Multidirectional Chromosomal Painting in the Harpy Eagle (Harpia harpyja): Conservation of Breakpoints in Accipitriformes

Diurnal birds of prey (Falconiformes and Accipitriformes) often display karyotypes that diverge markedly from the putative ancestral avian condition (2n = 80), with reduced diploid numbers and fewer microchromosome pairs driven by extensive chromosomal rearrangements. The harpy eagle (Harpia harpyja) was the first raptor analyzed by chromosome painting, revealing a karyotype (2n = 58) shaped by both microchromosome fusions and macrochromosome fissions followed by secondary fusions. However, these earlier analyses were limited in probe coverage. Here, we present a comprehensive chromosomal map of H. harpyja using multidirectional chromosome painting combined with chromosome-level genome assembly data. We integrated cross-species probes from Gallus gallus and Leucopternis albicollis with high-resolution genomic data to refine syntenic relationships and identify fission–fusion hotspots. G. gallus probes confirmed most previously described and genomically inferred associations but revealed novel features, including a new GGA1/GGA3 association and an increased number of GGA1-derived segments (five to six). Genomic data did not support previously suggested fusions involving GGA20–HHA1 or GGA12–Z. Dual-probe FISH further uncovered lineage-specific rearrangements, indicating rapid chromosomal evolution within Accipitriformes. This integrative approach clarifies harpy eagle genome organization and highlights dynamic evolutionary restructuring in raptor chromosomes.