The frameshift hypothesis is a widely accepted model of bird wing evolution. This hypothesis postulates a shift in positional values, or molecular-developmental identity, that caused a change in digit phenotype. The hypothesis synthesized developmental and paleontological data on wing digit homology. The "most anterior digit" (MAD) hypothesis presents an alternative view based on changes in transcriptional regulation in the limb. The molecular evidence for both hypotheses is that the MAD expresses Hoxd13 but not Hoxd11 and Hoxd12. This digit I "signature" is thought to characterize all amniotes. Here, we studied Hoxd expression patterns in a phylogenetic sample of 18 amniotes. Instead of a conserved molecular signature in digit I, we find wide variation of Hoxd11, Hoxd12, and Hoxd13 expression in digit I. Patterns of apoptosis, and Sox9 expression, a marker of the phalanx-forming region, suggest that phalanges were lost from wing digit IV because of early arrest of the phalanx-forming region followed by cell death. Finally, we show that multiple amniote lineages lost phalanges with no frameshift. Our findings suggest that the bird wing evolved by targeted loss of phalanges under selection. Consistent with our view, some recent phylogenies based on dinosaur fossils eliminate the need to postulate a frameshift in the first place. We suggest that the phenotype of the Archaeopteryx lithographica wing is also consistent with phalanx loss. More broadly, our results support a gradualist model of evolution based on tinkering with developmental gene expression.

Animal Science and Health Institute of Biology Leiden Leiden University Leiden The Netherlands

Departamento de Genética Universidad de Granada Lab 127 Centro de Investigación Biomédica Granada Spain

Institute of Vertebrate Biology Czech Academy of Sciences Brno Czech Republic

Kirksville College of Osteopathic Medicine A T Still University of Health Sciences Kirksville MO USA

Laboratory for Evolutionary Morphology RIKEN Center for Biosystems Dynamics Research Kobe Japan

Museum für Naturkunde Leibniz Institut für Evolutions und Biodiversitätsforschung Berlin Germany

Naturalis Biodiversity Center Leiden The Netherlands

Paläontologisches Institut und Museum Universität Zürich Zürich Switzerland

RIKEN Cluster for Pioneering Research Kobe Japan

School of BioSciences The University of Melbourne Melbourne VIC Australia

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Baron MG, Norman DB, Barrett PM.. 2017. A new hypothesis of dinosaur relationships and early dinosaur evolution. Nature 543(7646):501–506. PubMed

Barta DE, Nesbitt SJ, Norell MA.. 2018. The evolution of the manus of early theropod dinosaurs is characterized by high inter- and intraspecific variation. J Anat. 232(1):80–104. PubMed PMC

Brakefield PM.2011. Evo-devo and accounting for Darwin’s endless forms. Phil Trans R Soc B. 366:2069–2075. PubMed PMC

Carruthers AC, Thomas ALR, Taylor GK.. 2007. Automatic aeroelastic devices in the wings of a steppe eagle Aquila nipalensis. J Exp Biol. 210(Pt 23):4136–4149. PubMed

Chen C-H, Cretekos CJ, Rasweiler JJ, Behringer RR.. 2005. Hoxd13 expression in the developing limbs of the short-tailed fruit bat, Carollia perspicillata. Evol Dev. 7(2):130–141. PubMed

Chew KY, Yu H, Pask AJ, Shaw G, Renfree MB.. 2012. HOXA13 and HOXD13 expression during development of the syndactylous digits in the marsupial Macropus eugenii. BMC Dev Biol. 12:2. PubMed PMC

ChiariY, , CahaisV, , GaltierN, , Delsuc F.. 2012. Phylogenomic analyses support the position of turtles as the sister group of birds and crocodiles (Archosauria). BMC Biol. 10:65. PubMed PMC

Cooper KL, Sears KE, Uygur A, Maier J, Baczkowski K-S, Brosnahan M, Antczak D, Skidmore JA, Tabin CJ.. 2014. Patterning and post-patterning modes of evolutionary digit loss in mammals. Nature 511(7507):41–45. PubMed PMC

Crowley LC, Marfell BJ, Waterhouse NJ.. 2016. Detection of DNA fragmentation in apoptotic cells by TUNEL. Cold Spring Harb Protoc. 2016(10):pdb.prot087221. PubMed

Cuvier G., . 1840. Cuvier’s animal kingdom: arranged according to its organization… [Eng. transl. of: le règne Animal Distribué d'après son Organisation etc.]. London: Orr and Smith.

Davis DD.1964. The giant panda: a morphological study of evolutionary mechanisms. In: Ross LA, Williams PM, Nash EG. editors. Fieldiana: zoological memoirs. Vol. 3. Chicago: Chicago Natural History Museum Press. p. 339.

de Almeida HM, Sousa RP, Bezerra DO, Olivindo RFG, das Neves Diniz A, de Oliveira SC, Feitosa MLT, de Moura Fortes EA, Ferraz MS, de Carvalho YKP, et al.2015. Greater rhea (Rhea americana) external morphology at different stages of embryonic and fetal development. Anim Reprod Sci. 162:43–51. PubMed

de Bakker MAG, Fowler DA, den Oude K, Dondorp EM, Navas MCG, Horbanczuk JO, Sire J-Y, Szczerbińska D, Richardson MK.. 2013. Digit loss in archosaur evolution and the interplay between selection and constraints. Nature 500(7463):445–448. PubMed

Delacour J.1951. The significance of the number of toes in some woodpeckers and kingfishers. Auk Ornithol Adv. 68:49–51.

Delfino M, Fritz U, Sanchez-Villagra MR.. 2010. Evolutionary and developmental aspects of phalangeal formula variation in pig-nose and soft-shelled turtles (Carettochelyidae and Trionychidae). Organ Divers Evol. 10:69–79.

Duboule D, Tarchini B, Zàkàny J, Kmita M.. 2007. Tinkering with constraints in the evolution of the vertebrate limb anterior-posterior polarity. Novartis Found Symp. 284:130–137. PubMed

Endo S, Yamagiwa U, Kurohmaru H.. 1996. Functional anatomy of the radial sesamoid bone in the giant panda (Ailuropoda melanoleuca). J Anat. 189(Pt 3):587–592. PubMed PMC

Fabre PJ, Benke A, Joye E, Nguyen Huynh TH, Manley S, Duboule D.. 2015. Nanoscale spatial organization of the HoxD gene cluster in distinct transcriptional states. Proc Natl Acad Sci U S A. 112(45):13964–13969. PubMed PMC

Gavrieli Y, Sherman Y, Ben-Sasson SA.. 1992. Identification of programmed cell-death in situ via specific labeling of nuclear-DNA fragmentation. J Cell Biol. 119(3):493–501. PubMed PMC

Gould J1980. The panda’s thumb: more reflections in natural history. New York: Norton.

GreenRE, , BraunEL, , ArmstrongJ, , EarlD, , NguyenN, , HickeyG, , VandewegeMW, , St JohnJA, , Capella-GutiérrezS, , Castoe TA, . et al.2014. Three crocodilian genomes reveal ancestral patterns of evolution among archosaurs. Science 346(6215): 1335–1346. PubMed PMC

Hiscock TW, Tschopp P, Tabin CJ.. 2017. On the formation of digits and joints during limb development. Dev Cell. 41:459–465. PubMed PMC

Huang BL, Trofka A, Furusawa A, Norrie JL, Rabinowitz AH, Vokes SA, Taketo MM, Zakany J, Mackem S.. 2016. An interdigit signalling centre instructs coordinate phalanx-joint formation governed by 5'Hoxd-Gli3 antagonism. Nat Commun. 7:12903. PubMed PMC

Humphrey PS, Clark GA.. 1961. Pterylosis of the mallard duck. Condor 63(5):365–385.

Jacob F.1977. Evolution and tinkering. Science 196:1161–1166. PubMed

JarvisED, , MirarabS, , AbererAJ, , LiB, , HoudeP, , LiC, , HoSYW, , FairclothBC, , NabholzB, , Howard JT, . et al.2014. Whole-genome analyses resolve early branches in the tree of life of modern birds. Science 346(6215):1320–1331. PubMed PMC

JohnsonM, , ZaretskayaI, , RaytselisY, , MerezhukY, , McGinnisS, , Madden TL.. 2008. NCBI BLAST: a better web interface. Nucleic Acids Res. 36(Web Server issue):W5–W9. PubMed PMC

Kmita M, Duboule D.. 2003. Organizing axes in time and space. Science 301(5631):331–333. PubMed

Kundrát M.2009. Primary chondrification foci in the wing basipodium of Struthio camelus with comments on interpretation of autopodial elements in Crocodilia and Aves. J Exp Zool. 312:30–41. PubMed

KundráT M, Seichert V, Russell AP, Smetana K.. 2002. Pentadactyl pattern of the avian wing autopodium and pyramid reduction hypothesis. J Exp Zool. 294(2):152–159. PubMed

Kvon EZ, Kamneva OK, Melo US, Barozzi I, Osterwalder M, Mannion BJ, Tissières V, Pickle CS, Plajzer-Frick I, Lee EA, et al.2016. Progressive loss of function in a limb enhancer during snake evolution. Cell 167(3):633–642.e611. PubMed PMC

Langer MC, Ezcurra MD, Rauhut OWM, Benton MJ, Knoll F, McPhee BW, Novas FE, Pol D, Brusatte SL.. 2017. Untangling the dinosaur family tree. Nature 551(7678):E1–E3. PubMed

Larsson A.2014. AliView: a fast and lightweight alignment viewer and editor for large datasets. Bioinformatics. 30(22):3276–3278. PubMed PMC

Leal F, Cohn MJ.. 2016. Loss and re-emergence of legs in snakes by modular evolution of sonic hedgehog and HOXD enhancers. Curr Biol. 26(21):2966–2973. PubMed

Lee S-I, Kim J, Park H, Jabłoński PG, Choi H.. 2015. The function of the alula in avian flight. Sci Rep. 5:9914. PubMed PMC

Lopez-Rios J, Duchesne A, Speziale D, Andrey G, Peterson KA, Germann P, Unal E, Liu J, Floriot S, Barbey S, et al.2014. Attenuated sensing of SHH by Ptch1 underlies evolution of bovine limbs. Nature 511(7507):46–51. PubMed

Lorda-Diez CI, Montero JA, Diaz-Mendoza MJ, Garcia-Porrero JA, Hurle JM.. 2011. Defining the earliest transcriptional steps of chondrogenic progenitor specification during the formation of the digits in the embryonic limb. PLoS One. 6(9):e24546. PubMed PMC

Lucas AM, Stettenheim PR.. 1972. Avan anatomy: integument, Part 1. Washington (DC: ): United States Department of Agriculture.

Maxwell EE, Larsson HCE.. 2007. Osteology and myology of the wing of the Emu (Dromaius novaehollandiae), and its bearing on the evolution of vestigial structures. J Morphol. 268(5):423–441. PubMed

McGinnisS, , Madden TL.. 2004. BLAST: at the core of a powerful and diverse set of sequence analysis tools. Nucleic Acids Res. 32(Web Server issue):W20–W25. PubMed PMC

Mitgutsch R, Bakker D, Ezequiel Mart¡n K.. 2012. A molecular-morphological study of a peculiar limb morphology: the development and evolution of the mole's ‘thumb’. In: Asher RJ, Müller J, editors. From clone to bone - the synergy of morphological and molecular tools in palaeobiology. Cambridge: Cambridge University Press. p. 301–327.

Müller GB, Alberch P.. 1990. Ontogeny of the limb skeleton in Alligator mississippiensis: developmental invariance and change in the evolution of archosaur limbs. J Morphol. 203(2):151–164. PubMed

Nelson CE, Morgan BA, Burke AC, Laufer E, DiMambro E, Murtaugh LC, Gonzales E, Tessarollo L, Parada LF, Tabin C.. 1996. Analysis of Hox gene expression in the chick limb bud. Development 122(5):1449–1466. PubMed

Owen R.1835. On the osteology of the chimpanzee and orang utan. Trans Zool Soc Lond. 1:343–379.

Prud’homme B, Gompel N, Carroll SB.. 2007. Emerging principles of regulatory evolution. Proc Nat Acad Sci. 104(1 Suppl):8605–8612. PubMed PMC

Rauhut OWM, Foth C, Tischlinger H.. 2018. The oldest archaeopteryx (Theropoda: Avialiae): a new specimen from the Kimmeridgian/Tithonian boundary of Schamhaupten, Bavaria. PeerJ 6:e4191. PubMed PMC

Reno PL, McCollum MA, Cohn MJ, Meindl RS, Hamrick M, Lovejoy CO.. 2008. Patterns of correlation and covariation of anthropoid distal forelimb segments correspond to Hoxd expression territories. J Exp Zool B Mol Dev Evol. 310(3):240–258. PubMed

Richardson 2012. Manus horribilis: the chicken wing skeleton. In: Asher RJ, Müller J, editors. From clone to bone - the synergy of morphological and molecular tools in palaeobiology. Cambridge: Cambridge University Press.

Richardson MK, Jeffery JE, Tabin CJ.. 2004. Proximodistal patterning of the limb: insights from evolutionary morphology. Evol Dev. 6(1):1–5. PubMed

Salinas-Saavedra M, Gonzalez-Cabrera C, Ossa-Fuentes L, Botelho JF, Ruiz-Flores M, Vargas AO.. 2014. New developmental evidence supports a homeotic frameshift of digit identity in the evolution of the bird wing. Front Zool. 11(1):33. PubMed PMC

Santa Luca AP.1980. The postcranial skeleton of Heterodontosaurus tucki (Reptilia, Ornithischia) from the Stormberg of South Africa. Ann S Afr Mus. 7:159–211.

Santa Luca AP, Crompton AW, Charig AJ.. 1976. A complete skeleton of the Late Triassic ornithischian Heterodontosaurus tucki. Nature 264(5584):324–328.

Schaller NU, D'Août K, Villa R, Herkner B, Aerts P.. 2011. Toe function and dynamic pressure distribution in ostrich locomotion. J Exp Biol. 214(7):1123–1130. PubMed

Seki R, Li C, Fang Q, Hayashi S, Egawa S, Hu J, Xu L, Pan H, Kondo M, Sato T, et al.2017. Functional roles of Aves class-specific cis-regulatory elements on macroevolution of bird-specific features. Nat Commun. 8(1):14229. PubMed PMC

Sereno PC.1993. The pectoral girdle and forelimb of the basal theropod Herrerasaurus Ischigualastensis. J Vert Paleontol. 13:425–450.

Sereno PC.2012. Taxonomy, morphology, masticatory function and phylogeny of heterodontosaurid dinosaurs. ZooKeys 226:1–225. PubMed PMC

Sordino P, van der Hoeven F, Duboule D.. 1995. Hox gene expression in teleost fins and the origin of vertebrate digits. Nature 375(6533):678–681. PubMed

SpiekmanSNF, , NeenanJM, , FraserNC, , FernandezV, , RieppelO, , NosottiS, , Scheyer TM.. 2020. Aquatic Habits and Niche Partitioning in the Extraordinarily Long-Necked Triassic Reptile Tanystropheus. Curr Biol. 30(19):3889–3895.e2. PubMed

SteinRW, , BrownJW, , Mooers AØ.. 2015. A molecular genetic time scale demonstrates Cretaceous origins and multiple diversification rate shifts within the order Galliformes (Aves). Mol Phylogenet Evol. 92:155–164. PubMed

Stewart TA, Liang C, Cotney JL, Noonan JP, Sanger TJ, Wagner GP.. 2019. Evidence against tetrapod-wide digit identities and for a limited frame shift in bird wings. Nat Commun. 10(1):3244. PubMed PMC

Suzuki T, Hasso SM, Fallon JF.. 2008. Unique SMAD1/5/8 activity at the phalanx-forming region determines digit identity. Proc Natl Acad Sci U S A. 105(11):4185–4190. PubMed PMC

Tamura K, Nomura N, Seki R, Yonei-Tamura S, Yokoyama H.. 2011. Embryological evidence identifies wing digits in birds as digits 1, 2, and 3. Science 331(6018):753–757. PubMed

Tickle C, Towers M.. 2017. Sonic hedgehog signaling in limb development. Front Cell Dev Biol. 5:14. PubMed PMC

Towers M.2018. Evolution of antero-posterior patterning of the limb: insights from the chick. Genesis 56: PubMed PMC

Towers M, Signolet J, Sherman A, Sang H, Tickle C.. 2011. Insights into bird wing evolution and digit specification from polarizing region fate maps. Nat Commun. 2(1):Article number 426. PubMed

van der VosW, , SteinK, , Di-PoïN, , Bickelmann C.. 2018. Ontogeny of Hemidactylus (Gekkota, Squamata) with emphasis on the limbs. Zoosyst Evol. 94(1):195–209.

Vargas AO, Fallon JF.. 2005. Birds have dinosaur wings: the molecular evidence. J Exp Zool. 304B(1):86–90. PubMed

Vargas AO, Kohlsdorf T, Fallon JF, Vandenbrooks J, Wagner GP.. 2008. The evolution of HoxD-11 expression in the bird wing: insights from Alligator mississippiensis. PLoS One. 3(10):e3325. PubMed PMC

Wagner GP, Gauthier JA.. 1999. 1,2,3 = 2,3,4: a solution to the problem of the homology of the digits in the avian hand. Proc Natl Acad Sci U S A. 96(9):5111–5116. PubMed PMC

Welten MCM, Verbeek FJ, Meijer AH, Richardson MK.. 2005. Gene expression and digit homology in the chicken embryo wing. Evol Dev. 7(1):18–28. PubMed

Wild R.1973. Die Triasfauna der Tessiner Kalkalpen XXII. Tanystropheus longobardicus (Bassani) (Neue Ergebnisse). Schweiz Paläontol Abhand. 95:1 A 162.

Woltering JM, Duboule D.. 2010. The origin of digits: expression patterns versus regulatory mechanisms. Dev Cell. 18(4):526–532. PubMed

Xu X, Mackem S.. 2013. Tracing the evolution of avian wing digits. Curr Biol. 23(12):R538–R544. PubMed PMC