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Mutvei. Harry 1981 10 01: Exoskeletal structure in the Ordovician trilobite Flexicalymene. Lethaia. Vol. 14. pp. 225-234. Oslo. ISSN 0024-1164. Scanning Electron Microscope study of the exoskeletal ultrastructure of secondarily phosphatized material of Nexicalymene sp. from the Upper Ordovician Maquoketa Shale, Iowa, USA, shows that the exocuticle, comprising 20% of the total exoskeletal thickness, is composed of horizontal laminar units between 0.2 and I pm thick. These units consist of primarily mineralized organic fibres which form horizontal laminae interconnected by inter-laminae. The endocuticle is considerably more mineralizer than the exocuticle, and its original organic structure cannot be observed in untreated preparations. Etching with chromium sulphate reveals: ( 1 ) horizontal organic laminar units, 0.2 to 2 pm thick, and (2) pore canals with non-twisted walls about 0.3 pm in diameter. Exuvia cannot be distinguished from the exoskeletons of dead animals. The exoskeletal ultrastructure in trilobites agrees essentially with that in crustaceans. 0 Trilobita, Flexicalymene, exoskeleton, cuticle, ultrastructure, Ordovician.
Harry Mutvei, Department o Palueozoology, Swedish Museum o Natural History, Box50007, S-104 05 f f Stockholm. and Palaeontological Institute, Box 558, S-75 122 Uppsala. Sweden: 28th January, 1981.
Previous studies on exoskeletal structure in trilobites have been reviewed by Dalingwater (1973), and therefore only studies after this date are discussed below. Teigler & Towe ( 1975) studied exoskeletal structure and composition in twenty trilobite species ranging in age from Cambrian through Devonian. In twelve species two layers were distinguished: an inner thick layer and an outer thin layer. The inner layer was found to be composed of calcite crystals which have their c-axes oriented more or less perpendicular to the exoskeletal surface. In six species this layer showed fine lamellae. N o lamination was recognized in the outer layer, which was found to be prismatic in structure, pigmented, or composed of apatite. In the remaining eight species, the exoskeleton consisted of the inner layer only. Dalingwater & Miller ( 1977) distinguished three zones in the exoskeleton of the Lower Ordovician (Kundan) trilobite Asaphus (Asaphus) raniceps: (1) a narrow outer zone comprising up to one-fifth of the total exoskeletal thickness and consisting of laminar units about 5 pm thick; (2) a wide central zone with four to five laminar units 50-70 pm thick; and (3) a narrow innermost zone with laminar units 5 pm thick. The exoskeletal surface was found to be covered occasionally by a very thin prismatic layer. Three exoskeletal zones similar to those in Asaphus were distinguished by St@rmer(1980)
in chasmopinid and phacopid trilobites. In this material, the laminar units in the outer zone had a thickness of about 5 pm, in the middle zone 20-30 pm, and in the inner zone about 10 pm.
Material and methods
In this paper the exoskeletal ultrastructure is described in Flexicalymene sp. from the Upper Ordovician Maquoketa Shale, Iowa, USA, and compared with that in crustaceans and chelicerates. In the material studied the state of preservation of exoskeletal structures appears to exceed that in any other trilobites in which these structures have been studied previously. All specimens described here were collected from the lowermost Elgin coquina beds of the Maquoketa Shale, at Graf, Iowa, USA. The coquina consists largely of specimens of the cephalopod Isorthoceras sociule (e.g. Tasch 1955; Flower 1962), but about 50 fragments of trilobite exoskeletons belonging to Flexicalymene sp. were found by crushing about 5 kg of rock samples into small pieces (0.5-1.0 cm3). The exoskeleton was fractured vertically and studied either unetched, or etched for three to four hours with a chromium sulphate solution (Mutvei 1972). The specimens were mounted on specimen stubs, coated with evaporated gold, and studied with the scanning electron micro-
226 Harry Mutvei
Fig. I . Fkxicdymrne sp. Block diagram of the exoskeleton. Note difference in mineralization between exocuticle ( e m )and endocuticle ( e n d o ) .b, endocuticular bulb; d , ducts; f , tubercles.
scope. The original mineral composition of the trilobite exoskeleton was mostly calcitic (Dalingwater 1973; Teigler & Towe 1975). However, in the trilobites studied here the calcite has been replaced by a hydrated phosphate (dahllite according to Grandjean et al. 1964579) and other minerals, as in the cephalopod shells from the same locality, and this has facilitated preservation of structures down to ultrastructural level. There are clear indications that this replacement took place at an early diagenetic stage.
Exoskeleton in Flexicalymene
Two layers can usually be distinguished on vertical fracture planes of the exoskeleton. These layers are here termed the exocuticle and the endocuticle (exo, endo, Fig. l ) , in agreement with the terminology adopted for structurally similar layers in the exoskeleton of extant decapod crustaceans (e.g. Mutvei 1974). The exocuticle was probably covered by a thin epicuticle which is not preserved.
Exocuticle. - The exocuticle forms the outer layer of the exoskeleton, comprising about 20% of the total exoskeletal thickness in most preparations (exo, Fig. 1). It is occasionally thinner, probably because its outermost part has been worn off. Studies of untreated vertical fracture planes
show that the exocuticle has a horizontally laminate organic structure (4x0, Figs. I , ?A, B). The total number of laminar units seems to vary between 30 and 40 (Fig. 2A); the individual units are usually 0.2-0.5 pm thick. Close to the endocuticular boundary the laminar units may attain a thickness of about 1 pm (Figs. 2A, B). The laminar units consist of organic fibres which are primarily mineralized and probably aggregated to form bundles. These fibre-bundles are distinctly visible only in some preparations close to the endocuticular boundary, where the laminar units increase in thickness. In untreated preparations their diameter is about 0.2 pm (Fig. 2B). Sheets of these fibre-bundles form consecutive horizontal laminae (Fig. 2B) which are interconnected by parabolically arched sheets of fibres forming the inter-laminae (indicated by arrows in Fig. 2B). In a preparation etched for three hours with a chromium sulphate solution, the organic fibres became wholly or partially demineralized, and their diameter decreased considerably to less than 0.1 pm (exo, Fig. 3, and the separate fragment in the lower right corner of the same Fig.). In most preparations, however, the mineralized fibre-bundles have been diagenetically altered and form granulae, usually less than 0.5 pm in diameter. The occurrence of the horizontal laminar units is still indicated by a more or less distinct horizontal arrangement of the granulae. The exocuticle of Flexicalyrnene sp. has the same thickness as that in Asaphus raniceps (Dalingwater & Miller 1977, PI. 9: 1.2 and Fig. 2). In both trilobites it is horizontally laminate, but the laminar units seem to be much thinner in Flexicalymene. The calcified organic fibres and their arrangement into laminae and inter-laminae have not been observed in previous studies.
Endocuticle. - The boundary between the endocuticle and the exocuticle is distinct because the former is always considerably more mineralized than the latter. The endocuticle constitutes the principal inner layer of the exoskeleton, comprising about 80% of the total exoskeletal thickness (endo, Fig. 1). On unetched vertical fracture planes its structure appears to be clearly different from that of the exocuticle. It is composed of crystal blocks with a distinct cleavage oriented obliquely to the exoskeletal surface. These blocks have no distinct boundaries, and their size and shape are highly variable. Their diameter usually ranges between 50 and 100 pm.
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Exoskeletal structure in FLEXICALYMENE 227
Fig. 2. Flexiculymene sp. 0 A . Untreated exoskeletal surface and vertical fracture plane within the glabella. Note distinct horizontal lamina of the exocuticle (exo) at the boundary with an endocuticular bulb ( b ) .0 B. Untreated vertical fracture plane of
the exocuticle, showing bundles of calcified organic fibres in the horizontal laminar units. Fibre-bundles forming parabolic arcs in the inter-laminae are indicated by arrows.
Unetched endocuticle lacks distinct horizontal organic laminae. At the boundary with the exocuticle, the endocuticle forms numerous bulbs which protrude outwards into the exocuticle (Fig. 1, 6, Fig. 2A). Each bulb seems to contain a large duct which opens into a tubercle on the exoskeletal surface (Fig. 1 ) . Similar endocuticular bulbs occur in the extant decapod crustacean Cancer pagurus (Hegdahl et al. 1977a, Figs. 1-3).
O n the vertical fracture planes of the endocuticle, etched for four hours with a chromium sulphate solution, distinct horizontal organic laminae are often observed (Figs. 4A, B). These laminae extend through the crystal blocks. In a preparation shown in Fig. 4A (endo), the horizontal laminae seem to attain a maximum thickness of 2 pm. These thick laminae are found in the endocuticular bulbs around the large ducts, where they curve strongly outwards. In another
228 Harry Mutvei
LETHAIA 14 (1981)
Fig. 3 . Flexicalymene sp. Vertical fracture plane of the exocuticle (exo) etched for three hours with chromium sulphate solution. Note partially decalcified organic fibres in the inter-laminae, close to the endocuticular (endo)boundary, and on a separate fragment in the lower right comer.
preparation (Fig. 4B), thin horizontal laminae are arranged regularly around smaller ducts (indicated by arrows in Fig. 4B). These laminae, which have a thickness less than 0.5 pm, usually disappear about halfway between adjacent ducts. Remains of thin horizontal laminae were also found attached to the pore canal walls (Fig. 5B). In none of the endocuticular laminae can the organic fibres be distinguished. The endocuticle is traversed vertically by ducts of various diameters. Ducts’of about 3-7 pm (Fig. I , indicated by arrows in Fig. 4B) and 15-20 pm (Fig. 1) in diameter were observed
most frequently. In the cephalon, the larger of these ducts seem to extend through prominent tubercles to the exoskeletal surface (Fig. 1; see also Evitt & Whittington 1953; Stqirmer 1980). In previous studies (e.g. Osmolska 1975; Teigler & Towe 1975; Dalingwater & Miller 1977; Stqirmer 1980), the pore canals in trilobites have been observed only in light microscopic preparations, where their diameters have been estimated to be 1-2 pm. In Flexicalymene sp., the pore canak could not be observed on untreated vertical fracture planes of the exoskeleton. However, on the vertical fracture planes that were
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Exoskeletal structure in FLEXICAL YMENE 229
Fig. 4 . Flexicalymene sp. Vertical fracture planes of the endocuticle (endo) etched for four hours with chromium sulphate solution. 0 A. Horizontal organic lamellae curve strongly outwards in the bulbs. Between the bulbs they are absent. 0B. Thin horizontal organic lamellae occur around the ducts.
etched for four hours with a chromium sulphate solution, pore canals with well preserved walls were seen occasionally (Fig. SA, B). These canals have a diameter of about 0.3 p,m, and their walls are not helical or twisted as those in some extant crustaceans. A series of disc-like exten-
sions project horizontally from the wall of each canal (Fig. 5B). These extensions are situated about 0.2 pm from each other, and represent remains of horizontal organic laminae of the endocuticle (see above). In summary, the endocuticle is similar to the
230 Harry Mutvei
Fig. 5. Flexicalymene sp. Pore canals on a vertical fracture plane etched for four hours with chromium sulphate solution. Note the horizontal disc-like extensions from the pdre canal walls.These extensions are remains of horizontal organic lamellae.
LETHAIA 14 (1981)
E.xoskeleta1 structure in FLEXICAL YMENE 23 1 exuvium cannot be distinguished therefore from the exoskeleton of a dead animal (see also Henningsmoen 1975:181 ).
exocuticle in being horizontally laminate (exo, endo, Fig. 1). The organic laminar units are mostly very thin and the endocuticle is considerably more mineralized than the exocuticle. Moulting. - Prior to moulting in arthropods, much of the old exoskeleton is broken down enzymatically. The products of digestion are resorbed and re-used in the formation of the new exoskeleton. Among crustaceans, only ostracodes seem not to resorb their heavily mineralized carapaces before moulting (Turpen & Angel1 1971). In extant decapod crustaceans, a resorption from exuvia occurs generally, but its total amount is variable (Passano 1960). For example, in Palinurus the resorption before moulting amounts to 23% of the organic matter and 13% of the mineral matter, and in Carcinus to 79% of the organic matter and 18% of the mineral matter (Passano 1960). However, the resorption seems not to effect the thickness of the exuvia to any greater extent. The ultrastructural changes due to resorption are not known. Among chelicerates, the extant Lirnulus and probably also fossil eurypterids resorb practically all the endocuticle. The exuvia of Limulus are therefore thin, forming about 20% of the original exoskeletal thickness (Mutvei 1977). A similar magnitude of resorption takes place in insect exoskeletons before moulting (Wigglesworth 1959). It is generally believed that most trilobite exoskeletons in the fossil record represent exuvia, and it is reasonable to assume that the majority of Flexicalymene exoskeletons also represent such moult stages. If resorption has taken place, then the endocuticle in the trilobite exuvia would be characterized by ( 1 ) a reduction in thickness, and/or (2) a formation of pores and cavities, particularly around ducts and pore canals, as a result of digestion of the organic matter and dissolution of the mineral matter. However, none of these characteristics could be observed in the trilobite exoskeletons, including those of Flexicalymene. It is therefore probable that before moulting, no resorption took place from the heavily calcified exoskeleton, other than a softening along the sutures where the exoskeleton split open. As in extant ostracodes, trilobites were probably incapable of reusing organic and mineral matter from the old exoskeleton during the formation of a new exoskeleton. With regard to its structure, a trilobite
Comparisons with crustaceans and chelicerates
Towe (1978:459) expressed doubts as to whether ‘trilobites have a typical arthropod cuticle’. He pointed out that fine horizontal laminae, observed sporadically in a limited number of trilobites, ‘lack any evidence of microfibrils or parabolic structures’, the latter structures referring to the inter-laminae. As demonstrated here, both the exo- and endocuticle in Flexicalymene sp. have a horizontal, fine-laminate organic structure. Typical inter-laminae composed of parabolic arcs of organic fibres occur in the exocuticle. These organic fibres probably consisted of protein and chitin, as similar fibres in extant arthropods (e.g. Hackman 1971). During diagenesis, the fibres lost their chemical characteristics (Teigler & Towe 1975:139) but in places retained their shape and arrangement. Consequently, the trilobite exoskeleton possesses a typical arthropod structure. The exoskeleton in trilobites agrees with that in extant crustaceans in that it is composed of horizontal organic laminar units, and it is mineralized. In podocope ostracodes, the organic fibres in the heavily mineralized carapaces usually form a reticulate structure (Jgrgensen 1970; Bate & East 1975). However, a typical organic lamination has been observed in less mineralized juvenile carapaces of Podocopina (Rosenfeld 1979, Figs. 8, 9) and in non-mineralized carapaces of Myodocopina (Bate & East 1975). Among chelicerates, adult Limulus (Xiphosura, Merostomata) has a thin, horizontally laminate exocuticle and a thick, vertically laminate endocuticle. During ontogeny, the organic endocuticular structure seems to change considerably. Thus, in juvenile Limulus only the marginal parts of the carapace are vertically laminate (‘non-laminate’), whereas the remaining parts are horizontally laminate (Dalingwater 1980). The exoskeleton in the fossil eurypterids studied so far (Dalingwater 1973, 1980) has a similar structure to that in adult Lirnufus: the thin exocuticle is horizontally laminate and the thick endocuticle vertically laminate (Mutvei
232 Harry Mutvei
LETHAIA 14 (1981)
Fig. 6 . Nephrops norvegicus. Untreated vertical fracture plane of the exoskeleton in distal end of the chela showing structure of the exocuticle (em), vertically laminate layer ( v n , and endocuticle (endo).
1977). In extant scorpions, the distal ends of the pedipalps have a layer with a vertically laminate structure (Mutvei 1977) corresponding to the ‘non-laminate zone’ of Dalingwater (1980). The rest of the scorpion exoskeleton is horizontally laminate but the inter-laminae are often conspicuously thick (Mutvei 1977). In extant spiders the exoskeleton is horizontally laminate (Barth 1969). As seen in Fig. 6, a layer with vertically laminate organic structure (vf) also occurs in the distal end of the chela of the extant decapod crustaceans Homarus gammarus and Nephrops norvegicus. This layer replaces the outer part of the endocuticle, whereas the inner part (endo) has retained its normal, horizontally laminate structure. It is obvious that the development of the vertically laminate organic structure does not have a great taxonomic and phylogenetic significance
among arthropods. As pointed out previously (Mutvei 1977), there is no fundamental difference between the vertically and horizontally laminate structures, because the vertical laminae seem to correspond to the parabolic fibrous sheets in the inter-laminae. This condition is also clearly indicated in the right lower corner of Fig. 6. In trilobite exoskeletons a vertically laminate organic structure has not yet been observed. As in crustaceans, the exoskeleton in trilobites is mineralized. In the endocuticle, which is considerably more mineralized than the exocuticle, the c-axes of the calcite crystals are oriented essentially perpendicular to the exoskeletal surface (Teigler & Towe 1975). A similar orientation of the c-axes is found in the heavily mineralized exo- and endocuticles of ostracode carapaces (e.g., Jgrgensen 1970). The high degree of mineralization of the exoskeletons in ostracodes and trilobites explains their resistance against dia-
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Exoskeletal structure in FLEXICAL YMENE 233
Bergstrom, J . 1979: Morphology of fossil arthropods as a guide to phylogenetic relationships. I n Gupta, A.P. (ed.): Arthropod Phvlogenv, 3-53, Van Nostrand Reinhold Co,, New Yo&. Dalinewater, J. E. 1973: Trilobite cuticle microstructure and composition. Palaeontcjlogy 16, 827-839. Dalingwater, J . E. 1975: Further observations on eurypterid cuticles. Fossils and Struta 4 , 271-279. Dalingwater, J . E. 1980: SEM observations on the cuticles of some chelicerates. 8 . fnternationer Arachnologen-Kongress, W i m 285-289. Dalingwater. J . E. & Miller, J . 1977: The laminae and cuticular organization of the trilobite Asaphus raniceps. Palaeontolopv20, 21-32, < , * Evitt, W. & Whittington, H. B. 1953: The exoskeleton of R. Nexiculymene (Trilobita). J . Paleontol. 27, 49-55. Flower, R . H. 1962: Notes on the Michelinoceratida. N e w Mexico Bureau ojMines, Mem. 10, 2 W 2 . Grandjean, J . , Gregoire, Ch. & Lutts. A. 1964: On the mineral components and the remnants of organic structures in shells of fossil molluscs. Bull. Acad. Roy. Belg., Cl. Sci. 50,
genetic changes (Bergstram 1979:29). ln the less mineralized trilobite exocuticle, the original crystallographic orientation is still unknown. The exocuticle seems to aeree in its deeree of mineralization to the exo- and endocuticles of extant decapod crustaceans. For example, in Cancer pagurus small calcite crystals in the exocuticle have a variable and irregular arrangement, whereas in the endocuticle they
are Oriented main’y to the long Of the protein-chitin fibres (Hegdahl et al. 1977a, b). Travis (1970) did not find any Dreferred -~ crystallographic orientation of small calcite crystals in the exo- and endocuticles of the freshwater crayfish Orconectes ririlis. The following two factors explain some of the difficulties encountered in the study of trilobite exoskeletons at an ultrastructural level: ( 1 ) Organic fibres and their arrangement into horizontal laminae and inter-laminae can be expected to be observed practically only in the exocuticle, where mineralization was less extensive. However, the exocuticle is comparatively thin and is therefore easily worn off. In the heavily mineralized endocuticle the organic laminae are partially absent, and the organic fibres cannot be distinguished. (2) The horizontal organic laminar units in trilobite exo- and endocuticles are mostly extremely thin (0.2-0.5 pm). In extant arthropods with equally thin laminar units, e.g., many insects, the organic fibres and their arrangement cannot be observed distinctly with SEM. The latter condition can also be expected in trilobites. The mineralized organic fibres of the laminae and inter-laminae in Flexicalymene sp. could therefore be observed only in some of the thick laminar units which occur close to the endocuticular boundary.
Acknowledgements. - This study was financially supported by Grants 287-102 and 103 from the Swedish Natural Science Research Council. I wish to thank Professor W. M . Furnish and Professor 9. F. Glenister for supplying me with the Maquoketa material and for guidance in field.
Hackman, R. H. 1971: The integument of Arthropoda. I n Florkin, M. & Scheer, 9. T. (eds.): Chemical Zoology 4 , 1-53. Academic Press, New York and London. Hegdahl, T., Silness. J. & Gustavsen, F. 1977a:‘The structure and mineralization of the carapace of the crab (Cuncer pagurus L.). 1. The endocuticle. Zoologica Scripta 6, 89-99. Hegdahl, T.. Gustavsen, F. & Silness. J . 1977b: The structure and mineralization of the carapace of the crab (Cancer pagurus L.). 2. The exocuticle. Zoologicu Scripta 6, 101105.
Henningsmoen, G. 1975: Moulting in trilobites. Fossils and Struta 4 , 179-200. Jdrgensen, N. 0. 1970: Ultrastructure of some ostracods. Bull. Geoi. Soc. Denmurk 20, 79-92. Mutvei, H. 1972: Ultrastructural studies on cephalopod shells. Part 1: The septa and riphonal tube in Nautilus. Bull. Ceol. Instn. Univ. Uppsalu N . S . 3 , 237-261. Mutvei, H. 1974: SEM studies on arthropod exoskeletons. Part 1: Decapod crustaceans, Homarus gammarus L. and Curcinus maenas (L.). Bull. Geol. Instn. Univ. Uppsaln N . S . 4, 73-80. Mutvei. H. 1977: SEM studies on arthropod exoskeletons. 2. Horseshoe crab Limulus polvphemus (L.) in comparison with extinct eurypterids and recent scorpions. Zoologica Scripta 6, 203-2 13. Osmolska. Halszka. 1975: Fine morphological characters in some Upper Palaeozoic trilobites. Fossils and Srratu 4 ,
Barth, F. G. 1969: Die Feinstruktur des Spinneninteguments. I. Die Cuticula des Laufbeins adulter hautungsferner Tiere (Cupiennius s a k i Keys.). 2. Zellforsch. mikrosk. Anat. 97,
Passano, L. M. 1960: Molting and its control. I n Waterman, T. H. (ed.): The Physiology ofcrustarea I , 473-536. Academic Press, New York and London. Rosenfeld, A. 1979: Structure and secretion of the carapace in some living ostracodes. Lefhaia 12, 353-360. Stbrmer. L. 1980: Sculpture and microstructure of the exoskeleton in chasmopinid and phacopid trilobites. Palaeontology 23, 237-271. Tasch, P. 1955: Paleoecologic observations on the orthoceratid coquina beds of the Maquoketa at Graf, Iowa. J . Paleontol. 29, 51%518. Teigler, D. J. & Towe, K. M. 1975: Microstructure and composition of the trilobite exoskeleton. Fossils and Strata
4 , 137-149.
Bate, R. H. & East, 9. H . 1975: The ultrastructure of the ostracode (Crustacea) integument. Bulls. A m . Puleont. 65,
Towe, K . M. 1978: Do trilobites have a typical arthropod cuticle? Palaeontology 21, 459461. Travis, D. F. 1970: The comparative ultrastructure and orga-
16 Lethaia 3/81
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nization of five calcified tissues. I n Schraer, H . (ed.): Biological Calcificurion: Cellular and Molecular Aspects, 203-31 1. North-Holland Publishing Company, Amsterdam. Turpen, J. B. & Angell, R. W. 1971: Aspects of molting and calcification in the ostracod Heterocypris. B i d . Bull. 140, 33 1-338.
LETHAIA 14 (198 I ) Wigglesworth, V . B. 1959: The Control o Growrh undForm: a f Study of the Epidermal Cell in an Insect. 140 pp. Cornell University Press, Ithaca, New York.
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