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 (<i>Harpia harpyja</i>) 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 <i>H. harpyja</i> using multidirectional chromosome painting combined with chromosome-level genome assembly data. We integrated cross-species probes from <i>Gallus gallus</i> and <i>Leucopternis albicollis</i> with high-resolution genomic data to refine syntenic relationships and identify fission-fusion hotspots. <i>G. gallus</i> 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.