We have a limited understanding of the genetic and molecular basis of evolutionary changes in the size and proportion of limbs. We studied wing and pectoral skeleton reduction leading to flightlessness in the Galapagos cormorant (Phalacrocorax harrisi). We sequenced and de novo assembled the genomes of four cormorant species and applied a predictive and comparative genomics approach to find candidate variants that may have contributed to the evolution of flightlessness. These analyses and cross-species experiments in Caenorhabditis elegans and in chondrogenic cell lines implicated variants in genes necessary for transcriptional regulation and function of the primary cilium. Cilia are essential for Hedgehog signaling, and humans affected by skeletal ciliopathies suffer from premature bone growth arrest, mirroring skeletal features associated with loss of flight.
Bibliographical noteFunding Information:
Supported by the Jane Coffin Childs Memorial Fund for Medical Research (A.B.), the Howard Hughes Medical Institute (L.K.), the U.S. Geological Survey through the Wildlife Program of the Ecosystems Mission Area (A.M.R.), a Gruss-Lipper postdoctoral fellowship from the EGL Charitable Foundation (E.B.-D.), FONDECYT grant 11130305 (C.V.), and the National Institute of Arthritis and Musculoskeletal and Skin Diseases (K.L.). Field collection permits and export permits were by the Galapagos National Park, facilitated by the Charles Darwin Foundation. Any use of trade names is for descriptive purposes only and does not imply endorsement by the U.S. government. We thank C. Koo and M. Arbing at the UCLA-DOE Protein Expression Laboratory Core Facility for protein purification; S. Feng at the Broad Stem Cell Research Center High Throughput Sequencing Core for assistance; C. Cicero for granting access to cormorant specimens (134079 and 151575) at the Museum of Vertebrate Zoology at Berkeley; C. Valle (Universidad San Francisco de Quito) for advice; and K. Garrett (Natural History Museum of Los Angeles), K. Burns (San Diego Natural History Museum and San Diego State University Museum of Biodiversity), and R. Duerr (International Bird Rescue) for providing samples used in preliminary stages of this study. Author contributions: A.B. and L.K. conceived the study; A.B. coordinated the collection of samples, prepared libraries, assembled and annotated genomes, and performed analyses and experiments; E.B.-D. and A.B. performed the accelerated evolution analysis; P.C.W., A.M.R., C.V., and P.G.P. provided DNA or tissue samples; W.W. and A.B. carried out ATDC5 cell line experiments supervised by K.L.; A.B. and L.K. wrote the manuscript; and all authors discussed and agreed on the final version of the manuscript. All sequencing data from this study is available through the NCBI Sequence Read Archive under Bioproject accession number PRJNA327123. Alignments used for phylogenetic analysis and selection test are available at DRYAD doi:10.5061/dryad.8m2t5. The authors declare no competing financial interests.
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