Bird genomes are among the most stable in terms of synteny and gene content across vertebrates. However, germline-restricted chromosomes (GRCs) represent a striking exception where programmed DNA elimination confines large-scale genomic changes to the germline. GRCs are known to occur in songbirds (oscines), but have been studied only in a few species of Passerides such as the zebra finch, the key model for passerine genomics. Their presence and evolutionary dynamics in most major passerine lineages remain largely unexplored, with suboscines entirely unexamined by cytogenetic or genomic methods. Here, we present the most comprehensive comparative analysis of GRCs to date, spanning 44 million years of passerine evolution. By generating the first germline reference genomes of an oscine and a suboscine, 22 novel germline draft genomes spanning nearly all major passerine lineages and a germline draft genome of a parrot outgroup, we show that the GRC is likely present in 6,700 passerine species. Our results reveal that the GRC evolves rapidly and distinctly from the standard A chromosomes (autosomes and sex chromosomes), yet retains functionally important, selectively maintained genes. We observed gene and repeat turnover occuring orders of magnitude faster than on the A chromosomes. Some GRC genes, such as cpeb1 and pim1, are widespread from an ancient duplication. In contrast, other GRC genes, like mfsd2b and bmp15, have been independently duplicated onto the GRC multiple times, suggesting adaptive constraints. The discovery of zglp1 on the zebra finch GRC, initially copied from chromosome 30 and subsequently lost from it, indicates functional replacement, where the GRC permits gene loss from the standard genome. As the GRC harbors the only zglp1 copy in most of the ~4000 Passerides species, GRC loss would compromise essential germline functions. Our findings establish the GRC as a genomic innovator driving rapid germline evolution. This fact highlights its evolutionary significance for passerine diversification and suggests that programmed DNA elimination may be an overlooked yet phylogenetically widespread mechanism in many understudied animal lineages.

Australian National Wildlife Collection National Research Collections Australia CSIRO Australia

Bonn Institute for Organismal Biology Animal Biodiversity University of Bonn Germany

Bragato Research Institute Lincoln New Zealand

Centre for Molecular Biodiversity Research Leibniz Institute for the Analysis of Biodiversity Change Museum Koenig Bonn Germany

Department of Behavioural Neurobiology Max Planck Institute for Biological Intelligence Germany

Department of Biochemistry and Biophysics National Bioinformatics Infrastructure Sweden Science for Life Laboratory Stockholm University Sweden

Department of Bioinformatics and Genetics Swedish Museum of Natural History Sweden

Department of Biological Sciences Macquarie University Australia

Department of Biology and Museum of Southwestern Biology University of New Mexico USA

Department of Biology Duke University USA

Department of Biosciences University of Oslo Norway

Department of Ecology and Genetics Uppsala University Sweden

Department of Organismal Biology Systematic Biology Science for Life Laboratory Uppsala University Sweden

Department of Ornithology Max Planck Institute for Biological Intelligence Germany

Department of Zoology Faculty of Science Charles University Czech Republic

Embark Veterinary Inc USA

German Centre for Integrative Biodiversity Research Halle Jena Leipzig Leipzig 04103 Germany

Institute of Cytology and Genetics Russian Academy of Sciences Russia

Institute of Vertebrate Biology Czech Academy of Sciences Czech Republic

Instituto de Ciências Exatas e Naturais Universidade Federal do Pará Belém PA Brazil

Laboratory of Neurogenetics of Language The Rockefeller University USA

Natural History Museum of Denmark University of Copenhagen Denmark

Novosibirsk State University Russia

Population Ecology Group Institute of Biodiversity Ecology and Evolution Friedrich Schiller University of Jena Germany

School of Biological Sciences University of Auckland New Zealand

School of Biological Sciences University of East Anglia UK

Seção de Meio Ambiente Instituto Evandro Chagas Ananindeua PA Brazil

State Scientific Research Institute Nature Research Centre Vilnius Lithuania

Swiss Ornithological Institute Switzerland

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