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Ontogenetic development of the otic region in the new model organism, Leucoraja erinacea (Chondrichthyes; Rajidae)

Published online by Cambridge University Press:  18 January 2019

Cathrin PFAFF
Affiliation:
University of Vienna, Department of Palaeontology, Althanstrasse 14, 1090 Vienna, Austria. Email: cathrin.pfaff@univie.ac.at
Jürgen KRIWET
Affiliation:
University of Vienna, Department of Palaeontology, Althanstrasse 14, 1090 Vienna, Austria. Email: cathrin.pfaff@univie.ac.at
Kyle MARTIN
Affiliation:
Department of Earth Sciences, Natural History Museum, Cromwell Road, London SW75BD, UK.
Zerina JOHANSON
Affiliation:
Department of Earth Sciences, Natural History Museum, Cromwell Road, London SW75BD, UK.
Corresponding

Abstract

Cartilaginous fishes have a long evolutionary history dating back 440 million years and include model organisms in a number of fields of biological research. However, comparative developmental studies of these organisms, particularly neuroanatomical investigations, still remain sparse. Here, pre-hatching to adult developmental stages of the Little Skate, Leucoraja erinacea, are investigated using micro-computed tomography scanning in conjunction with staining procedures designed to improve visualisation of soft tissues. Within the ear, the anatomy of the skeletal labyrinth changes during ontogeny and differs substantially from the underlying membranous system, contrary to previous observations in sharks. Additionally, substantial morphological remodelling characterises the parietal fossa, which appears initially as a massive and hook-like structure and subsequently becomes slender and surrounded by soft tissue. The sizes of the vestibular system and neurocranium increase isometrically from pre- to post-hatching phases, and then exponentially after the post-hatching stages.

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Articles
Copyright
Copyright © The Royal Society of Edinburgh 2019 

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References

Aschliman, N. C., Nishida, M., Miya, M., Inoue, J. G., Rosana, K. M. & Naylor, G. J. P. 2012a. Body plan convergence in the evolution of skates and rays (Chondrichthyes: Batoidea). Molecular Phylogenetics and Evolution 63, 2842.CrossRefGoogle Scholar
Aschliman, N. C., Claeson, K. M. & McEachran, J. D. 2012b. Phylogeny of batoidea. In Carrier, J. C., Musick, J. A. & Heithaus, M. R. (eds) Biology of sharks and their relatives, 2nd edn, 5796. Boca Raton, FL: CRC Press.CrossRefGoogle Scholar
Ayers, H. 1892. Vertebrate cephalogenesis. II. A contribution to the morphology of the vertebrate ear, with reconsideration of its function. Journal of Morphology 6, 1–360.CrossRefGoogle Scholar
Banner, A. 1967. Evidence of sensitivity to acoustic displacements in the lemon shark, Negaprion brevirostris (Poey). In Cahn, P. (ed.) Lateral line detectors, 265273. Bloomington, IN: Indiana University Press.Google Scholar
Barber, V. C., Yake, K. I., Clark, V. F. & Pungur, J. 1985. Quantitative analyses of sex and size differences in the macula neglecta and ramus neglectus in the inner ear of the skate, Raja ocellata. Cell and Tissue Research 241, 597605.CrossRefGoogle Scholar
Benoit, J., Lehmann, T., Vatter, M., Lebrun, R., Merigeaud, S., Costeur, L. & Tabuce, R. 2015. Comparative anatomy and three-dimensional geometric-morphometric study of the bony labyrinth of Bibymalagasia (Mammalia, Afrotheria). Journal of Vertebrate Paleontology 35, e930043.CrossRefGoogle Scholar
Bertozzi, T., Lee, M. S. Y. & Donnellan, S. C. 2016. Stingray diversification across the end-Cretaceous extinctions. Memoirs of Museum Victoria 74, 379390.CrossRefGoogle Scholar
Bleckman, H. & Hoffman, M. H. 1999. Special senses. In Hamlett, W. C. (ed.) Sharks, skates and rays: the biology of elasmobranch fishes, 300328. Baltimore, MD: John Hopkins Press.Google Scholar
Braun, C. B. & Grande, T. 2008. Evolution of peripheral mechanisms for the enhancement of sound reception. In Webb, J. F., Fay, R. R. & Popper, A. N. (eds) Fish bioacoustics, 99144. New York: Springer.CrossRefGoogle Scholar
Breschet, G. 1838. Recherches anatomique et physiologique sur l'organe de l'ouie des poissons. Des Mémoires de l'Académie des Sciences. Paris: Imprimerie Royale.Google Scholar
Bustamate, C., Lamilla, J., Concha, F., Ebert, D. A. & Benett, M. B. 2012. Morphological characters of the thickbody skate Amblyraja frerichsi (Krefft 1968) (Rajiformes. Rajidae), with notes on its biology. Plos One 7, e39963.Google Scholar
Carlström, D. 1963. A crystallographic study of vertebrate otoliths. Biological Bulletin 125, 441463.CrossRefGoogle Scholar
Casper, B. M. & Mann, D. A. 2006. Evoked potential audiograms of the nurse shark (Ginglymostoma cirratum) and the yellow stingray (Urobatis jamaicensis). Environmental Biology of Fishes 76, 101108.CrossRefGoogle Scholar
Clack, J. A. 2016. Vertebrate diversity in a sensory system: the fossil record of otic evolution. In Clack, J. A., Fay, R. R. & Popper, A. N. (eds) Evolution of the vertebrate ear: evidence from the fossil record, 117. Cham: Springer.CrossRefGoogle Scholar
Clack, J. A., Ahlberg, P. E., Finney, S. M., Dominiquez, A. P., Robinson, J. & Ketcham, R. A. 2003. A unique specialised ear in a very early tetrapod. Nature 425, 6569.CrossRefGoogle Scholar
Clack, J. A. & Anderson, J. S. 2016. Early tetrapods: experimenting with form and function. In Clack, J. A., Fay, R. R. & Popper, A. N. (eds) Evolution of the vertebrate ear: evidence from the fossil record, 71107. Cham: Springer.CrossRefGoogle Scholar
Coates, M. I., Gess, R. W., Finarelli, J. A., Criswell, K. E. & Tietjen, K. 2017. A symmoriiform chondrichthyan braincase and the origin of chimaeroid fishes. Nature 541, 208211.CrossRefGoogle ScholarPubMed
Corwin, J. T. 1977. Morphology of the macula neglecta in sharks of the genus Carcharhinus. Journal of Morphology 152, 341362.CrossRefGoogle ScholarPubMed
Corwin, J. T. 1978. The relation of inner ear structure to feeding behavior in sharks and rays. In Johari, O. (ed.) Scanning electron microscopy, 11051112. Chicago, IL: SEM Inc.Google Scholar
Corwin, J. T. 1981. Audition in sharks. In Tavolga, W. N., Popper, A. N. & Fay, R. R. (eds) Hearing and sound communication in fishes, 81105. New York: Springer.CrossRefGoogle Scholar
Corwin, J. T. 1983. Postembryonic growth of the macula neglecta auditory detector in the ray, Raja clavata: continual increases in hair cell number, neural convergence, and physiological sensitivity. Journal of Comparative Neurology 217, 345356.CrossRefGoogle Scholar
Corwin, J. T. 1989. Functional anatomy of the auditory system in sharks and rays. Journal of Experimental Zoology Supplement 5, 6675.Google Scholar
Coutier, F., Hautier, L., Cornette, R., Amson, E. & Billet, G. 2017. Orientation of the lateral semicircular canal in Xenarthra and its links with head posture and phylogeny. Journal of Morphology 278, 704717.CrossRefGoogle ScholarPubMed
Criswell, K. E., Coates, M. I. & Gillis, J. A. 2017. Embryonic development of the axial column in the little skate, Leucoraja erinacea. Journal of Morphology 278, 300320.CrossRefGoogle ScholarPubMed
Cuvier, G. 1799. Lecons d'anatomie comparée. Paris: Baudouin, Imprimeur de l'institut national des sciences et des arts. 595 pp.Google Scholar
Cuvier, G. & Valenciennes, A. 1828. Histoire naturelle des poissons. Paris: Levraut. 598 pp.Google Scholar
Daniel, J. F. 1934. The elasmobranch fishes. Berkeley, CA: University of California Press.CrossRefGoogle Scholar
Davis, S. P., Finarelli, J. A. & Coates, M. I. 2012. Acanthodes and shark-like conditions in the last common ancestor of modern gnathostomes. Nature 486, 247250.CrossRefGoogle ScholarPubMed
De Beer, G. R. 1931. The development of the skull in Scyllium (scyliorhinus) canicula l. Quarterly Journal of Microscopial Science 74, 591652.Google Scholar
De Burlet, H. M. 1934. Vergleichende Anatomie des stato-akustischen Organs. In Bolk, L., Göppert, E., Kallius, E. & Lubosch, W. (eds) Handbuch der vergleichenden anatomie der wirbeltiere, 12931432. Berlin, Wien: Urban & Schwarzenberger.Google Scholar
DiSanto, V. 2015. Ocean acidification exacerbates the impacts of global warming on embryonic little skate, Leucoraja erinacea (Mitchill). Journal of Experimental Marine Biology and Ecology 493, 7278.CrossRefGoogle Scholar
Ekdale, E. G. 2016. Morphological diversity among the inner ears of extinct and extant baleen whales (Cetacea: Mysticeti). Anatomical Record 299, 15991619.Google Scholar
Elger, M., Hentschel, H., Litteral, J., Wellner, M., Kirsch, T., Luft, F. C. & Haller, H. 2003. Nephrogenesis is induced by partial nephrectomy in the elasmobranch Leucoraja erinacea. Journal of the American Society of Nephrology 14, 15061518.CrossRefGoogle ScholarPubMed
Evangelista, C., Mills, M., Siebeck, U. E. & Collin, S. P. 2010. A comparison of the external morphology of the membranous inner ear in elasmobranchs. Journal of Morphology 271, 483495.Google ScholarPubMed
Fänge, R. 1982. Exogenous otoliths of elasmobranchs. Journal of the Marine Biological Association of the United Kingdom 66, 225.Google Scholar
Fay, R. R., Kendall, J. I., Popper, A. N. & Tester, A. L. 1974. Vibration detection by the macula neglecta of sharks. Comparative Biochemistry and Physiology – Part A 47, 12351240.CrossRefGoogle ScholarPubMed
Fay, R. R. & Popper, A. N. 2000. Evolution of hearing in vertebrates: the inner ears and processing. Hearing Research 149, 110.CrossRefGoogle Scholar
Federal Committee on Anatomical Terminology. 1998. Terminologia Anatomica. Thieme: Stuttgart.Google Scholar
Gardiner, J. M., Hueter, R. E., Maruska, K. P., Sisneros, J. A., Casper, B. M., Mann, D. A. & Demski, L. S. 2012. Sensory physiology and behaviour in elasmobranchs. In Carrier, J. C., Musick, J. A. & Heithaus, M. R. (eds) Biology of sharks and their relatives, 350401. Boca Raton, FL: CRC Press.Google Scholar
Giles, S., Rogers, M. & Friedman, M. 2016. Bony labyrinth morphology of early neopterygian fishes (Actinopterygii: Neopterygii). Journal of Morphology 279, 426440.CrossRefGoogle Scholar
Gillis, J. A., Dahn, R. D. & Shubin, N. H. 2009. Chondrogenesis and homology of the visceral skeleton in the little skate, Leucoraja erinacea (Chondrichthyes: Batoidea). Journal of Morphology 270, 628643.CrossRefGoogle Scholar
Grande, L. & Bemis, W. E. 1998. A comprehensive phylogenetic study of amiid fishes Amiidae) based on comparative skeletal anatomy. An empirical search for interconnected patters of Natural History. Society of Vertebrate Palaeontology Memoirs 4, 1–690.Google Scholar
Grohé, C., Tseng, Z. J., Lebrun, R., Boistel, R. & Flynn, J. J. 2016b. Bony labyrinth shape variation in extant Carnivora: a case study of Musteloidea. Journal of Anatomy 228, 366383.CrossRefGoogle Scholar
Hasse, C. 1873a. Die Lymphbahnen des inneren Ohres der Wirbelthiere. Anatomische Studien, vol. 1, 19 pp.Google Scholar
Hasse, C. 1873b. Die vergleichende Morphologie und Histologie des häutigen Gehörorganes der Wirbelthiere. Suppl. Zeitschrift der Anatomischen Studien, vol. 1, 102 pp.Google Scholar
Holmgren, N. 1940. Studies on the head in fishes: embryological, morphological and phylogenetical researches. Acta Zoologica 21, 51267.CrossRefGoogle Scholar
Hyrtl, J. 1845. Vergleichend-anatomische untersuchungen über das innere gehörorgan des menschen und der säugethiere. Prague: Ehrlich.Google Scholar
Hyrtl, J. 1873. Die corrosions-anatomie und ihre ergebnisse. Vienna: Braumüller.Google Scholar
Jollie, M. 1971. Some developmental aspects of the head skeleton of the 35–37 mm Squalus acanthias foetus. Journal of Morphology 133, 1740.CrossRefGoogle ScholarPubMed
Kasumyan, A. O. 2004. The vestibular system and sense of equilibrium in fish. Journal of Ichthyology 44, 224268.Google Scholar
Ladich, F. & Popper, A. N. 2004. Parallel evolution in fish hearing organs. In Manley, G. A., Fay, R. R., Popper, A. N. (eds) Evolution of the vertebrate auditory system, 95127. New York: Springer.CrossRefGoogle Scholar
Ladich, F. & Schulz-Mirbach, T. 2016. Diversity in fish auditory systems: one of the riddles of sensory biology. Frontiers in Ecology and Evolution 4, 126.CrossRefGoogle Scholar
Last, P., White, W., de Carvalho, M., Séret, B., Stehmann, M. & Naylor, G. 2016. Rays of the world. Clayton North, NC: Cornell University Press. 790 pp.CrossRefGoogle Scholar
Lebrun, R., De León, M. P., Tafforeau, P. & Zollikofer, C. 2010. Deep evolutionary roots of strepsirrhine primate labyrinthine morphology. Journal of Anatomy 216, 368380.CrossRefGoogle ScholarPubMed
Lebrun, R. & Orliac, M. J. 2016. MorphoMuseumM: an online platform for publication and storage of virtual specimens. The Palaeontological Society Papers 22, 183195.CrossRefGoogle Scholar
Leydig, F. 1852. Beiträge zur mikroskopischen anatomie und entwicklungsgeschichte der rochen und haie. Leipzig: Engelmann. 140 pp.Google Scholar
Maisey, J. G. 1983. Cranial anatomy of Hybodus basanus Egerton from the Lower Cretaceous of England. American Museum Novitates 2758, 164.Google Scholar
Maisey, J. G. 1985. Cranial morphology of the fossil elasmobranch Synechodus dubrisiensis. American Museum Novitates 2804, 128.Google Scholar
Maisey, J. G. 1987. Cranial anatomy of the lower Jurassic shark Hybodus reticulatus (Chondrichthyes: Elasmobranchii), with comments on hybotid systematics. American Museum Novitates 2878, 139.Google Scholar
Maisey, J. G. 1999. A supraotic bone on neopterygian fishes (Osteichthyes, Actinopterygii). American Museum Novitates 3267, 152.Google Scholar
Maisey, J. G. 2001. Remarks on the inner ear of elasmobranchs and its interpretation from skeletal labyrinth morphology. Journal of Morphology 250, 236264.CrossRefGoogle ScholarPubMed
Maisey, J. G. 2004. Morphology of the braincase in the broadnose sevengill shark Notorynchus (elasmobranchii, Hexanchiformes), based on CT scanning. American Museum Novitates 3429, 152.2.0.CO;2>CrossRefGoogle Scholar
Maisey, J. G. & Lane, J. A. 2010. Labyrinth morphology and the evolution of low-frequency phonoreception in elasmobranchs. Comptes Rendus Palevol 9, 289309.CrossRefGoogle Scholar
Maxwell, E. E., Fröbisch, N. B. & Heppleston, A. C. 2008. Variability and conservation in late chondrichthyan development: ontogeny of the winter skate (Leucoraja ocellata). Anatomical Record 291, 10791087.CrossRefGoogle Scholar
Miles, R. S. 1973. Relationships of acanthodians. In Miles, P. H., Patterson, R. S. & Greenwood, C. (eds) Interrelationships of fishes. Zoological Journal of Linnean Society (Suppl. 1), 63103.Google Scholar
Naylor, G. J. P., Caira, J. N., Jensen, K., Rosana, K. A. M., White, W. T. & Last, P. R. 2012a. A DNA sequence based approach to the identification of shark and ray species and its implications for global elasmobranch diversity and parasitology. Bulletin of the American Museum of Natural History 367, 1–262.CrossRefGoogle Scholar
Naylor, G. J. P., Caira, J. N., Jensen, K., Rosana, K. A. M., Straube, N. & Lakner, C. 2012b. Elasmobranch phylogeny: a mitochondrial estimate based on 595 Species. In Carrier, J. C., Musick, J. A. & Heithaus, M. R. (eds) Biology of sharks and their relatives, 2nd edn, 3156. Boca Raton, FL: CRC Press.CrossRefGoogle Scholar
Norris, H. W. 1929. The parietal fossa and related structures in the plagiostome fishes. Journal of Morphology and Physiology 48, 543561.CrossRefGoogle Scholar
Pfaff, C., Martin, T. & Ruf, I. 2015. Bony labyrinth morphometry indicates locomotor adaptations in the squirrel-related clade (Rodentia, Mammalia). Proceedings of the Royal Society B: Biological Science 282, 19.CrossRefGoogle Scholar
Pfaff, C., Nagel, D., Gunnell, G., Weber, G. W., Kriwet, J., Morlo, M. & Bastl, K. 2017a. Palaeobiology of Hyaenodon exiguus (Hyaenodonta, Mammalia) based on morphometric analysis of the bony labyrinth. Journal of Anatomy 230, 282289.CrossRefGoogle Scholar
Pfaff, C., Czerny, S., Nagel, D. & Kriwet, J. 2017b. Functional morphological adaptations of the bony labyrinth in marsupials (Mammalia, Theria). Journal of Morphology 278, 742749.CrossRefGoogle Scholar
Platt, C. & Popper, A. N. 1981. Structure function in the ear. In Tavolga, W. N., Popper, A. N., Fay, R. R. (eds) Hearing and sound communication in fishes, 338. New York: Springer.CrossRefGoogle Scholar
Pradel, A., Langer, M., Maisey, J. G., Geffard-Kuriyama, D., Cloetens, P., Janvier, P. & Tafforeau, P. 2009. Skull and brain of a 300-million-year-old chimaeroid fish revealed by synchrotron holotomography. PNAS 106, 52245228.CrossRefGoogle ScholarPubMed
Rayner, D. H. 1952. On the cranial structure of an early palaeoniscid, Kentuckia, gen. nov. Earth and Environmental Science Transactions of the Royal Society of Edinburgh 62, 5383.CrossRefGoogle Scholar
Retzius, G. 1881. Das gehörorgan der wirbelthiere: morphologisch-histologische studien. Stockholm: Samson & Wallin.Google Scholar
Roberts, B. L. 1978. Mechanoreception and the behaviour of elasmobranch fishes with special reference to the acoustico-lateralis system. In Hodgon, E. S. & Mathewson, R. R. (eds) Sensory biology of sharks, skates, and rays, 331390. Washington, DC: US Government Printing Office.Google Scholar
Scarpa, A. 1800. Anton Scarpa's anatomische untersuchungen des gehörs und des geruchs. Nuremberg: Raspe.Google Scholar
Schaeffer, B. 1981. The xenacanth shark neurocranium, with comments on elasmobranch monophyly. Bulletin of the American Museum of Natural History 169, 366.Google Scholar
Schnetz, L., Kriwet, J. & Pfaff, C. 2017. Virtual reconstruction of the skeletal labyrinth of two lamnid sharks (Elasmobranchii, lamniformes). Journal of Fish Biology 90, 10831089.CrossRefGoogle Scholar
Sipla, J. S. & Spoor, F. 2008. The physics and physiology of balance. In Thewissen, J. G. M. & Nummela, S. (eds) Sensory evolution on the threshold: adaptations in secondarily aquatic vertebrates, 227232. Berkeley & Los Angeles, CA: University of California Press.Google Scholar
Spoor, F., Garland, T. Jr., Krovitz, G., Ryan, T. M., Silcox, M. T. & Walker, A. 2007. The primate semicircular canal system and locomotion. Proceedings of the National Academy of Sciences 104, 10808–12.CrossRefGoogle ScholarPubMed
Stensiö, E. A. 1950. La cavité labyrinthique, l'ossification sclérotique et l'orbite de Jagorina. In Jorge, A. (ed.) Paléntologie et transformisme, 943. Paris: Albin Michel.Google Scholar
Stensiö, E. A. 1963. The brain and the cranial nerves in fossil lower craniate vertebrates. Skrifter utgitt av det Norske Videnskaps-Akademi i Oslo 13, 5–120.Google Scholar
Stolte, H., Galaske, R. G., Eisenbach, G. M., Lechene, C., Schmidt-Nielson, B., Boylan, J. W., Antkowiak, D., Patel, Y. & Niermann, U. 1977. Renal tubule ion transport and collecting duct function in the elasmobranch little skate, Raja erinacea. Journal of Experimental Zoology 199, 403410.CrossRefGoogle ScholarPubMed
Tester, A. L., Kendall, J. I. & Milisen, W. B. 1972. Morphology of the ear of the shark genus Carcharhinus with particular reference to the macula neglecta. Pacific Science 26, 264274.Google Scholar
Torrey, T. M. 1962. Morphogenesis of the vertebrates. New York: Wiley. 600 pp.Google Scholar
Weber, E. H. 1820. De aure et auditu hominis et animalium. Pt.1: Se aure animalium aquatilium. Lipsidae. 198 pp.Google Scholar
Wyffels, J., King, B. L., Vincent, J., Chen, C., Wu, C. H. & Polson, S. W. 2014. Skatebase, an elasmobranch genome project and collection of molecular resources for chondrichthyan fishes. F1000Research 12, 191.Google Scholar

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Ontogenetic development of the otic region in the new model organism, Leucoraja erinacea (Chondrichthyes; Rajidae)
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