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

## TL;DR

The study maps the harpy eagle's chromosomes using advanced painting techniques, revealing new insights into raptor genome evolution.

## Contribution

A comprehensive chromosomal map of the harpy eagle using multidirectional chromosome painting and genomic data.

## Key findings

- The harpy eagle genome shows extensive structural reorganization, including novel GGA1/GGA3 associations.
- Previously proposed fusions involving GGA20–HHA1 or GGA12–Z are not supported by genomic data.
- Lineage-specific rearrangements indicate rapid chromosomal evolution in Accipitriformes.

## Abstract

Compared with most avian species, diurnal birds of prey, including hawks and eagles, frequently exhibit atypical karyotypes, with lower diploid numbers, and lower number of microchromosomes. The mechanisms and evolutionary pathways underlying these chromosomal changes remain incompletely understood. However, comparative chromosome painting has significantly advanced the understanding of avian karyotype evolution, revealing recurrent chromosomal macrochromosome fissions and fusions with microchromosomes. The harpy eagle was the first bird of prey to undergo partial comparative chromosome painting analysis using a pool containing chicken chromosome paints for pairs 1–6. The present study aimed to expand this analysis by employing the full set of available chicken chromosome painting probes (GGA1–GGA11), generating a detailed chromosomal map of the harpy eagle through the integration of chromosome painting results with high-quality chromosome-level genome sequence data. Our results confirm several previously reported chromosomal rearrangements and reveal novel structural changes, while also indicating that certain previously proposed chromosomal fusions are unlikely to have occurred. Overall, our findings demonstrate that the harpy eagle genome has undergone extensive structural reorganization during its evolutionary history. These results contribute to a deeper understanding of avian genome evolution, providing an improved cytogenomic framework to support future evolutionary studies.

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.