A Palaeozoic shark with osteichthyan-like branchial arches

LETTER doi:10.1038/nature13195 A Palaeozoic shark with osteichthyan-like branchial arches Alan Pradel1, John G. Maisey1, Paul Tafforeau2, Royal H. Mapes3 & Jon Mallatt4 The evolution of serially arranged, jointed endoskeletal supports internal to the gills—the visceral branchial arches—represents one of the key events in early jawed vertebrate (gnathostome) history, because it provided the morphological basis for the subsequent evolution of jaws1–5. However, until now little was known about visceral arches in early gnathostomes6–17, and theories about gill arch evolution were driven by information gleaned mostly from both modern cartilaginous (chondrichthyan) and bony (osteichthyan) fishes. New fossil discoveries can profoundly affect our understanding of evolutionary history, by revealing hitherto unseen combinations of primitive and derived characters18,19. Here we describe a 325 million year (Myr)-old Palaeozoic shark-like fossil that represents, to our knowledge, the earliest identified chondrichthyan in which the complete gill skeleton is three-dimensionally preserved in its natural position. Its visceral arch arrangement is remarkably osteichthyan-like, suggesting that this may represent the common ancestral condition for crown gnathostomes. Our findings thus reinterpret the polarity of some arch features of the crown jawed vertebrates and invert the classic hypothesis, in which modern sharks retain the ancestral condition3,20. This study underscores the importance of early chondrichthyans in resolving the evolutionary history of jawed vertebrates. The visceral skeleton of jawed vertebrates consists of a series of jointed arches including the jaws, hyoid arch and gill arches. This fundamental arrangement is shared by all chondrichthyans (sharks, rays and chimaeroids) and osteichthyans (bony fishes and their limbed relatives), as well as the extinct ‘placoderm’ and ‘acanthodian’ fishes. It thus represents a highly conserved feature of gnathostomes. Important differences in arch structure between modern chondrichthyans and osteichthyans5,9,21,22 could reflect their long independent evolutionary history (over 420 Myr). The modern osteichthyan arrangement is already recognizable in early actinopterygian (‘ray-finned’) and sarcopterygian (‘lobe-finned’) osteichthyans (for example, Pteronisculus8, Mimipiscis9, Eusthenopteron12) and is commonly assumed to be derived, whereas the modern shark pattern is viewed as primitive3,20. Crucially, little was known about visceral arches in Palaeozoic chondrichthyans11,13–15,17 (Supplementary Notes), precluding a detailed comparison with other early jawed vertebrates. Articulated, three-dimensionally preserved specimens of a small symmoriiform shark (stem chondrichthyan23) from the Lower Carboniferous period of Arkansas, United States, were investigated by propagation phase contrast X-ray synchrotron microtomography, which revealed the complete series of visceral arches on both sides (Fig. 1 and Supplementary Video 1). Although several specimens were examined, the most complete example (American Museum of Natural History (AMNH) FF20544) is used here to illustrate our findings (Fig. 1 and Extended Data Fig. 1). The specimens possess tessellated calcified cartilage, which is considered to be the hallmark character of ‘conventionally defined’ chondrichthyans19, a group that we informally name ‘euchondrichthyans’. Class Chondrichthyes Huxley, 1880 Order Symmoriiformes Zangerl, 1981 Family Falcatidae Zangerl, 1990 Ozarcus mapesae gen. et sp. nov. Etymology. The generic name derives from Ozark (a highland region of Arkansas where the specimens were found) and ultimately from arcus (Latin for arch). The species is named after G. K. Mapes in recognition of her work collecting and describing fossils from Palaeozoic strata in the United States for more than 40 years, and who found the holotype specimen. Holotype. AMNH FF20544 (Fig. 1 and Extended Data Fig. 1). Referred material. Articulated heads: AMNH FF20525, 20528 and 20542. Locality and horizon. Fayetteville Formation (lower shale member), Chesterian, Upper Mississippian, from the ARC-07 locality24 (section (sec.) 22, township (T.) 14 N, range (R.) 15 W) near Leslie, Searcy County, Arkansas, United States. Diagnosis. Falcatidae possessing small cladodont, pentacuspid and symmetrical teeth; ten upper and lower tooth families; palatoquadrate lacks a continuous posterior quadrate margin; no dermal denticles covering the top of the head; no ‘spine–brush’ complex; no laterally extended supraorbital shelf; laterally extended antorbital process that overlies a suborbital process possessing a series of four ridges and grooves for the articulation with the palatoquadrate (Supplementary Notes and Extended Data Fig. 2). The visceral skeleton of Ozarcus has the same basic organization as in most other piscine jawed vertebrates, with a series of paired, jointed arches including a mandibular arch (jaw), hyoid arch (behind the jaw) and five branchial arches (Fig. 1). The palatoquadrate articulates with the postorbital process of the braincase (Fig. 1b) as in many other Palaeozoic sharks. No traces of labial cartilages were identified. The hyoid arch of Ozarcus includes paired epihyals and ceratohyals, but neither pharyngohyals nor interhyals. Unlike in modern chondrichthyans22, anteriorly directed, osteichthyan-like21 paired hypohyals are present instead of a median basihyal (Figs 1f, 2 and Extended Data Fig. 3). Some other Palaeozoic euchondrichthyans also possess hypohyals, but the distribution of basihyals and hypohyals among most early gnathostomes is poorly resolved (Supplementary Notes). The epi- and ceratohyals are morphologically similar to the corresponding branchial elements (Figs 1b, e, 2a), as in modern chimaeroids but unlike in elasmobranchs, where the hyoid arch is specialized towards jaw suspension5. The epihyal extends forward to just behind the orbit, but both the epihyal and the braincase lack any articular processes or recesses, suggesting that the epihyal was weakly connected to the braincase (Extended Data Fig. 3b, c). The epihyal meets the ceratohyal well posterior to the jaw joint, leaving a space between the mandibular and hyoid arches that, even allowing for compaction of the fossil, is far wider than those between the branchial arches (Fig. 1b and Extended Data Fig. 3a); a similar space has been inferred in the symmoriiform Cobelodus15. Its conjunctionwiththerecessedpalatoquadratemargininOzarcusmayindicate thepresenceof a larger version of thenon-respiratory, pseudobranch-bearing 1 Department of Vertebrate Paleontology, American Museum of Natural History, Central Park West at 79th Street, New York, New York 10024, USA. 2European Synchrotron Radiation Facility, BP 220, 6 rue Jules Horowitz, 38043 Grenoble Cedex, France. 3Department of Geological Sciences, Ohio University, Athens, Ohio 45701, USA. 4School of Biological Sciences, Washington State University, Pullman, Washington 99164-4236, USA. 0 0 M O N T H 2 0 1 4 | VO L 0 0 0 | N AT U R E | 1 ©2014 Macmillan Publishers Limited. All rights reserved RESEARCH LETTER d a postp e b br br eh pq eh pq mc ch mc ch eb1–5 c sb4 bb2 sb3 ib4+5 ib3 sb2 ib2 sb1 eb1–5 bb3 f ib1 cp eb3 eb5 eb2 eb1 hh hb1 eb4 mc hb3 hb2 cb1 ch cb2 cb3 cb4 cb5 ac4 ac3 ac2 ac1 Figure 1 | Three-dimensional reconstructions of Ozarcus mapesae AMNH FF20544. a, Concretion in right lateral view. b, Braincase and associated visceral skeleton in right lateral view. c, Branchial skeleton in right lateral view (mandibular and hyoid arches removed). d, Concretion in left lateral view. e, Braincase and associated visceral skeleton in left lateral view. f, Braincase and the right elements of the visceral skeleton in medial aspect, left lateral view. Colour coding of the skeletal elements: yellow, epi-; blue, cerato-; green, hypo-; orange, infrapharyngo-; turquoise, suprapharyngo-; purple, accessory elements; red, basi-; peachy pink, braincase. The colours of the mandibular elements are lightened and those of the hyoid are darkened. The white circle indicates the space between the mandibular and hyoid arches. ac1–4, accessory elements of branchial arches 1–4; bb2–3, basibranchials 2–3; br, braincase; cb1–5, ceratobranchials 1–5; cp, copula of posterior basibranchials; eb1–5, epibranchials 1–5; eh, epihyal; hb1–3, hypobranchials 1–3; hh, hypohyal; ib1–4, infrapharyngobranchials 1–5; mc, Meckel’s cartilage/lower jaw; postp, postorbital process; pq, palatoquadrate/upper jaw; sb1–4, suprapharyngobranchials 1–4. Scale bar, 10 mm. spiracular pouch seen in modern jawed vertebrates5, or perhaps even a fully respiratory hyomandibular gill pouch1,3,16,25 (Supplementary Notes and Extended Data Fig. 4). The branchial arches include the basi-, hypo-, cerato-, epi- and pharyngobranchials (Figs 1, 2), arranged in a way not previously observed in a euchondrichthyan. Instead of a single pharyngobranchial as in modern chondrichthyans, arches 1–4 each have two pharyngobranchials (Figs 1c, 2). We regard these as homologues of the infra- and suprapharyngobranchials in osteichthyans6,21 (Supplementary Notes). The single pharyngobranchial in modern chondrichthyans is topographically homologous with osteichthyan infrapharyngobranchials6,10, not with the suprapharyngobranchials26, because both chondrichthyan pharyngobranchials and osteichthyan infrapharyngobranchials6 support the roof of the pharynx. Unlike in modern chondrichthyans, the infrapharyngobranchials of Ozarcus are directed anteriorly, as in osteichthyans. A separate, fifth epibranchial element is present in Ozarcus (Figs 1b, c, e, 2), as well as in Debeerius13, some extinct hybodont sharks (for example, Tribodus27) and Acanthodes28. By contrast, epibranchial element 5 is fused with pharyngobranchial 4 in modern chondrichthyans and is absent in crown osteichthyans20. The last ceratobranchial in Ozarcus is not broadened laterally, a feature shared with xenacanths, Debeerius13 and chimaeroids. By contrast, in many modern sharks and the Carboniferous shark Tristychius17, the last ceratobranchial is much wider than those farther anteriorly. Ventrally, arches 3–5 meet the lateral margins of the basibranchial copula, as in osteichthyans (Figs 1f, 2), whereas only the last ceratobranchial is attached to the copula in modern chondrichthyans. All the hypobranchials are directed anteriorly in Ozarcus (Figs 1c, f, 2), an arrangement shared with osteichthyans5, Acanthodes10, modern chimaeroids7 and Debeerius13. By contrast, only hypobranchial 1 is anteriorly directed in some modern elasmobranchs (for example, Scyliorhinus canicula; Fig. 3). Small ‘accessory cartilages’ are present between the ceratobranchial and epibranchial on each of the first four gill arches (Figs 1c, e, 2). These were previously unknown in chondrichthyans, but similar elements are a b Figure 2 | Reconstructions of the branchial skeleton of O. mapesae. a, Right elements of the branchial skeleton (mandibular arch removed) in lateral aspect, right lateral view. b, Branchial skeleton reconstructed as horizontally spread, viewed dorsally and with anterior above (mandibular arch removed). Same colours and abbreviations as in Fig. 1. Not to scale. 2 | N AT U R E | VO L 0 0 0 | 0 0 M O N T H 2 0 1 4 ©2014 Macmillan Publishers Limited. All rights reserved LETTER RESEARCH Crown gnathostomes Osteichthyans Chondrichthyans Crown chondrichthyans Elasmobranchs Holocephalans Scyliorhinus canicula Callorhinchus milii Actinopterygians † Pteronisculus stensioi Sarcopterygians † Eusthenopteron foordi hb post. sb lost ib post. ac lost hh lost ?bh ?eb5 fused to ib 4+5 † Ozarcus mapesae † Acanthodes bronni sb ib ant. hb ant. ac hh Figure 3 | Evolution of the branchial skeleton in the crown gnathostomes, mapped onto a tree compiled from the most recent phylogenetic analyses19,23,28. Only the right and median parts of the branchial skeletons of Scyliorhinus, Callorhinchus, Ozarcus, Acanthodes10,28, Pteronisculus8 and Eusthenopteron12 are shown, as viewed dorsally and with anterior above. Diagrammatic right views of one branchial arch are shown at the crown gnathostome, chondrichthyan and osteichthyan nodes of the tree. To maximize clarity, the branchial skeletons are reconstructed as horizontally spread and the opercular cartilage of Callorhinchus is not shown. Some elements of Acanthodes, Pteronisculus and Eusthenopteron are outlined in dashes or are uncoloured, because of their uncertain existence or uncertain homologies (Supplementary Notes). hb ant., hypobranchials anteriorly directed; hb post., hypobranchials posteriorly directed; ib ant., infrapharyngobranchial anteriorly directed; ib post., infrapharyngobranchial posteriorly directed. Light orange, compound infrapharyngobranchial 4, 5 (Scyliorhinus) or 3, 4, 5 (Callorhinchus) plus epihyal 5 (Scyliorhinus) or 4, 5 (Callorhinchus). Other colours and abbreviations are the same as in Fig. 1. Shared derived characters of each clade are listed at the corresponding nodes. The layout is not intended to reflect strict chronological history. Not to scale. Daggers indicate fossil taxa. present in modern basal actinopterygians (Polypteriformes, Lepisosteiformes, possibly Acipenseriformes)20 and in the Triassic actinopterygian Pteronisculus8. It has been suggested that these accessory cartilages are serially homologous with the symplectic or the interhyal of the hyoid arch8,20. Their occurrence in a stem chondrichthyan considerably expands their distribution, suggesting that they are a gnathostome symplesiomorphy. No traces of extrabranchial cartilages3 were found in Ozarcus. Ozarcus considerably broadens our understanding of visceral arch morphology in jawed vertebrates. Several features can now be reinterpreted as primitive for crown gnathostomes (Fig. 3 and Supplementary Notes), including: presence of supra- and infrapharyngobranchials, anteriorly directed infrapharyngobranchials, chevron arrangement of branchial arches along the basibranchial copula, all hypobranchials directed anteriorly, accessory cartilages between the cerato- and epibranchials, and paired hypohyals. Additionally, several features are reformulated here as potential synapomorphies of crown chondrichthyans, including a single (infra)pharyngobranchial element per arch, infrapharyngobranchials directed posteriorly, hypobranchials of the branchial arches concentrated at the anterior part of the basibranchial copula, absence of accessory cartilages, and absence of paired hypohyals anterior to the ceratohyals. The presence of a basihyal and fusion of epibranchial 5 with pharyngobranchial 4 may be apomorphic for, or within, crown chondrichthyans (Supplementary Notes). Having most of the hypobranchials directed posteriorly is interpreted as a synapomorphy of living elasmobranchs and extinct hybodont sharks (collectively known as euselachians29), not shared with modern chimaeroids. Ozarcus challenges a widely held opinion that the ancestral state of the gnathostome branchial arch resembled the S-shape arches of 0 0 M O N T H 2 0 1 4 | VO L 0 0 0 | N AT U R E | 3 ©2014 Macmillan Publishers Limited. All rights reserved RESEARCH LETTER modern sharks, which have posteriorly directed pharyngo- and hypobranchial elements. Instead, the ,-shaped arrangement of the bony fishes, with anteriorly directed pharyngo- and hypobranchial elements, is likely to be primitive (Fig. 3). Ozarcus thus shows a novel combination of chondrichthyan and osteichthyan characters, thereby demonstrating that the most recent common ancestor of crown gnathostomes possessed an osteichthyan-like branchial apparatus. Our findings cast doubt on the traditional view of visceral arch evolution that modern chondrichthyans mirror the ancestral morphotype of jawed vertebrates. Bony fishes and stem chondrichthyans may have more to tell us about our first jawed ancestors than do living sharks. 14. 15. 16. 17. 18. 19. 20. METHODS SUMMARY The O. mapesae specimens were scanned on the ID19 beamline of the European Synchrotron Radiation Facility (ESRF). The scan parameters were as follows: voxel size 30.3 mm; single propagation distance of 3 m; BM5 beam, filtered by 90 mm of aluminium and 0.1 mm of molybdenum; effective energy 106 KeV; attenuation protocol with beam profiler; CCD FreLon 2K14 detector camera; LuAG 750 mm cintillator; double scan, half acquisition, with 5,000 projections of 0.1 s, phase retrieval using a Paganin process. The final reconstruction (60 mm voxel size) was obtained after binning. Volumes were reconstructed using ESRF software PyHST. Segmentation and three-dimensional rendering were performed with MIMICS 15.01 64-bit software. Details of both the S. canicula and Callorhinchus milii specimens are provided elsewhere30. Online Content Any additional Methods, Extended Data display items and Source Data are available in the online version of the paper; references unique to these sections appear only in the online paper. Received 11 December 2013; accepted 3 March 2014. 21. 22. 23. 24. 25. 26. 27. 28. Published online 16 April 2014. 29. 1. 2. 3. 30. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Gegenbaur, C. Elements of Comparative Anatomy (Macmillan, 1878). Janvier, P. Early Vertebrates (Oxford Univ. Press, 1996). Mallatt, J. Ventilation and the origin of jawed vertebrates: a new mouth. Zool. J. Linn. Soc. 117, 329–404 (1996). Kuratani, S. Evolution of the vertebrate jaw from developmental perspective. Evol. Dev. 14, 76–92 (2012). De. Beer, G. R. The Development of the Vertebrate Skull (Clarendon, 1937). Nelson, G. J. in Nobel Symposium 4, Current Problems of Lower Vertebrate Phylogeny (ed. Ørvig, T.) 129–143 (Almqvist & Wiksell, 1968). Nelson, G. J. Gill arches and the phylogeny of fishes, with notes on the classification of vertebrates. Bull. Am. Mus. Nat. Hist. 141, 477–552 (1969). Nielsen, E. Studies on Triassic fishes from East Greenland. I. Glaucolepis and Boreosomus Vol. 1 (C. A. Reitzel, 1942). Gardiner, B. G. The relationships of the palaeoniscid fishes, a review based on new specimens of Mimia and Moythomasia from the Upper Devonian of Western Australia. Bull. Br. Mus. Nat. Hist. Geol. 37, 173–428 (1984). Miles, R. S. In Interrelationships of Fishes (eds Greenwood, P. H., Miles, R. S. & Patterson, C.) 63–103 (Academic, 1973). Heidtke, U. H. J. & Krätschmer, K. Gladbachus adentatus nov. gen. et sp., a primitive shark from the Upper Givetian Upper Middle Devonian of Bergisch Gladbach Paffrath Basin Rhenish Slate Mountains [in German]. Mainzer Geowissenschaftliche Mitteilungen 30, 105–122 (2001). Jarvik, E. Basic Structure and Evolution of Vertebrates Vol. 1 (Academic, 1980). Grogan, E. D. & Lund, R. Debeerius ellefseni (fam. nov., gen. nov., spec. nov.), an autodiastylic chondrichthyan from the Mississippian Bear Gulch Limestone of Montana (USA), the relationships of the Chondrichthyes, and comments on gnathostome evolution. J. Morphol. 243, 219–245 (2000). Coates, M. I. & Sequeira, S. E. K. A new stethacanthid chondrichthyan from the Lower Carboniferous of Bearsden, Scotland. J. Vertebr. Paleontol. 21, 438–459 (2001). Zangerl, R. & Williams, M. E. New evidence of the nature of the jaw suspension in Palaeozoic anacanthus sharks. Palaeontology 18, 333–341 (1975). Watson, D. M. S. The acanthodian fishes. Philos. T. Roy. Soc. B 228, 49–146 (1937). Dick, J. R. On the Carboniferous shark Tristychius arcuatus Agassiz from Scotland. T. Roy. Soc. Edin. 70, 63–108 (1978). Patterson, C. Significance of fossils in determining evolutionary relationships. Annu. Rev. Ecol. Syst. 12, 195–223 (1981). Zhu, M. et al. A Silurian placoderm with osteichthyan-like marginal jaw bones. Nature 502, 188–193 (2013). Carvalho, M., Bockmann, F. A. & de Carvalho, M. R. Homology of the fifth epibranchial and accessory elements of the ceratobranchials among gnathostomes: insights from the development of Ostariophysans. PLoS ONE 8, e62389 (2013). Friedman, M. & Brazeau, M. D. A reappraisal of the origin and basal radiation of the Osteichthyes. J. Vertebr. Paleontol. 30, 36–56 (2010). Shirai, S. Squalean Phylogeny: A New Framework of ‘‘Squaloid’’ Sharks and Related Taxa (Hokkaido Univ. Press, 1992). Pradel, A., Tafforeau, P., Maisey, J. G. & Janvier, P. A new Paleozoic Symmoriiformes (Chondrichthyes) from the Late Carboniferous of Kansas (USA) and cladistic analysis of early chondrichthyans. PLoS ONE 6, e24938 (2011). Mapes, R. H. Carboniferous and Permain Bactritoidia (Cephalopoda) in North America (Univ. of Kansas Paleontological Institute, 1979). Mallatt, J. Shark pharyngeal muscle and early vertebrate evolution. Acta Zool. 78, 279–294 (1997). Holmgren, N. Studies on the head in fishes. Part III. The phylogeny of elasmobranch fishes. Acta Zool. 23, 129–261 (1942). Lane, J. A. & Maisey, J. G. The visceral skeleton and jaw suspension in the durophagous hybodontid shark Tribodus limae from the Lower Cretaceous of Brazil. J. Vertebr. Paleontol. 86, 886–905 (2012). Davis, S. P., Finarelli, J. A. & Coates, M. I. Acanthodes and shark-like conditions in the last common ancestor of modern gnathostomes. Nature 486, 247–250 (2012). Maisey, J. G. What is an ‘elasmobranch’? The impact of palaeontology in understanding elasmobranch phylogeny and evolution. J. Fish Biol. 80, 918–951 (2012). Pradel, A., Didier, D. A., Casane, D., Tafforeau, P. & Maisey, J. G. Holocephalan embryo provides new information on the evolution of the glossopharyngeal nerve, metotic fissure and parachordal plate in gnathostomes. PLoS ONE 8, e66988 (2013). Supplementary Information is available in the online version of the paper. Acknowledgements We thank staff at the ID19 beamline at the ESRF for assistance, and F. Ippolito (AMNH) for photographs of the specimens. The main work was supported by the H. R. & E. Axelrod Research Chair in paleoichthyology at the AMNH. Author Contributions J.G.M. and A.P. conceived the project. A.P. performed computerized microtomography restorations. A.P., J.G.M. and J.M. interpreted the results and prepared the manuscript. P.T. performed synchrotron computerized microtomography on the material. R.H.M. did the fieldwork. Author Information Data have been deposited in ZooBank under the following LSIDs: urn:lsid:zoobank.org:pub:D80955F3-7C21-457C-BA68-9C70F466E913 (article); urn:lsid:zoobank.org:act:F7EDD4EB-6700-4583-8569-9BAC89B3A945 (genus); and urn:lsid:zoobank.org:act:6ACCB5D2-E0D7-4BFD-85D6-9C8250515614 (species). Reprints and permissions information is available at www.nature.com/ reprints. The authors declare no competing financial interests. Readers are welcome to comment on the online version of the paper. Correspondence and requests for materials should be addressed to A.P. (apradel@amnh.org) or J.G.M. (maisey@amnh.org). 4 | N AT U R E | VO L 0 0 0 | 0 0 M O N T H 2 0 1 4 ©2014 Macmillan Publishers Limited. All rights reserved LETTER RESEARCH METHODS 24 Locality. The specimen was collected from the ARC-07 locality (section 22, T. 14 N., R. 15 W.), from black dysoxic/anoxic shale of the Fayetteville Formation (Carboniferous, upper Mississippian, Chesterian, Pendleian stage, E1 zone 5 lower Namurian A), near Leslie, Searcy County, Arkansas. Other fossils from the same locality include invertebrates preserved by calcite, pyrite and phosphate, plants preserved as permineralizations, coprolites, and vertebrates (mostly chondrichthyans). The chondrichthyan remains are mostly preserved in a hard phosphatic matrix that is difficult to prepare by conventional means. Synchrotron. The Ozarcus specimens were scanned on the ID19 beamline of the European Synchrotron Radiation Facility (ESRF), supported by glass balls contained in a cylinder; voxel size 30.3 mm, with a single propagation distance of 3 m; BM5 beam, filteredby90 mmof aluminiumand0.1 mmof molybdenum; effectiveenergy106 KeV. We used an attenuation protocol with beam profiler. The detector camera was a CCD FreLon 2K14, and the scintillator a LuAG 750 mm. We performed a double scan, half acquisition, with 5,000 projections of 0.1 s, with phase retrieval using a Paganin process. The final reconstruction (60 mm voxel size) was obtained after binning. Volumes were reconstructed using ESRF software PyHST. Subvolumes were corrected for ring artefacts and concatenated to generate a single stack of 16-bit tif slices. Segmentation and three-dimensional rendering were performed with MIMICS 15.01 64-bit software (Materialise). Details about the origin, preparation and scanning of both the S. canicula and Callorhinchus milii specimens shown in Fig. 3 are provided elsewhere30. ©2014 Macmillan Publishers Limited. All rights reserved RESEARCH LETTER Extended Data Figure 1 | Photographs of O. mapesae AMNH FF 20544 (holotype). a, Right lateral view. b, Left lateral view. Scale bar, 10 mm. ©2014 Macmillan Publishers Limited. All rights reserved LETTER RESEARCH Extended Data Figure 2 | The diagnostic features of O. mapesae. a, b, Part of right palatoquadrate in Ozarcus (a) and Akmonistion14 (b) showing smaller size of teeth (t) relative to the dental bullae (db) in Ozarcus. Anterior to left. Not to scale. c, One tooth family (top) composed of tiny cladodont, pentacuspid and symmetrical teeth in lingual (left) and labial (right) views. d, e, f, g, The ventral (d) and lateral (e) surfaces of the palatoquadrate (right palatoquadrate shown here) and the dorsal (f) and medial (g) surface of the Meckel’s cartilage (left Meckel’s cartilage shown here) show ten concavities that each housed a tooth family (tfc). The quadrate part of the palatoquadrate (q) lacks a continuous posterior margin. h, The neurocranium (top, right lateral view; bottom, ventral view) possesses a laterally extended antorbital process (ap) that overlies a suborbital process (subp) which displays a series of four ridges and grooves for articulation with the palatoquadrate. ©2014 Macmillan Publishers Limited. All rights reserved RESEARCH LETTER Extended Data Figure 3 | Three-dimensional reconstructions of O. mapesae showing the relationship of the hyoid arch with the surrounding cartilages. a, Braincase, right mandibular and hyoid arches in lateral right view. b, Braincase and right hyoid arch in lateral right view. c, Braincase and right hyoid arch in oblique ventral view. d, Dorsal (epi-, supra- and infra-) branchial cartilages of the right hyoid and branchial arches in oblique dorsal view. Colour coding of the skeletal elements: yellow, epi-; blue, cerato-; green, hypo-; orange, infrapharyngo-; turquoise, suprapharyngo-; peachy pink, braincase. The colours of the mandibular elements are lightened and those of the hyoid are darkened. Asterisk indicates the space between the mandibular and hyoid arches. ©2014 Macmillan Publishers Limited. All rights reserved LETTER RESEARCH Extended Data Figure 4 | The aphetohyoidean hypothesis and jaw evolution. a, c, d, The stage shown in a leads to c and d, both of which have a jaw-supporting hyoid. b, Ozarcus may fit just after a, because of its possible aphetohyoidean aspects. However, it is impossible to know whether Ozarcus had a respiratory gill pore between its mandibular and hyoid arches (indicated View publication stats by a question mark). Colours are as in Fig. 1, plus: light pink, gill pouches; dark pink, otic capsule of braincase; purple, interhyal. gp, gill pore; gp1, first (mandibulohyoid) gill pore; ma, mandibular arch; mh, mandibulohyoid (first) gill pouch; oc, otic capsule; sp., spiracle; spp., spiracular pouch. Not to scale. ©2014 Macmillan Publishers Limited. All rights reserved