Weiguang Wang
Eyal Ben-David
Paul C. Wolf
Andrew M. Ramey
Claudio Verdugo
Karen Lyons
Patricia G. Parker
Leonid Kruglyak
Alejandro Burga
2017
<div id="sec-1" class="subsection"><p><strong>INTRODUCTION</strong></p><p id="p-4">Changes in the size and proportion of limbs and other structures have played a key role in the evolution of species. One common class of limb modification is recurrent wing reduction and loss of flight in birds. Indeed, Darwin used the occurrence of flightless birds as an argument in favor of his theory of natural selection. Loss of flight has evolved repeatedly and is found among 26 families of birds in 17 different orders. Despite the frequency of these modifications, we have a limited understanding of their underpinnings at the genetic and molecular levels.</p></div><div id="sec-2" class="subsection"><p><strong>RATIONALE</strong></p><p id="p-5">To better understand the evolution of changes in limb size, we studied a classic case of recent loss of flight in the Galapagos cormorant (<i>Phalacrocorax harrisi</i>). Cormorants are large water birds that live in coastal areas or near lakes, and<span> </span><i>P. harrisi</i><span> </span>is the only flightless cormorant among approximately 40 extant species. The entire population is distributed along the coastlines of Isabela and Fernandina islands in the Galapagos archipelago.<span> </span><i>P. harrisi</i><span> </span>has a pair of short wings, which are smaller than those of any other cormorant. The extreme reduction of the wings and pectoral skeleton observed in<span> </span><i>P. harrisi</i><span> </span>is an attractive model for studying the evolution of loss of flight because it occurred very recently; phylogenetic evidence suggests that<span> </span><i>P. harrisi</i><span> </span>diverged from its flighted relatives within the past 2 million years. We developed a comparative and predictive genomics approach that uses the genome sequences of<span> </span><i>P. harrisi</i><span> </span>and its flighted relatives to find candidate genetic variants that likely contributed to the evolution of loss of flight.</p></div><div id="sec-3" class="subsection"><p><strong>RESULTS</strong></p><p id="p-6">We sequenced and de novo assembled the whole genomes of<span> </span><i>P. harrisi</i><span> </span>and three closely related flighted cormorant species. We identified thousands of coding variants exclusive to<span> </span><i>P. harrisi</i><span> </span>and classified them according to their probability of altering protein function based on conservation. Variants most likely to alter protein function were significantly enriched in genes mutated in human skeletal ciliopathies, including<span> </span><i>Ofd1</i>,<span> </span><i>Evc</i>,<span> </span><i>Wdr34</i>, and<span> </span><i>Ift122</i>. We carried out experiments in<span> </span><i>Caenorhabditis elegans</i><span> </span>to confirm that a missense variant present in the Galapagos cormorant IFT122 protein is sufficient to affect ciliary function. The primary cilium is essential for Hedgehog (Hh) signaling in vertebrates, and individuals affected by ciliopathies have small limbs and ribcages, mirroring the phenotype of<span> </span><i>P. harrisi</i>. We also identified a 4–amino acid deletion in the regulatory domain of<span> </span><i>Cux1</i>, a highly conserved transcription factor that has been experimentally shown to regulate limb growth in chicken. The four missing amino acids are perfectly conserved in all birds and mammals sequenced to date. We tested the consequences of this deletion in a chondrogenic cell line and showed that it impairs the ability of CUX1 to transcriptionally up-regulate cilia-related genes (some of which contain function-altering variants in<span> </span><i>P. harrisi</i>) and to promote chondrogenic differentiation. Finally, we show that positive selection may have played a role in the fixation of the variants associated with loss of flight in<span> </span><i>P. harrisi</i>.</p></div><div id="sec-4" class="subsection"><p><strong>CONCLUSION</strong></p><p id="p-7">Our results indicate that the combined effect of variants in genes necessary for the correct transcriptional regulation and function of the primary cilium likely contributed to the evolution of highly reduced wings and other skeletal adaptations associated with loss of flight in<span> </span><i>P. harrisi</i>. Our approach may be generally useful for identification of variants underlying evolutionary novelty from genomes of closely related species.</p></div>
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10.1126/science.aal3345
en
American Association for the Advancement of Science
A genetic signature of the evolution of loss of flight in the Galapagos cormorant
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