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

Ribosomal DNA (rDNA) clusters are important cytogenetic markers that can inform both taxonomic delimitation and chromosomal evolution in ants. In this study, we molecularly characterize and validate the widely used 18S rDNA probe applied in cytogenetic studies of Hymenoptera and provide new FISH-based chromosomal data for two previously unstudied leaf-cutting ant species (<i>Acromyrmex ambiguus</i> (Emery, 1888) and <i>Ac. crassispinus</i> (Forel, 1909)). While the general distribution of 45S rDNA loci in leafcutting is relatively well documented (copy number and site), we expand the comparative framework by testing the phylogenetic structure of rDNA positioning across genera. Our results confirm the conserved number of rDNA loci per species but reveal lineage-specific variation in chromosomal location, including both subterminal and pericentromeric arrangements. Phylogenetic signal analyses suggest non-random patterns consistent with evolutionary constraints in locus positioning. Together, our findings refine current cytogenetic models for leafcutting ants and demonstrate the utility of rDNA as a cytotaxonomic character and evolutionary marker for assessing chromosomal diversification.