ARTICLE IN PRESS ZOOLOGY Zoology 109 (2006) 164–168 www.elsevier.de/zool Mineralized C artilage in the skeleton of chondrichthyan fishes Mason N. Dean, Adam P. Summers Ecology and Evolutionary Biology, University of California – Irvine, 321 Steinhaus Hall, Irvine, CA 92697-2525, USA Received 10 February 2006; received in revised form 2 March 2006; accepted 3 March 2006 Abstract The cartilag inous endoskeleton of chondrichthyan fishes (shark s, rays, and chimaeras) exhibits complex arrangements and morphologies of calcifi ed tissues that vary with age, species, feeding behavior, and location in the body. Unders tanding of the development, evolutionary history and function of these tissue types has been hampered by the lack of a unifying terminology. I n order to facilitate reciprocal illu mination between disparate fields with convergent interests, we present levels of organization in which crystal orientation/size delimits three calcificati on types (areol ar, globular, and prismatic) that interact in two distinct skeletal types, vertebral and tessellated carti lage. The tessellated skeleton is composed of small blocks (tesserae) of calcified cartilag e (both prismatic and globular) overlying a core of unmineralized cartilag e, while vertebral cartilag e usually contains all three types of calcification. r 2006 Elsevier GmbH . All rights reserved. Keywords: Elasmobranch skeleton; Mineralization; Calcified cartilage; Tesserae Introduction The breadth of morphological varia tion of the mineralized cartilag e of the endoskeleton of chondrichthyan fishes (shar ks, rays, and chimaeras) has led to a confusion of terminology in the literature. Confli cting descriptive terms make it difficul t to define, let alone answer, questions of the evolution, homology and function of the skeleton and skeletal tissues. Here we lay out the most accepted terminology for mineraliz ed tissue, sometimes called ‘cal cified cartilag e’ in the literature, in carti laginous fishes and propose a hierarchical fram ework for future descriptive work. To integrate the convergent evolutionary (Smith and Hall , 1990; Sansom et al., 1992; Hall, 2005), paleontological (Coat es et al., 1998; Janvier et al., 2004), developmental (Davi s et al., 2004) and biomechanical Correspond ing author. E-mail address: asummers@uci.edu (A.P. Summers). 0944-2006/$ - see front matter r doi:10.1016/j.zool.2006 .03.002 2006 Elsevier GmbH. All rights reserved. (Summers, 2000; Schaefer and Summers, 2005; Dean et al., 2006) interests in cartilag inous skeletons, we need a common language. Furtherm ore, the current terminology masks unappreciated complexity in morphology that will fuel futur e research in several of these fields. Classification of elasmobranch cartilage tissue types The entire endoskeleton of sharks, chimaeras and rays is cartilagino us, composed of chondrocytes in an extracell ular matrix (ECM ) surrounded by a fibrous perichondri um. The ECM may be mineralized to varying degrees with crystals of calcium phosphate hydroxyapat ite (Apple gate, 1967; Dean et al., 2005). Ther e are no nanostructural data on the orientation or size of these crystals , but the higher-level organization of the mineral has inspired several useful descriptive terms. ARTICLE IN PRESS M.N. Dean, A.P. Summers / Zoology 109 (2006) 164–168 From these terms and concepts we recognize three types of calcification (synthesized originall y by Ørvi g, 1951) in a natural classification based on anatomy and location, that will provide a framework for appreciating the true complexity at this level of organization. 165 Globular calcifica tion (var. spherulitic, e.g., Ørvig, 1951; Janvier et al., 2004) is a moderately well mineralized tissue formed of nanoscale (40–55 nm) spherules of hydroxyap atite fused together (Fig . 1c) (Ørvig, 1951; Dean et al., 2005). Mesostructure: tesserae Microstructure: calcification types Areolar calcification (var. alveolar, M oss, 1977) is a densely calcified tissue that occurs in the vertebral centra of most cartilag inous fishes, forming what has been called the ‘double cone’ of the vertebral body (Ridewood, 1921; Ørvig, 1951) (as illustrated by the hemisected centra in Fig. 1a). Thi s form of minerali zation is laid down in concentric rings and has been successfully used to age cartilag inous fishes (Jones and Geen, 1977; Lessa et al., 1999). Prismati c calcificati on is always perichondrally associated (Fig. 1b) and is so dense as to specularly refr act light (Wurmbach, 1932; Ørvig, 1951). Prism atic and globular calcifi cation (Figs. 1b,c) typically co-occur in block s of calcified tissue called tesserae that form a continuous mosaic between the perichondriu m and uncalcified core of most endoskeletal elements (Fig. 2). The gradation within a single tessera from globular calcificati on on the inner surfa ce to prismati c calcification on the perichondral surfa ce (Figs. 1b,c and Fi g. 2 lower inset) raises the possibility that they represent ontogenetic stages of progressively more mineraliz ed tissue, though there is no published developmental evidence (Ørvi g, 1951; Moss, 1977; Kemp, 1979). It has also been proposed that these two forms of calcification are of completely different cellular Fig. 1. Schematic of a generalized shark endoskeleton showing occurrences of areolar (a), prismatic (b) and globular (c) calcification types. Prismatic (pris calc, PC) and globular calcification (glob calc, GC) form tessellated skeletal tissue in all areas color coded green, while areolar calcification (AC) forms the centra of the vertebral column, color coded orange. The majority of the skeleton is tessellatedcartilage, comprised of a cortex of mineralized blocks (tesserae), lying just beneath the fibrous perichondrium (peri chon) and overlying the uncalcified ECM. Prismatic and globular calcification, respectively, form the outer and inner surfaces of ‘surface’ tesserae (see text). Calcification is apparently periodic, entombing chondrocytes in their lacunae (lac) and forming concentric Liesengang lines (lies line). Prismatic and globular calcification often ornament the vertebral structures surrounding areolar-calcified vertebral centra; this interaction of all three calcification types is apparently only found in the vertebral cartilage of cartilaginous fishes (a). ARTICLE IN PRESS 166 M.N. Dean, A.P. Summers / Zoology 109 (2006) 164–168 Fig. 2. Uncalcified and calcified phases of tessellated elasmobranch cartilage, depicted as a schematic of a cross-sectional backscatter electron micrograph of the lower jaw of the round stingray, Urobatis halleri (upper inset; white regions are calcified tissue). The lower inset provides an expanded view of the region bounded by the blue box, showing the arrangement of prismatic (PC) and globular (GC) calcification relative to the uncalcified cartilage matrix (ECM) and fibrous perichondrium. origin, with prismatic and globular calcificati on being chondrally and perichondrally derived, respectively (Kemp, 1977; Kemp and Westrin, 1979). Chondrocy tes on the inner side (i.e. adjacent to unmineralized ECM) of tesserae are still aliv e and surroun ded by characteristic ‘Lies engang lines’ (Fig. 1c) that signify varyin g density of calcificati on (Apple gate, 1967). Some authors (e.g., Moss, 1977) believe entombed chondrocytes to be vital in both prismati c and globular types, while others (e.g., Summers, 2000) only observe them in the latter. Macrostructure: skeletal types The three calcification types are associated with uncalcified cartilag e and organized into two skeletal types: vertebral cartilag e and tessellated cartilag e (Fig. 3). Vertebral cartilag e can contain all three calcification types (illust rated by the vertebra in Fig. 1a). Areol ar carti lage comprises the centrum (‘doubl e cone’) of each vertebra, with all surroun ding structure (e.g., neural/ hemal arches, the vertebral body) formed by uncalcifi ed carti lage, typically sheathed with tesserae. In addition to forming the chondral surfa ce of tesserae, globular calcification may also be present on the outer surfa ce of the centra (Fig. 1a) (Ride wood, 1921; Clement, 1992). Wh ile the areolar part of the centrum is well known in sagittal section by biologists interested in aging sharks, there is nothing known of the mechanics of the centra, nor of the interaction between the diff erent forms of calcificati on found in the rest of the vertebral cartilag e (Summers and Long, 2006). Ther e is extensive morphological variation in calcification at the level of species, but there has beenno examination of this varia tion in an evolutionary context (Hasse, 1879; Ride wood, 1921; Ørvi g, 1951). Tessellated carti lage forms the remainder of the axial and the appendicular skeleton and consists of a mosaic of tesserae (composed of both prismati c and globular calcificati on) overlaying a core of unmineralized ECM (Kemp and Westr in, 1979) (Fig . 2). The arrangement of tesserae appears to be functionally important in stiffening the skeleton with several layers present at the corners of the jaws in large sharks and throughout the core of jaws of species with highly demanding feeding modes (e.g., durophagy, prey excavation) (Dingerkus et al., 1991; Summers, 2000; Dean et al., 2006). Where there are multiple tesseral layers, ‘surfa ce’ and ‘sub-surface’ tesserae exhibit different calcification types, perhaps reflective of their tissue associations. ‘Sub -surface’ tesserae do not have contact with the perichondrium and do not have any prismati c calcification but rather are composed entirely of globular calcification (Summers, 2000). Thi s rais es the possibility that the perichondri um is either the origin of, or induces the ARTICLE IN PRESS M.N. Dean, A.P. Summers / Zoology 109 (2006) 164–168 167 Fig. 3. Distribution of types of cartilage and mineralization in sharks and rays. appearance of, prismati c calcification (Kemp and Westr in, 1979). Fro m thin sections or micro-CT scans of fossil material it should be possible to distinguish subsurfa ce tesserae from the more common perichondrally associated ones. Discussion The development of tesserae and the role of the uncalcified inner core are of particular interest because, in contrast to endochondral bone, the signaling that produces this calcification happens in the absence of any vascular tissue. Though all extant sharks and rays have tessellated skeletons, the evolutionary history of this character is unknown. Since bone has been secondarily lost in sharks the history of the tessellated skeleton is of considerable functional importance because of selective pressures to maintain a stiff skeleton (Coat es et al., 1998). Previous studies have ignored the interaction of different calcification types by equating ‘tesserae’ with ‘prismat ic calcifi cation’ or layering of ‘prismat ic calcium phosphate’ (Ørvig, 1951; Apple gate, 1967; Cappetta, 1987; Dingerkus et al., 1991; Curr ey, 2002; Hall , 2005) or referring to the entire skeleton as ‘pri smatic’ (Halstead, 1974). Tessera e contain two types of calcification; Kemp and W estrin (1979) made a vital contribution by recognizing the association between globular and prismatic calcifi cation in tesserae. To speak of chondrichthyan skeleton as ‘prismat ically calcified’ is to refer only to the perichondra l portion of surface tesserae, ignoring sub-surface tesserae as well as vertebral cartilag e. To further clari fy the situation we have described four distinct levels of organization in the calcified portions of the chondrichthyan skeleton–hydroxy apatite crystal orientation and size (nanostructur e); calcification type (microstructur al arrangement and density of mineral phase); tesserae (mesostructure); and skeletal type (macrostru ctural arrangement and association of calcification types) (Fig. 3). While the first of these levels has not beeninvestigated at all, the third and fourt h levels of organization have the most immediate potential to infor m our understanding of the evolution of the vertebrate endoskeleton. Acknowledgments This research was supported by a grant from the National Science Foundat ion (IBN- 0317155) to APS and a Journ al of Expe rimental Biology Traveling Fellow ship to MND. References Applegate, S.P., 1967. A survey of shark hard parts. In: Gilbert, P.W., Mathewson, R.F., Rall, D.P. (Eds.), Sharks, Skates and Rays. Johns Hopkins Press, Maryland, pp. 37–66. Cappetta, H. (Ed.), 1987. Chondrichthyes II: Mesozoic and Cenozoic Elasmobranchii. Handbook of Paleoichthyology. Gustav Fischer Verlag, New York. Clement, J.G., 1992. Re-examination of the fine structure of endoskeletal mineralization in Chondricthyes: implications for growth, ageing and calcium homeostasis. Aust. J. Mar. Fresh. Res. 43, 157–181. Coates, M.I., Sequeira, S.E.K., Sansom, I.J., Smith, M.M., 1998. Spines and tissues of ancient sharks. Nature 396, 729–730. C urrey, J.D ., 2002. B ones. Princeton U niversity Press, Princeton. Davis, M.C., Shubin, N.H., Force, A., 2004. Pectoral fin and girdle development in the basal actinopterygians Polyodon ARTICLE IN PRESS 168 M.N. Dean, A.P. Summers / Zoology 109 (2006) 164–168 spathula and Acipenser transmontanus. J. Morphol. 262, 608–628. Dean, M.N., Chiou, W.-A., Summers, A.P., 2005. Morphology and ultrastructure of prismatic cartilage calcification. Microsc. Microanal. 11, 1196–1197. Dean, M.N., Huber, D.R., Nance, H.A., 2006. Functional morphology of jaw trabeculation in the lesser electric ray Narcine brasiliensis, with comments on the evolution of structural support in the Batoidea. J. Morphol., in press. DOI:10.1002/jmor.10302. Dingerkus, G., Séret, B., Guilbert, E., 1991. Multiple prismatic calcium phosphate layers in the jaws of present-day sharks (Chondrichthyes; Selachii). Experientia 47, 38–40. Hall, B.K., 2005. Bones and Cartilage: Developmental Skeletal Biology. Elsevier/Academic Press, London. Halstead, L.B., 1974. Vertebrate Hard Tissues. Wykeham Science Publications, Ltd., London. Hasse, J.C.F., 1879. Über den Bau und über die Entwickelung des Knorpels bei den Elasmobranchiern. Zool. Anz. 2, 325–374. Janvier, P., Arsenault, M., Desbiens, S., 2004. Calcified cartilage in the paired fins of the osteostracan Escuminaspis laticeps (Traquair 1880), from the late Devonian of Miguasha (Québec, Canada), with consideration of the early evolution of the pectoral fin endoskeleton in vertebrates. J. Vert. Paleontol. 24, 773–779. Jones, B.C., Geen, G.H., 1977. Age-determination of an Elasmobranch (Squalus acanthias) by X-ray spectrometry. J. Fish. Res. Board Can. 34, 44–48. Kemp, N.E., 1977. Calcification of endoskeleton in Elasmobranchs. Am. Zool. 17, 932. Kemp, N.E., 1979. Growth of calcified tissue in the endoskeleton of Elasmobranchs. Am. Zool. 19, 977. Kemp, N.E., Westrin, S.K., 1979. Ultrastructure of calcified cartilage in the endoskeletal tesserae of sharks. J. Morphol. 160, 75–102. Lessa, R., Santana, F.M., Paglerani, R., 1999. Age, growth and stock structure of the oceanic whitetip shark, Carcharhinus longimanus, from the southwestern equatorial Atlantic. Fish. Res. 2, 21–30. Moss, S.A., 1977. Skeletal tissues in sharks. Am. Zool. 17, 335–342. Ørvig, T., 1951. Histologic studies of Placoderms and fossil Elasmobranchs. I: The endoskeleton, with remarks on the hard tissues of lower vertebrates in general. Arkiv. Zool. 2, 321–454. Ridewood, W.G., 1921. On the calcification of the vertebral centra in sharks and rays. Philos. Trans. R. Soc. London B. Biol. Sci. 210, 311–407. Sansom, I.J., Smith, M.P., Armstrong, H.A., Smith, M.M., 1992. Presence of the earliest vertebrate hard tissues in conodonts. Science 256, 1308–1311. Schaefer, J.T., Summers, A.P., 2005. Batoid wing skeletal structure: novel morphologies, mechanical implications, and phylogenetic patterns. J. Morphol. 264, 298–313. Smith, M.M., Hall, B.K., 1990. Development and evolutionary origins of vertebrate skeletogenic and odontogenic tissues. Biol. Rev. 65, 277–373. Summers, A.P., 2000. Stiffening the stingray skeleton – an investigation of durophagy in myliobatid stingrays (Chondrichthyes, Batoidea, Myliobatidae). J. Morphol. 243, 113–126. Summers, A.P., Long, J.H., 2006. Skin and bone, sinew and gristle: the mechanical behavior of fish skeletal tissues. In: Lauder, G.V., Shadwick, R.E. (Eds.), Fish Biomechanics: Fish Phsyiology, vol. 23. Academic Press, San Diego, pp. 141–178. Wurmbach, H., 1932. Das Wachstum des Selachierwirbels und seiner Gewebe. Zool. Jahrb. (Abt. Anat. Ont. Tiere) 55, 1–136.