A recent study by Xu et al. (Nature, 2021, 594, 535–540) provided strong evidence that cryptochrome 4 (Cry4) is a key protein to endow migratory birds with the magnetic compass sense. The investigation compared the magnetic field response of Cry4 from migratory and nonmigratory bird species and suggested that a difference in magnetic sensitivity could exist. This finding prompted an in-depth investigation into Cry4 protein differences on the structural and dynamic levels. In the present study, the pigeon Cry4 (ClCry4) crystal structure was used to reconstruct the missing avian Cry4 protein structures via homology modeling for carefully selected bird species. The reconstructed Cry4 structure from European robin, Eurasian blackcap, zebra finch, chicken, and pigeon were subsequently simulated dynamically and analyzed. The studied avian Cry4 structures show flexibility in analogous regions pointing to similar activation mechanisms and/or signaling interaction partners. It can be concluded that the experimentally recorded difference in the magnetic field sensitivity of Cry4 from different birds is unlikely to be due to solely intrinsic dynamics of the proteins but requires additional factors that have not yet been identified.

Recombination is responsible for breaking up haplotypes, influencing genetic variability, and the efficacy of selection. Bird genomes lack the protein PRDM9, a key determinant of recombination dynamics in most metazoans. The historical recombination maps in birds show an apparent stasis in the positioning of recombination events. This highly conserved recombination pattern over long timescales should constrain the evolution of recombination in birds, but extensive variation in recombination rate across the genome and between different species has been reported. Here, we characterise a fine-scale historical recombination map of an iconic migratory songbird, the European blackcap (Sylvia atricapilla) using a LD-based approach which accounts for population demography. We found variable recombination rates among and within chromosomes, which associate positively with nucleotide diversity and GC content, and a negatively with chromosome size. The recombination rates increased significantly at regulatory regions and not necessarily at high-dense gene regions. CpG islands associated strongly with recombination rates; however, their specific position and local DNA methylation patterns likely influenced this relationship. The association with retrotransposons varied according to specific family and location. Our results also provide evidence of a heterogeneous conservation of recombination maps between the blackcap and its closest sister taxon, the garden warbler at the intra-chromosomal level. These findings highlight the considerable variability of recombination rates at different scales and the role of specific genomic features at shaping this variation. This study opens the possibility of further investigating the impact of recombination in specific population-genomic features.

Migratory birds possess remarkable accuracy in orientation and navigation, which involves various compass systems including the magnetic compass. Identifying the primary magnetosensor remains a fundamental open question. Cryptochromes (Cry) have been shown to be magnetically sensitive, specifically Cry4 shows enhanced magnetic sensitivity in migratory songbirds compared to resident species. Here, we investigate cryptochromes and their potential involvement in magnetoreception in a phylogenetic framework, integrating molecular evolutionary analyses with protein dynamics modeling. We base our analysis on 363 bird genomes and associate different selection regimes with migratory behaviour. We show that Cry4 is characterized by strong positive selection and high variability, typical characteristics of sensor proteins. We identify key sites that likely facilitated the evolution of a highly optimized sensory protein for night time compass orientation in songbirds and a potential functional shift or specialisation. Additionally, we show that Cry4 was lost in hummingbirds, parrots and Tyranni (Suboscines) and thus identified a natural comparative gene knockout, which can be used to test the function of Cry4 in birds. In contrast, the other two cryptochromes Cry1 and Cry2, were highly conserved in all species, indicating basal, non-sensory functions. Our results strengthen the hypothesised role of Cry4 as sensor protein in (night)-migratory songbirds.

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