive, transcriptionally active and prone to recombination, their number and position on chromosomes can shift over evolutionary time, and those shifts can be read as signatures of chromosomal rearrangement. In ants, surveys across many subfamilies have found that a single rDNA-bearing chromosome pair per haploid genome is the common condition, likely the ancestral state not just for ants but for insects as a whole. Yet the precise location of the cluster, whether terminal, interstitial or pericentromeric, varies considerably between lineages, and that variation is exactly what the new study set out to interpret in an evolutionary framework.

The team began with a methodological problem that has quietly undermined reproducibility in the field. The 18S rDNA probes routinely used in FISH experiments on Neotropical Hymenoptera had never been formally molecularly characterized. FISH depends on precise nucleotide complementarity between probe and target, so unknown probe sequence means unknown specificity. To close this gap, the researchers amplified the 18S rDNA fragment from genomic DNA of the ant Mycetophylax morschi and the stingless bee Melipona quadrifasciata, using the primers 18SF and 18SR, and sequenced the resulting products. The ant-derived probes yielded fragments of 741 to 750 base pairs corresponding to the initial segment of the 18S gene, beginning at position 80 of the complete 18S sequence of the chalcid wasp Aphelinus gossypii, which served as a reference. BLASTn searches confirmed 99 percent sequence identity to ant 18S rDNA sequences, validating the probe’s taxonomic origin and suitability for chromosomal mapping.

The sequencing also produced a reassuring finding about cross-hybridization. Although ant and stingless bee 18S sequences differ at scattered positions along the probe’s length, the probes still hybridize effectively across these distantly related Hymenoptera, supporting the robustness of the primers. Base composition analysis showed balanced nucleotide proportions with a slight thymine enrichment in Melipona probes of roughly one percent. Since thymine is the base labeled in the digoxigenin-11-dUTP detection system, the authors note that minor PCR adjustments might be considered, but the deviation is too small to compromise labeling efficiency. With the probe now sequenced, deposited in GenBank and characterized, the protocol gains a standard of reproducibility it previously lacked.

With the validated probe in hand, the team turned to two leaf-cutting ant species that had never been cytogenetically mapped for rDNA: Acromyrmex ambiguus, collected at Ilha Comprida in São Paulo, Brazil, and Acromyrmex crassispinus, from Ouro Preto in Minas Gerais. Metaphase chromosomes were prepared from the brain ganglia of pre-pupal larvae using a colchicine-based protocol, with at least five individuals and fifteen high-quality metaphase plates examined per species. Both species proved to have 38 chromosomes, predominantly subtelocentric in shape, and both carried a single 45S rDNA cluster located in the telomeric region of the short arm of the largest subtelocentric pair. The researchers designated this configuration ST1-t, a code that would become central to the phylogenetic analysis.

The picture changed when the same probe was applied to species of Amoimyrmex, a genus of leaf-cutting ants sampled in Argentina. Amoimyrmex bruchi and Amoimyrmex silvestrii have only 22 chromosomes, mostly metacentric, and their single rDNA locus sits interstitially on the second metacentric pair, a configuration labeled M2-i. Compiled with previously published data, the full dataset now spans twenty species across three genera. Every Acromyrmex species examined shows the ST1-t pattern, with one species, Acromyrmex balzani, showing a slightly displaced short-arm variant, while all Atta species carry the rDNA cluster in the pericentromeric region of the fourth metacentric pair, coded M4-i. The number of loci never varies: one per haploid genome, everywhere.

To test whether these positional patterns are evolutionarily structured rather than random, the researchers coded each species’ rDNA location as a discrete character and mapped it onto a dated, multilocus phylogeny of fungus-farming ants. Using an equal-rates transition model in a maximum likelihood framework, they found that the distribution of signal codes across the tree is non-random, with closely related species sharing the same rDNA position. Transition rates between states were symmetrical and low, and ancestral state reconstruction strongly supported the terminal ST1-t configuration as the ancestral condition for Acromyrmex and the interstitial M4-i configuration as ancestral for Atta. Interstitial localization, the analysis suggests, is the retained ancestral state in Amoimyrmex and Atta, while terminal positioning is a derived condition that arose in the Acromyrmex lineage.

Correlation analyses reinforced this interpretation. The terminal ST1-t signal showed a significant positive correlation with divergence time, with a correlation coefficient of 0.57 and a p-value of 0.009, consistent with its being a derived apomorphy that accumulated as Acromyrmex diversified. The short-arm ST1-sa signal showed a moderate negative correlation that approached significance, potentially marking an ancestral or transitional state, while the genus-restricted M2-i and M4-i signals showed no significant correlation with divergence time, reflecting rearrangements specific to Amoimyrmex and Atta respectively. The authors are candid about one limitation: the phylogeny was pruned to include only species with known cytogenetic data, and future work with fuller taxon sampling could refine the ancestral reconstructions. Even so, the genus-level clustering of positional states is strong enough to treat them as putative evolutionary signatures.

What emerges is a dual portrait of the ribosomal DNA locus as both a conservative and a dynamic element. The number of rDNA sites is evidently under strong evolutionary constraint, remaining fixed at one per haploid genome even as diploid chromosome numbers range from 22 to 38 and chromosome morphologies shift between metacentric and subtelocentric forms. The position of the locus, by contrast, is more labile, and its shifts track lineage-specific histories of chromosomal change. The researchers suggest mechanisms such as pericentric inversions and centromere repositioning to explain the genus-specific patterns, and they note that the repeated localization of rDNA to pericentromeric or interstitial regions may reduce the risk of deleterious ectopic recombination, which is more frequent at chromosome ends. There may also be functional consequences: because rDNA clusters form the nucleolus, their position could influence nuclear architecture, transcriptional regulation and chromatin compartmentalization, with interstitial placement potentially favoring nucleolar stability while terminal placement accompanies the greater karyotypic plasticity seen in Acromyrmex.

For a group of ants that reshapes ecosystems and costs agriculture billions of dollars each year, leaf-cutting ants have now gained a sharper cytogenetic toolkit. The molecular validation of the 18S rDNA probe standardizes a method used across dozens of studies, and the demonstration that rDNA positioning carries genuine phylogenetic signal elevates the marker from descriptive curiosity to cytotaxonomic character. Whether the terminal arrangement in Acromyrmex is merely structural or carries adaptive significance remains an open question, one that the authors propose to address through expanded sampling and integration with chromatin topology and gene expression data. What is already clear is that the chromosomes of these fungus farmers preserve a legible record of their evolutionary history, written in the language of ribosomal genes.

Subject of Research: Chromosomal mapping and phylogenetic analysis of 18S rDNA loci in leaf-cutting ants

Article Title: Phylogenetic conservation and diversification of 18S rDNA loci in leaf-cutting ants ( Hymenoptera , Formicidae ): insights from molecular validation and chromosomal mapping using FISH

Article References: Cardoso, D. C., & Cristiano, M. P. (2026). Phylogenetic conservation and diversification of 18S rDNA loci in leaf-cutting ants (Hymenoptera, Formicidae): insights from molecular validation and chromosomal mapping using FISH. Comparative Cytogenetics, 20, 1-20. https://doi.org/10.3897/compcytogen.20.162616

Image Credits: AI Generated

DOI: 10.3897/compcytogen.20.162616

Keywords: leaf-cutting ants, ribosomal DNA, FISH, cytogenetics, Acromyrmex, Atta, Amoimyrmex, chromosomal evolution, phylogenetic signal, Hymenoptera, karyotype, 18S rDNA