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Home , Batoidea, Benthobatis, Breviraja, Diplobatis, Narcine, Narcine brasiliensis

Biological Journal of the Linnean Society (1992), 46: 259-298. With 17 figures

Development and morphology of rostral in batoid (: ), with comments on homology within

TSUTOMU MIYAKE, JOHN D. McEACHRAN*, PETER J. WALTON? AND BRIAN K. HALL

Department of , Dalhousie University, Halifax, Nova Scotia B3H 431, Canada, *Department of Wildlge and Sciences, Texas A@M University, College Station, Texas 77843, and ?Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas 77030, U.S.A.

Received 30 August 1990, acceptedfor publication 28 November 1990

The rostral cartilages of batoid fishes were examined to elucidate their development, morphology and homology. Comparison of a variety of rostral cartilages among elasmobranchs with other groups of vertebrates shows that rostral cartilages originate embryologically from the trabecula and/or lamina orbitonasalis. Because different morphogenetic patterns of the derivatives of the two embryonic cartilages give rise to a wide variety of forms of rostral cartilages even within elasmobranchs, and because morphogenesis involves complex interactions among participating structures in the ethmo-orbital area, we put forward conceptual and empirical discussions to elucidate the homology of the rostral cartilages in batoid fishes. With six assumptions given in this study and based on recent discussions of biological and historical homology, our discussions centre on: ( 1 ) recognition of complex interactions of participating biological entities in development and evolution; (2) elucidation of a set of interacting biological and evolutionary factors to define a given morphological structure; (3) assessment of causal explanations for similarities or differences between homologous structures by determining genetic, epigenetic and evolutionary factors. Examples of conceptual approaches are given to make the approaches testable. Although a paucity of knowledge of rostral formation is the major obstacle to thorough analysis of the conceptual framework, several tentative conclusions are made on the homology of rostral cartilages that will hopefully attract more research on development and evolution in morphology. These are: (1) the rostral cartilage in each group of vertebrates examined can be defined by both developmentally associated and adult structural attributes, yet such data do not allow us to assess homology of a variety of forms of rostral cartilages at higher taxonomic categories; (2) the entire rostral cartilage in elasmobranchs is formed by the contribution of the embryonic trabecula and lamina orbitonasalis. The status of the development and homology of the rostral cartilage in holocephalans remains uncertain; (3) there is no simple picture of evolution of rostral cartilages among three putative monophyletic assemblages of elasmobranchs, galeomorphs, squaloids (possibly plus Squatina, Chlamydoselachus and hexanchoids as the orbitostylic group) and batoid fishes. It is highly likely that rostral cartilages in each subgroup or subgroups of these assemblages may be of phylogenetic significance but that it may not serve as a basis to unite these assemblages into much higher assemblages; (4) the tripodal rostral cartilage is unique in form in the group including some carcharhinoid and lamnoid . The status of the analogous tripodal cartilage in some squaloids remains uncertain. The unfused tripodal cartilage of the is interpreted as developmentally equivalent to, but not homologous with, the unfused or fused ones in the sharks; (5) the rostral cartilage in the electric ray is uniquely formed because of its embryonic origin solely from the ventro-medial part of the lamina orbitonasalis, but it is regarded as homologous with the rostral cartilages which are formed by the trabecula and other components of the lamina 259 0024-4066/92/070259 + 40 $03.00/0 0 1992 The Linnean Society of London 260 T. MIYAKE ETT AL.

orbitonasalis in other batoid fishes; (6) the cornu trabecula contributes to the formation of the ventral stem of the rostral cartilage at least in elasmobranchs, especially to a particular set of rostral cartilages, i.e. the tripodal rostral cartilage in the scyliorhinus and dorso-ventrally flattened rostral shaft in the narcinidid electric rays; (7) there is a unique form of a rostral shaft with rostral appendix in skates and probably ; (8) there is no rostral cartilage in adult benthic stingrays, pelagic stingrays Dasyatis violacea and Myliobatidae, although it is present in embryonic stages; (9) there is a unique form of the rostral cartilage as a rostral projection from the dorso-lateral part of the lamina orbitonasalis in pelagic stingrays Rhinopteridae and Mobulidae, which together with part of the pectoral fins, forms a pair of cephalic fins; (10) different developmental mechanisms may be responsible for the absence or loss of rostral cartilages in different groups, i.e. absence of the cartilage derived from the medial area of the trabecula in Torpedo vs absence of the rostral cartilage in benthic stingrays; (1 1) the rostral cartilages in some placental mammals (cetaceans and sirenians) arise only from the medial area of the trabecula because monotreme and placental mammals do not form the trabecula cranii; (12) some actinopterygians and sacropterygians possess a rostral cartilage which originates only from the medial area of the trabecula. One scombroid group, including Sardini and Thunnini, Scornberamorus, Acanthocybium, lstiophoridae and Xiphias, possesses a unique larval composed of the rostral cartilage, ethmoid cartilage and premaxillar . The development and homology of other rostral cartilages remain to be further elucidated; (13) urodeles possess a medial rostral process whose anlage is probably developmentally equivalent to that in batoid fishes but the occurrence in urodeles is either atavistic or unique (autapomorphic); (14) the upper jaw of tadpoles is unique in possessing the suprarostral cartilage; the anlage of the cartilage is probably developmentally equivalent to the outgrowth of the cornu trabecula in batoid fishes.

KEY WORDS:--Batoid fishes - craniofacial development - development of rostral cartilage

trabecula ~ lamina orbitonasalis - biological and historical homology.

CONTENTS Introduction ...... 260 Materials and methods ...... 261 Results ...... 26 1 Cranial embryonic cartilages and formation of rostral cartilages ..... 26 1 Rostral cartilages of skates ...... 263 Rostral cartilages of guitarfishes ...... 265 Rostral cartilages of stingrays ...... 266 Rostral cartilages of ...... 269 Rostral cartilages of electric rays ...... 270 Discussion...... 274 Development of the trabecula and cornu trabecula...... 274 Development of the lamina orbitonasalis ...... 276 Developmental perspectives ...... 279 Homology: assumptions ...... 280 Homology: historical approach ...... 281 Homology: biological approach ...... 286 Homology: conclusions ...... 289 Homology of rostral cartilages ...... 289 Acknowledgements ...... 292 References ...... 293 Abbreviations used in figures ...... 296 Appendix...... 297

INTRODUCTION Batoid fishes, one of three major groups of chondrichthyan fishes, comprise more than 450 and occupy a variety of habitats ranging from freshwater to pelagic and deep benthic marine waters. They consist of five monophyletic groups; electric rays (torpedinoids), sawfishes (pristoids), skates (rajioids), guitarfishes (rhinobatoids) and stingrays (myliobatoids) (Compagno, 1973, 1977). All groups have a dorso-ventrally depressed body with laterally expanded pectoral fins. Their palaeontological history dates back to the (Carroll, 1988) and their external morphologies give the impression that they have been morphologically conservative during their long evolution history. However, ROSTRAL CARTILAGES IN BATOID FISHES 26 1 recent systematic and anatomical studies have revealed a variety of anatomical features among the five major groups of batoid fishes (Compagno, 1977; Miyake, 1988; Miyake & McEachran, 1991 ), suggesting major morphological evolution. Recent chromosomal and molecular data support the dynamic nature of their evolutionary history (Ida et al., 1986; Ida, Sat0 & Miyawaki, 1986; Olmo et al., 1982; Schwartz & Maddock, 1986; Stingo, 1979; Stingo & Capriglione, 1986), suggesting considerable karyotypic and molecular changes even within subgroups of batoid fishes (Ida et al., 1986; Schwartz & Maddock, 1986; Stingo & Capriglione, 1986). Miyake (1988) undertook a broad anatomical survey of craniofacial morphologies of batoid fishes and brought attention to many of their evolutionary features. One which has been recognized as of systematic and evolutionary significance is the presence of a rostral cartilage (McEachran & Compango, 1979, 1982; Miyake, 1988). The rostral cartilage is an anterior extension of the cartilaginous neurocranium which is medially sandwiched by the anterior expansion of the pectoral fins (Compagno, 1977; Miyake, 1988). It displays a variety of sizes and shapes, in some cases with an additional cartilaginous appendix at the tip (McEachran & Compagno, 1979, 1982; Miyake, 1988). McEachran & Compagno ( 1982), therefore, placed systematic and phylogenetic emphasis on the morphology of the rostral cartilage in skates, and McEachran & Miyake (1990) showed that the cartilage and its associated morphological features display considerable variations during evolution among subgroups of skates. We have surveyed the morphology and development of the rostral cartilage in batoid fishes in to elucidate further its role in craniofacial development. Our study presents results based on our own observations of rostral cartilages in some representatives of batoid fishes, combined with a compilation of published data. Also we offer working hypotheses for further studies on development and morphology of rostral cartilages and provide conceptual and empirical discussions on homology of rostral cartilage in batoid fishes as well as in other chondrichthyan fishes.

MATERIALS AND METHODS Rostra1 cartilages were examined from gross dissection of specimens, cleared and stained specimens and X-radiographs. and juvenile specimens of selected batoid fishes were cleared and double stained based on Dingerkus & Uhler (1977). A list of the specimens examined and used in discussions is found in Appendix 1. Photomicrographs of the neurocranium were taken with the Wild MPS I2 System equipped with Wild MPD 05 Microphoto Automatic Exposure Meter. Data on earlier developmental stages of rostral cartilages are gleaned from published data and interpreted based on our views of development, morphology and homology.

RESULTS Cranial embryonic cartilages and formation of rostral cartilages The embryonic cartilages associated with the ethmo-orbital area of chondrichthyan fishes (batoid fishes, sharks and holocephalans) are the lamina 262 T. MIYAKE ET AL.

supob

lmhy Figure I. Schematic diagram of embryonic cranial cartilages in chondrichthyans, constructed based on Allis (1923), de Beer (1937), Holmgren (1940) and Miyake (1988).See abbreviations in the text.

orbitonasalis, orbital cartilage, three pilas of orbital cartilage, preoptic root, pila metoptica and pila antotica, supraorbital cartilage, trabecula and polar cartilage (Fig. 1). The rostral cartilage of elasmobranch fishes and possibly also that of holocephalans, arise from the trabecula and lamina orbitonasalis. As an example, we use the development of two embryonic cartilages in the shark Scyliorhinus to present generalized developmental pathways leading to the formation of the adult rostral cartilages in sharks and batoid fishes. The trabecula is initially formed as a pair of blastemata in front of the polar cartilage anlage (called here trabecular cranii) (de Beer, 1937; Holmgren, 1940). The area between the trabecular cranii becomes blastematic and forms a medial area ( = trabecula communis) Uollie, 1971). The medial area of the trabecula extends anteriorly and gives rise to a medial plate (Holmgren, 1940). As it chondrifies, it creates the cranial base in front of the hypophysial fenestra (Fig. 2). The medial area, in some cases, extends further antero-laterally to form a rostral plate (Holmgren, 1940; Jollie, 1971). In Scyliorhinus, th: initial chondrification in the medial area takes place denovo in the distal area of the blastema (Fig. 2A). The proximal area then chondrifies, grows anteriorly and meets the cartilage initially formed (de Beer, 1937; Holmgren, 1940). The fate of the cartilaginous differentiation and the extent and direction of the subsequent growth of the medial area, therefore, vary considerably at least among different groups of batoid fishes as well as sharks. The area also contributes to formation of the internasal septum and cornu trabecula ( = frontal wall of nasal capsules in de Beer, 1931); trabecular horn in de Beer & Moy-Thomas (1935); suprarostral process of Holmgren (1940). The latter cartilage of Scyliorhinus grows out from the anterior tip of the trabecula cranii, extends ventro-laterally along the ventro-medial margin of the nasal capsules and fuses with the anterior wall of the nasal capsules (Fig. 2). The lamina orbitonasalis arises from dorsal and ventro-lateral outgrowths of the trabecula (de Beer, 1937; Holmgren, 1941; Jollie, 1971). Although the lamina orbitonasalis appears to form a continuous blastematic sheet with the trabecula (Holmgren, 1940), it may chondrify as a separate centre (de Beer, 1937; Holmgren, 1940) or by extension from the trabecula Uollie, 1971). As it chondrifies, the dorsal outgrowth of the lamina orbitonasalis forms the hind and medial walls of the nasal capsules. The lamina orbitonasalis, as it grows dorso- medially, meets medio-laterally, the growth of the orbital component, preoptic ROSTRAL CARTILAGES IN BATOID FISHES 263

A B C D

b

Figure 2. Ventral view of early development of the neurocranium in the shark Scyliorhinus canicula showing the development of the tripodal rostral cartilage (after Holmgren, 1940, but modified based on de Beer, 1937). A, 30 mm in total length; B, 31 mm; C, 38 mm; D, 40 mm. Arrow head indicates the medial plate of the trabecula and arrow shows paired lateral rostral rods which are derived from the antero-dorsal growth of the lamina orbitonasalis. root, and creates the orbitonasal canal and anterior cerebral vein foramen at the boundary of two cartilages (Miyake, 1988). It meets the spheno-ethmoidal commissure dorsally, which connects the major portion of the orbital cartilage, and fuses with the anterior growth of the supraorbital cartilage (de Beer, 1926, 1931, 1937; de Beer & Moy-Thomas, 1935; Holmgren, 1940; Jollie, 1971). Further growth, especially anteriorly, of this component of the lamina orbitonasalis forms the medial wall of the nasal capsules, but the extent to which the growth contributes to the dorsal part of the rostral cartilage remains speculative in batoid fishes (de Beer, 1937; Holmgren, 1940; Miyake, 1988). In Scyliorhinus, the anterior growth of the lamina orbitonasalis gives rise to a long projection as paired lateral rostral rods from the nasal capsules and forms a tripodal rostral cartilage together with a single medial rostral rod (Fig. 2) (de Beer, 1931; Holmgren, 1940; Compagno, 1988). The ventro-lateral outgrowth of the lamina orbitonasalis composes the frontal and side walls of the nasal capsules. It also gives rise to both ethmoid process and antorbital cartilages (de Beer, 1937; Holmgren, 1940, 1941). Combined contributions of the medial area plus possibly the cornu trabecula, with mainly the dorsal component of the lamina orbitonasalis, thus give rise to rostral cartilage with a variety of sizes and shapes in batoid fishes, sharks and possibly holocephalans.

Rostra1 cartilages of skates In skates the rostral cartilage varies considerably in size and shape, and is associated with an additional structure, the rostral appendix (McEachran & Compagno, 1982; McEachran & Miyake, in press). However, the initial developmental pathway of formation of the rostral cartilage appears to vary little within the groups examined in this study (Figs 3,4). Holmgren (1940) described the development of the cranial cartilage in (Raja) clavata in which the rostral cartilage is formed mainly by an outgrowth, as a rostral plate, of the medial area of the trabecula. The medial plate of the trabecula initially is formed as a large blastematic condensation at the 38 mm embryonic stage which connects the paired prechondrogenic trabecular cranii. The medial plate starts 264 T. MIYAKE ET AL.

A B C D

Figure 3. Ventral view of development of the neurocranium in Raja (Raja) clauata (after Holmgren, 1940). A, 46 mm in total length; B, 50 rnm; C, 58 mm; D, 70 mm. chondrifying at the 46-50 mm stages, grows anteriorly as a rostral plate and forms the ventral stem of the rostral cartilage (Fig. 3). In most groups of skates, the rostral cartilage becomes continuous with the dorso-medial apex of both sides of the nasal capsules, forming a dorsal ridge continuous with the cartilage around the precerebral fontanelle. This continuity indicates that the connection of the rostral cartilage with the antero-dorsal apex of the nasal capsules is formed by the dorsal growth of the rostral plate, as suggested by Holmgren (1940). However, because the cornu trabecula grows from the medial area dorso- laterally and forms part of the ventro-frontal wall of the nasal capsules, this dorsal connection may have in part or solely a contribution from the cornu trabecula. In addition, the rostral cartilage is ornamented with a cartilaginous rostral appendix (Fig. 3C). The appendix is usually fused with the anterior apex of the rostral cartilage, but the relationships between the appendix and the rostral cartilage varies considerably among different subgroups of skates (McEachran & Miyake, 1990). The appendix is formed de nouo at both sides of the rostral shaft and is carried away by the anterior growth of the rostral shaft at later stages (Fig. 3). The medial area of the trabecula also makes a major contribution to the rostral cartilage of Raja (Leucoraja) erinacea (Fig. 4). Even in this specimen, it is difficult to determine the extent of the contribution of the lamina orbitonasalis to formation of the rostral cartlilage. Figure 5 shows a variety of rostral cartilages in skates. Size and shape seem to depend on the following combinations of development of the major areas of the rostral cartilage (called rostral shaft) and appendix: (1) fully developed rostral shaft and appendix, with appendix fused over its entire length to the shaft

Figure 4. Development of the neurocranium in Raja (Leucoraja) erinncea (TCWC uncat., 56 mm in total length). A, Lateral view. B, Ventro-frontal view. ROSTRAL CARTILAGES IN BATOID FISHES 265

0

Figure 5. Dorsal view of the neurocranium in adult skates showing the range of variation of rostral cartilages and appendices. A, Raja (Dipturus) johannisdauisi (BMNH 1939.5.24.19). B, Raja (Leucoraja) ocellata (TCWC uncat.). C, Breuiraja colesi (MCZ 41998). D, Gurgesiellafumescm (TCWC uncat.). E, Bathyraja peruana (USNM 267045). F, Arhynchobatis aperrimus (NMNZ 5200). G, Psammobatis scobina (TCWC uncat.). H, Pseudorajajischeri (TCWC uncat.). Arrow head indicates the rostral appendix and arrow locates the rostral shaft (rostral cartilage).

(Fig. 5A); (2) fully developed rostral shaft and appendix, with appendix free over much of its length from the rostral shaft (Fig. 5B); (3) distal section of the rostral shaft regressed, with appendix largely free of the rostral shaft (Fig. 5C), or appendix entirely free of the rostral shaft (Fig. 5D); (4) entire rostral shaft regressed and appendix fully developed (Fig. 5E); (5) entire rostral shaft regressed and separated from remainder of neurocranium by segment (Fig. 5F) or shaft separated from remainder of neurocranium by space (Fig. 5G); (6) rostral shaft vestigial and appendix fully developed (Fig. 5H). The rostral shaft varies in development and may be entirely absent but the appendix is always present at the tip of the snout. Developmental aspects of these diverse forms of the rostral cartilage remain to be elucidated.

Rostra1 cartilages of guitarJLrhes Guitarfishes display considerable variation in the structure of the rostral cartilage and rostral appendix but development of the cartilage has been described only for Rhinobatos (Hamdy, 1974). According to Hamdy (1974),the rostral cartilage of the Rhinobatos initially arises as three projections from the trabecula, which then supposedly fuse to produce a massive rostral cartilage. Although the exact origin and sequence of the chondrification of the three 266 T. MIYAKE ET AL

A

ns

Figure 6. Dorsal view of the neurocranium in adult guitarfishes. A, Rhinobatos productus (TCWC 6183.1). 9, siensis (after Holmgren, 1941). C, Plnbrhinaidis spp. (after Compagno, 1977). projections still remain unknown, it seems likely, based on his illustration and brief description, that the projections arise from the medial area of the trabecula and form a long, trough-shaped rostral cartilage. The condition of the rostral cartilage in Rhinobatos is essentially identical to that in the Raja, especially R. (Dipturus) (McEachran, unpublished data). It is speculated that the cornu trabecula would contribute to the formation of the ventral stem of the rostral cartilage. The rostral cartilage of guitarfishes are variable in size and shape but resemble those of skates. Those of , Rhina and

Rostral cartilages of stingrays Two embryonic cartilages form a vestige of the rostral cartilages in benthic stingrays (Dasyatoidea) and possibly in the pelagic stingrays of the family Myliobatidae. Holmgren ( 1940) described the early development of the rostral cartilage in the Urolophus halleri. According to his study, the medial area of the trabecula is formed as a blastema connecting a pair of trabecular cranii. The area chondrifies as a rostral plate and forms medial projection at the 49 mm ROSTRAL CARTILAGES IN BATOID FISHES 267

Figure 7. Microphotograph of dorsal view of early development of the neurocranium in Urotygon venezuelae (TCWC uncat., 28 mm in total length). A, Dorsal view of the whole area of the anterior neurocranium. B, Close-up view of the left side of the ethmo-orbital area. embryonic stage, that indicates the medial growth of the trabecula. However, the projection does not appear to grow and displays only a vestigial cartilaginous projection arising from the ventro-medial apex of the nasal capsules in adults. A small cartilage similar to the rostral appendix of skates develops medial to the anterior extension of the pectoral fins (Holmgren, 1940: figs 183, 184). Because of this location of the appendix Holmgren (1940) interpreted it as originating from the pectoral fin . Our study on the rostral cartilage of Urolophus 268 T. MIYAKE ET AL.

A

(V)

B Jmb

(D) (V) Figure 8. Early development of the anterior area of the neurocranium in stingrays. A, Urolrygon uenezuelae (TCWC uncat., 28 mm in total length). B, Rhinofitera bosanus (TCWC 2528.1, 56 mm in total length). Hatched area indicates the structures (including nasal capsules and part of the rostral cartilage) which the lamina orbitonasalis gives rise to. (D) Dorsal view; (V) ventral view. uenezuelae (Figs 7A, 8A) confirms Holmgren’s findings that the medial plate of the trabecula actually chondrifies and forms the internasal septum with a short medial projection, but in adults the projection grows little and eventually degenerates. The rostral appendix is found in the 60 mm , some adults of Urolophus halleri, adults of the freshwater stringray genera Potamotrygon and Plesiotrygon (Rosa, 1985; Rosa, Castello & Thorson, 1987) and some adult species of (Fig. 9) (Miyake, 1988). However, contrary to Holmgren’s claim, the appendix appears to originate in connection with the medial area of the trabecula; once formed, it seems to be carried away between the pectoral fins as the cranium grows anteriorly with the pectoral fin extension. The lamina orbitonasalis is also present as dorsal and ventro-lateral outgrowths of the trabecula (Figs 7, 8A), but it does not seem to contribute to the rostral cartilage. In the pelagic family Myliobatidae, as in other benthic stingrays, there is no rostral cartilage or paired projections from the antero-dorsal end of the nasal capsules (Figs 10, 11A) (Hamdy & Khalil, 1972). Nothing has been published on the development of the rostral cartilage in pelagic stingrays including the families Rhinopteridae and Mobulidae. However, the 56 mm embryo of bonasus reveals an interesting aspect of the early development of the ethmo-orbital area (Fig. 8B). The medial area of the trabecula chondrifies, possibly as a medial plate, but shows no sign of its antero-medial growth. The area forms only the internasal septum in adults. The lamina orbitonasalis develops ventrally and dorso-laterally from the trabecula. In adults of Rhinoptera, the nasal capsules are formed ventral to the main axis of the neurocranium (Fig. 1 l), yet the neurocranium possesses a pair of anterior projections arising from the dorso-medial area of the nasal capsules (Figs 10, 1 IB). Because the paired projections are formed rostral to the anterior limit of the neurocranium and since the projections appear to be the antero-dorsal ROSTRAL CARTILAGES IN BATOID FISHES 269

Figure 9. Microphotograph of the rostral appendix in Urolrygon nana (FMNH 72281). growth of the nasal capsules, we propose that the paired projections originate from the lamina orbitonasalis and that they represent at least the dorso-lateral component of the rostral cartilage. Similar rostral projections are found in the mobulid genus (Figs 10, 1 1C) . Our findings of the presumptive rostral cartilage in two families of pelagic stingrays Rhinopteridae and Mobulidae also reveal an interesting morphological complex which may relate to functional in pelagic waters. Species of the two families have a pair of cephalic fins which project from the anterior limit of the head (Garman, 1913). These cephalic fins are composed of a separated propterygium of the pectoral fin ornamented with fin rays and are supported dorsally by a pair of the presumptive rostral projections, as discussed above. In Rhinopteridae, the cephalic fins are continuous with the major portion of the propterygium, but the fin rays on the cephalic fins are discontinuous with those on the propterygium. The rostral projections lie ventral to the cephalic fins and morphologically and structurally become associated with the latter. Although the two morphological features are not externally observable, the space between their two features creates externally a deep groove running longitudinally from the anterior tip of the head to the posterior limit (Bigelow & Schroeder, 1953). In the mobulid genera Mobula and Manta, the cephalic fins are externally separated from the major portion of the pectoral fin and form a pair of paddle- shaped projections in front. Dorsally, the rostral projections become structurally associated with the proximal end of the cephalic fins (Fig. 11C). This morphological complex may be adapted for filter feeders and related to hydrodynamic adaptations to pelagic waters, yet no data exist to support this speculation.

Rostra1 cartilages of sawjshes The development of the neurocranium of two genera Pristis and Anoxypristis has been studied (Hoffmann, 19 13; Miller, 1974), but neither study 270 T. MIYAKE ET AL.

Other stingrays

Myliobctidae

Rhinopteridce

Mcbulidae

Figure 10. Neurocrania of adult stingrays. Left: lateral view; right: dorsal view. Hatched area indicates part of the rostral cartilage which is proposed to originate from the lamina orbitonasalis. discussed the early modes of development of the rostral cartilage. Features of the rostral cartilage in sawfishes include teeth along the lateral margin of the long and flat rostral cartilage (Hoffmann, 1913; Compagno, 1973, 1977) and enclosure, as a precerebral fossa, of three pairs of cranial nerves, dorsally the superficial ophthalmic trunk of the r. ophthalmicus superficialis V nerve and r. ophthalmicus lateralis and ventrally the r. buccalis lateralis (Hoffman, 1913; Holmgren, 1941; Miyake, 1988). The teeth along the rostral cartilage in sawfishes have been considered as not being replaced, unlike those in the rostral cartilage of (Pristiophoridae) (Miller, 1974). However, Slaughter & Springer (1968) observed that extant sawfishes and the Late Sclerorhpchus also possess replacement teeth, as suggested by Schaeffer (1963).

Rostra1 cartilage of electric rays In electric rays that comprise three monophyletic lineages, torpedinids, narcinidids and narkinids (Compagno, 1973, 1977), the early development of ROSTRAL CARTILAGES IN BATOID FISHES 27 1

A B C

Figure 11. Cranial area of stingrays showing the cephalic fins in Rhinopteridae and Mobulidae. A, Myliobatidae (after Garman, 1913 and Miyake, 1988). B, Rhinopteridae (after Garman, 1913). C, Mobulidae (after Garman, 1913). Arrow indicates the anterior part of the pectoral fins which form part of the cephalic fins in Rhinopteridae and Mobulidae. Hatched area represents the putative rostral cartilage. the rostral cartilage differs in several respects from that described for skates and stingrays. In Torpedo (), the medial area of the trabecula is formed as a blastema at the 17-21 mm embryonic stages. At the 24.5 mm stage, the trabecular cranii and the proximal portion of the medial area chondrify, yet the distal portion of the medial area remains blastematic (Fig. 12A). At subsequent stages, the trabecular cranii and the proximal portion of the medial area form the internasal septum and cranial base in front of the hypophysial fenestra. The greater portion of the medial area, however, remains blastematic and eventually contributes only to the formation of ligaments between a pair of cartilaginous projections; it does not produce any of the rostral cartilage. On the other hand, a pair of projections in adults of Torpedo, which Holmgren (1940) called the rostral appendix, arises from the lamina orbitonasalis. At the 24.5 mm stage, the anlage for the projections appears in the ventro-medial area of the lamina orbitonasalis which caudally is continuous with the future antorbital cartilage. The anlage starts chondrifying at the 31 mm stage and grows anteriorly as a pair of cartilaginous plates in the subsequent stages (Fig. 12B). That the projections are dorsally continuous with the dorsal apex of the nasal capsules in adults (Holmgren, 1940: figs 165, 166) indicates either a possible dorsal growth of the anlage to meet the apex of the nasal capsules, or a possible contribution of the antero-dorsal growth of the lamina orbitonasalis. However, neither Holmgren’s study in 1940 nor our own studies confirm clearly to what extent the antero-dorsal growth of the lamina orbitonasalis contributes to the dorsal portion of the rostral cartilage in Torpedo. Holmgren’s prenasal area 272 1’. MIYAKE ET AL.

A B

Figure 12. Two developmental stages of the neurocranium in Torpedo lorpedo (after Holmgren, 1940). A, 24.5 mm in total length; B, 35 mm. Arrow head indicates the area of the medial plate of the trabecula and arrow shows the rostral cartilage arising from the ventro-medial plate of the lamina orbi tonasalis.

(Holmgren, 1940: fig 162, p.n.a.) may be the whole area or parts of the antero- dorsal growth of the lamina orbitonasalis. Whether this pair of projections in Torpedo is developmentally equivalent and/or homologous to the rostral appendix of skates, as Holmgren (1940) argued, or represent the rostral cartilage proper will be discussed below. Nothing is known about the early development of the rostral cartilage in Hypnos (Hypnidae). The anterior margin of the precerebral fontanelle in adult Hypnos is not enclosed within a cartilaginous structure, but is merely covered with thin connective tissue (Fig. 13). Holmgren (1941: fig. 55) illustrated the rostral cartilage of Hypnos monopterygium ( = H. subnigrum) with several branches of cartilages medial to the antorbital cartilage which project from the anterior margin of the neurocranium. However, our observation of the same species reveals that the branched segments of his “rostral cartilage” are parts of the antorbital cartilage (Fig. 13). Thus, we conclude that Hypnos monoptevgiurn does not possess any portion of a rostral cartilage. The trabecula cartilage is a major component of the rostral cartilage in narcinidid electric rays. In Nurcine brusiliensis (Figs 14A, 15B), unlike any of the other batoids or sharks so far known, the initial chondrification is a pair of cartilaginous strips located just anterior to hypophysial fenestra; the area

A B anb

Figure 13. Ethmo-orbital area of adult Hypnos monopleiygium (MCZ 38602). A, Dorsal view. B, Lateral view. ROSTRAL CARTILAGES IN BATOID FISHES 273

A

rc

Figure 14. Early stage of development of the neurocranium in brasifiensis (TCWC 6777.1).A, 34 mm in total length; B, 36 mm. (V) ventral view. (L) lateral view. Arrow head indicates a pair of cartilaginous strips which are considered the cornu trabecula arising from the anterior tip of the trabecula cranii. Hatched area represents the dorsal component of the rostral cartilage. Dotted line indicated the location of the primary cranial flexure. between the two cartilaginous strips starts chondrifying later on. Because of the location and direction of the growth of the paired cartilaginous strips, they are considered to be the cornu trabecula which arises from the most anterior tip of the trabecular cranii and grows dorsally around the nasal capsules. Because the nasal capsules of narcinidid electric rays are set far apart and create a wide space as the internasal septum, the growth of the cornu trabecula may proceed more laterally to cover the antero-ventral area of rostral cartilage. Laterally, there is a pair of fenestra on the rostral cartilage in Narcine brusiliensis. Because the dorso- lateral area of the rostral cartilage is widely continuous with the anterior portion of the neurocranium, it is difficult to determine the boundary between two structures which are supposedly formed by the fusion of the lamina orbitonasalis and spheno-ethmoidal commissure (Fig. 15A). In Narcine, Diplobatis, and Discopyge, the genera of narcinidid electric rays, the rostral cartilage is dorso- ventrally flattened and trough-shaped (Fig. 16D, E). All the genera have lateral fenestra on both sides of the rostral cartilage and Diplobatis also possesses a pair of ventral fenestra (Fechhelm & McEachran, 1984). The presence of the ventral fenestrae indicates that the trabecula may not completely chondrify to close the mid-ventral area of the rostral cartilage. Nothing is known about the development of the rostral cartilage in narkinid electric rays. In adults of narkinid and narcinidid electric rays, the rostral 274 T. MIYAKE ET AL. cartilage differs considerably from that of torpedinid electric rays. According to Garman's ( 1913) illustration, the rostral cartilage of Narke japonica consists of a single medial rod-like structure. However, Holmgren (1941) and Nishida (1985) demonstrated that in Narke japonica, the rostral cartilage forms tri-podal cartilages, one medially and two laterally, without being fused together (Fig. 16B), the condition which is also found in the scyliorhinid sharks Scyliorhinus retzfer and Cephaloscyllium ventriosum (Compagno, 1988). The medial cartilage supposedly originates embryologically from the nasal septa1 area whereas the lateral cartilages are continuous with the nasal capsules. The condition is similar in Narke capensis except for the lateral cartilages which are completely detached from the nasal capsules (Holmgren, 1941). In the genus , another genus in narkinid electric rays, the rostral cartilage does not seem to chondrify, but the space is filled by dorso-laterally directed strong ligamentous fibres extending from the mid-dorsal area of the nasal capsules to both sides of the antorbital cartilage (Fig. 16C).

DISCUSSION Development of the trabecula and corm trabecula The trabecular cranii and medial area are initially involved in development of the rostral cartilage and other components of the ethmo-orbital area in batoid fishes and sharks. However, size and shape of the blastema of the two embryonic cartilages vary considerably among elasmobranch taxa (Holmgren, 1940), and the subsequent differentiation of cartilages from the two areas of the trabecula displays a variety of size and shape of rostral cartilages and other structural components in the ethmo-orbital area (Fig. 17). The trabecular cranii invariably show little participation in the formation of the rostral cartilages in batoid fishes. However, because it contributes to the formation of the cornu trabecula and cranial base including the nasal septum anterior to the hypophysial fenestra, and since the size and shape of the area involved exhibit a wide range of morphology in chondrichthyan fishes (de Beer, 1937; Holrngren, 1940, 1941; Compagno, 1977; Miyake, 1988; McEachran & Miyake, 1990), studies on morphogenesis of the trabecular cranii would shed light on the understanding of how, and to what extent, the formation of the area involved imposes its influence on morphogenesis of the rostral cartilage. The main contributor to the stem of the rostral cartilage in batoid fishes and sharks is the medial area and/or its anterior development as a medial plate. When these areas do not differentiate into a cartilaginous sheet, the rostral cartilage is absent, as in the electric rays Hypnos and Typhlonarke, or other cartilages contribute to the embryonic rostral cartilage, as in the electric rays Torpedo and Narke, and the pelagic stingrays Rhinopteridae and Mobulidae. If the area chondrifies, the timing, sequence and direction of chondrification play some role in the final shape of the rostral cartilage. As seen in Scyliorhinus, the first cartilage is formed de nouo at the tip of the medial area; then the latter area appears to grow anteriorly, fuse with the former cartilage and form a medial rostral rod (de Beer, 193 1; Holmgren, 1940). Differential chondrification of the medial area, as exemplified by Scyliorhinus, may also occur in the skate genera Rhinoraja, Sympterygia, Psammobatis, Irolita and Pavoraja, except that in the skate ROSTRAL CARTILAGES IN BATOID FISHES 275

Figure 15. Microphotograph of development of the neurocranium in the 34 mm embryo of Narc& brmiliensir (TCWC 6777.1). A, Oblique ventro-lateral view. B, Fronto-ventral view. Arrow head indicates a pair of putative cornu trabecula and arrow indicates the possible junction between the orbital and rostral cartilage. genera there is no fusion between the cartilages. In Rhinoraja, Sympterygia and Irolita the two cartilages are connected by a , and in Psammobatis and Pavoraja the two cartilages are separated by a distinct space. The shaft is always, however, ornamented with a rostral appendix. In the case of Pseudoraja Jischeri (Fig. 5H) and some stingrays, the rostral cartilage is devoid of most of the rostral shaft and is represented only by a rostral appendix. These instances indicate interesting developmental phenomena of differentiation, growth and remodelling of the rostral cartilage formed from a variety of size and shape of the blastema of the medial area of the trabecula. The contribution of the cornu trabecula to the formation of the rostral cartilage remains elusive. However, this cartilage may participate in the formation of the proximo-ventral area of the rostral cartilage in elasmobranchs. The cornu trabecula has structurally been considered homologous with the trabecular horn which separates the anterior from the posterior nasal fenestrae in other vertebrate groups (de Beer & Moy-Thomas, 1935). In some salamanders, for example, the cornu trabecula grows laterally from the anterior tip of the trabecula cranii and forms part of the nasal capsules as the trabecula horn (de Beer, 1937). In the shark Scyliorhinus, the cornu trabecula arises from the most 276 T. MIYAKE ET AL. rc A B rc

OC endfo oc endfo

D

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3:*. oc endfo enbfo Figure 16. Dorsal view of neurocrania in adult electric rays. A, 7orpedo mannorata (after Garman, 1913). B, Narke japonica (after Garman, 1913, but modified based on Nishida, 1985). C, Typhlonarke aysoni (FRSKU 46477). D, Benthobatis morcida (TCWC 1903.1). E, Narcine brasilienris (TCWC uncat.). anterior tip of the trabecular cranii just ventral to the proximal origin of the medial rostral rod and connects ventrally both sides of paired lateral rostral rods (Holmgren, 1940). This dorso-lateral growth pattern of the cornu trabecula is analogous to the growth of the rostral plate which results in the formation of the distal connection of the rostral cartilage with the anterior limit of the neurocranial proper in the sharks Squalus and Etmopterus (de Beer, 1937; Holmgren, 1940; Jollie, 1971) and most skates (Fig. 5). In the case of the electric ray Narcine, the most anterior tip of the trabecula cranii starts chondrifying as a pair of cartilaginous strips and grows laterally, as the narrow medial area between the two strips delays chondrifying and fills the ventro-medial area of the rostral cartilage (Fig. 14A, 15B). Although the cornu trabecula was proposed to form the ventro-medial wall of the nasal capsules (de Beer & Moy-Thomas, 1935), it is quite possible that the growth and contribution of the cartilage have changed during the evolution of batoid fishes, and that it may display a variety of patterns of morphogenesis at the proximo-ventral area of the trabecula. If this is the case, the cornu trabecula would play its special role in forming the ventral stem of the rostral cartilage in batoid fishes as well as sharks. The strips of cartilage seen in Narcine would thus represent the earlier development of the cornu trabecula.

Development of the lamina orbitonasalis The role of the lamina orbitonasalis in the formation of the nasal cartilage remains uncertain. In Torpedo the ventro-medial area of the lamina orbitonasalis ROSTRAL CARTILAGES IN BATOID FISHES 277 forms the nasal capsule without any involvement of the medial plate of the trabecula. Because of the topographic resemblance of the blastematic origin of the rostral cartilage of Torpedo to that of the rostral appendix of skates, Holmgren (1940) concluded that the rostral cartilage of Torpedo is not the rostral cartilage proper but the rostral appendix. However, our study reveals that the paired rostral cartilage of Torpedo (Holmgren’s rostral appendix) arises from the ventro- medial area of the lamina orbitonasalis, while the rostral appendix of skates, as seen in Raja clavata, is formed de nouo at the proximal sides of the growing rostral plate (Fig. 3C). As the rostral appendix chondrifies it is fused with the rostral cartilage and displaced anteriorly by the growth of the rostral cartilage. In addition, the last contribution of the lamina orbitonasalis is to form the medial wall of the nasal capsules (Fig. 3A) which in turn fuses the capsules with the rostral plate (Fig. 3B). Thus the lamina orbitonasalis makes no contribution to the formation of any cartilages near the medial plate of the trabecula. As discussed by de Beer (1937), the formation of the medial wall of the nasal capsules by the lamina orbitonasalis is found only in elasmobranchs and urodeles, whereas in other vertebrate groups, the medial wall is formed either by the internasal septum or by the rostral cartilage. Therefore, the rostral appendix appears to arise de nouo without any relationships with the lamina orbitonasalis and thus the paired cartilage of Torpedo represent the rostral cartilage proper. The identity of the dorsal component of the lamina orbitonasalis is problematic. There are four possibilities based on the growth pattern and its topographic relationships with other embryonic cartilaginous components (de Beer, 1926, 1931, 1937; Holmgren, 1941). It may be: (1) a dorsal outgrowth of the major component of the lamina orbitonasalis without any fused part with other embryonic cartilage; (2) an independent component which may chondrify de nouo in the dorsal ethmoid area; (3) an anterior extension of the supraorbital and/or orbital cartilage accompanied by the spheno-ethmoid commissure; (4) a fused component of the dorsal outgrowth of the major component of the lamina orbitonasalis with the spheno-ethmoidal commissure. Because the identity is critically important in relation to homology of rostral cartilages throughout chondrichthyans, further discussion is given below. The area concerned here is embryologically very interesting, and involves complex patternings of the participating embryonic cartilages and other structures such as cranial nerves, cranial vascular system and the development of ocular muscles (Miyake, 1988). Further complication of the morphogenesis of the lamina orbitonasalis stems from the fact that it also forms most of the nasal capsules. Based on the early development of this area in the sharks Scyliorhinus, Squalus and Etrnopterus (de Beer, 1931, 1937; Holmgren, 1940) and electric ray Torpedo and stingray Urolophus (de Beer, 1926, 1937; Holmgren, 1940), the lamina orbitonasalis arises from the dorsal margin of the trabecula and expands mainly dorso-medially and ventro-laterally or laterally. The dorso-medial expansion together with the dorsal outgrowth of the preoptic root of the orbital cartilage forms the posterior and medial walls of the future nasal capsules (de Beer, 1931). The distinction between the lamina orbitonasalis and preoptic root is marked by the appearance of two foramina, the anterior cerebral vein foramen dorsally and orbitonasal canal ventrally (de Beer, 1931; Miyake, 1988). The dorso-medial outgrowth of the lamina orbitonasalis then appears to meet the anterior outgrowth of the supraorbital and orbital cartilage together with the 278 T. MIYAKE ET AL. spheno-ethmoidal commissure. There is further dorso-anterior growth of this dorsally expanded lamina orbitonasalis which forms the dorso-medial portion of the future nasal capsules. Holmgren ( 1940) described further anterior extension of the lamina orbitonasalis after forming the portion of the nasal capsules in the shark Scyliorhinus and termed this extension a lateral rostral rod (Fig. 2). These paired rods projecting from the anterior margin of the nasal capsules have been identified as paired lateral rostral rods which together with a medial rostral rod constitute the tripodal rostral cartilage in some carcharhinoid and lamnoid sharks (Maisey, 1984a; Compagno, 1988). Some squaloid sharks, i.e. Centrophorus (Gegenbaur, 1872; Yano, 1985), Etmopterus (Holmgren, 1941; Yano, 1985) and Deania (Gegenbaur, 1872; Holmgren, 1941; Yano, 1985), possess a rod-like rostral cartilage which ends distally in a laterally projected either cartilaginous or ligamentous structure toward both sides of the nasal capsules. The structure resembles superficially paired lateral rods of tripodal rostral cartilages in some carcharhinoid and lamnoid sharks. Based on the development of rostral cartilages in Squalus and Etmopterus (de Beer, 1937; Holmgren, 1940), the projections represent part of lateral growths of the rostral plate possibly the cornu trabecula, as discussed above. The continuity of the projections with the nasal capsules thus indicates differential patternings of either or both these cartilaginous elements and/or lamina orbitonasalis. However, unlike Squalus in which the whole area of the rostral plate chondrifies, expands laterally and meets the lamina orbitonasalis, only the medial area of the trabecula in Etmopterus chondrifies and expands slightly laterally as the cornu trabecula where the lamina orbitonasalis as the nasal capsules outgrows more medially and meets the lateral growth of the cornu trabecula (Holmgren, 1940). Differences between the tripodal cartilage of galeomorphs and the analogous cartilage of these squaloid sharks thus reside in: 1) whether or not the anlage of the rostral plate is present; 2) how much the plate chondrifies and grows as the cornu trabecula; 3) how much the lamina orbitonasalis contributes to the formation of the rostral cartilage and nasal capsules in this area. We agree with de Beer (1937: 64) that the communicating canal has nothing to do with the space enclosed within rostral cartilages. The space is created as a result of differential patternings of the above mentioned embryonic cartilaginous anlagen. It may be also argued that the portion expanded laterally in these squaloid sharks is neither developmentally equivalent to nor homologous with the rostral appendix in batoid fishes, although they are superficially alike. We propose that the paired projections in two families of pelagic stingrays (Fig. 8) represents the anterior extension of the lamina orbitonasalis. The anterior growth of the lamina orbitonasalis also may account for formation of the lateral rostral rod in the electric ray Narke, presumably developmentally equivalent to the paired rods of some carcharhinoid and lamnoid sharks. In addition, the rostral projection in Rhinopteridae and Mobulidae may be developmentally equivalent to the paired lateral rods in Narke and some carcharhinoid and lamnoid sharks. Thus, the lamina orbitonasalis does actually grow dorsally and antero-dorsally, creating not only parts of the nasal capsules but also the dorso-lateral component of the rostral cartilage. It is, therefore, argued that the putative dorsal component of the lamina orbitonasalis would be the whole or parts of the dorsal outgrowth of the lamina orbitonasalis. The ROSTRAL CARTILAGES IN BATOID FISHES 279 question, however, still remains regarding whether this component solely or partially represents the lamina orbitonasalis, because the anterior growth of the spheno-ethmoidal commissure converges, sometimes with the supraorbital cartilage, becoming fused with the lamina orbitonasalis.

Developmental perspectives Extensive descriptive and experimental approaches are needed to elucidate the development of the rostral cartilage in batoid fishes. Areas of research which should be particularly emphasized are: (1) timing and pattern of formation of early blastematic condensation for the trabecula and lamina orbitonasalis; (2) subsequent differential patterns of chondrification for both cartilages; (3) interactions of these cartilages with other embryonic cartilages, as discussed above. Because the cartilage grows interstitially as a continuous sheet after independently chondrified embryonic cartilages fuse, it is difficult to assess to what extent embryonic cartilages contribute to the adult cartilaginous structures in chondrichthyans. Therefore, careful mapping of the embryonic cartilages by using different markers and three-dimensional reconstructions of the developing cartilages will be needed to circumvent this difficulty. As already described in development of several elasmobranchs (Gohar & Mazhar, 1964), morphogenetic movement of the anterior cranial area, accompanied by anterior extension of the pectoral fin, would drastically change the size and shape of embryonic configuration in the anterior cranial areas. In addition, it was noted that the primary cranial flexture of batoid fishes would affect, in some cases, the subsequent structural organization of the anterior cranial areas, as seen in the development of the pelagic stingrays Myliobatos and Rhinoptera (Miyake, 1988), suggesting that morphogenetic movements accom- panied by differentiation of the underlying structures would affect the patterning of the anterior cranial areas including the nasal capsules and rostral cartilage. Morphogenetic movements and differentiation of the craniofacial areas in mammals involve spatially and temporally controlled developmental pathways which would bring separated embryonic structures into closely juxtaposed, interacting structural components (Diewert, 1983; 1985; Sulik & Schoenwolf, 1985; Ferguson, 1988; Couly & Le Douarin, 1988). In addition, if embryos are tetratogenetically treated, movement and differentiation of the underlying structures can be drastically affected, partly because of concomitant cell death (Sulik et al., 1988) or because of abnormal differentiation and tissue interactions leading to craniofacial abnormality (Ferguson, 1988). This brings us to another area of research for the development of rostral cartilages: (1) morphogenetic movements to form rostral cartilages by using morphometrics, SEM and histochemical techniques; (2) correlating differentiation of rostral cartilages with the morphogenetic movement of the anterior area of neurocranium including the nasal capsules; (3) effects of primary cranial flexture on the development of rostral cartilages as well as the nasal capsules including the internasal septum. A series of extirpation studies on the cells in the amphibian Ambystoma mexicanum led Horstadius to compile the first comprehensive data on the nature of neural crest cells and their derivatives. (Horstadius, 1950). Subsequently, Hall (1988) summarized studies on neural crest cells, with 280 T. MIYAKE E'T AL. particular focus on their skeletal derivatives. According to these studies, the trabecula appears to originate from both neural crest and mesodermal cell populations. Earlier Holmgren (1940) also suggested a mixed origin of the trabecula in the development of the cranial skeleton in sharks and batoid fishes. Of particular interest in understanding the differentiation of neural crest cells is a series of extirpation studies of the neural plate and brain in the amphibian Ambystoma (Horstadius, 1950). His studies showed that the development of the anterior trabecula is very sensitive to reductions or abnormality of the forebrain. In addition, Tyler (1983) suggested an inhibitory effect of the neural ectoderm on cranial chondrogenesis in chicks. This sensitivity of the trabecula and effects of neural ectoderm on cranial chondrogenesis may indicate possible interactions between the cells associated with the trabecula and the forebrain leading to the differentiation of the cells into cartilaginous structures. As described above, a remodelling process may be responsible for the atrophy or resorption of the medial plate in benthic stingrays. In fact, a possible remodelling process of the rostral cartilage is indicated by hypercalcified rostral and other cartilages in several groups of sharks (Compagno, 1988) and the -like cells found in Scyliorhinus canicula (Peignoux-Deville et al., 1989). Therefore, our studies on the development of rostral cartilages should extend to another area of research: (1) interactions of the participating cartilages with other structures such as vascular and nervous system including the forebrain and cranial musculature; (2) causal mechanisms of patternings of two embryonic cartilages, the trabecula and lamina orbitonasalis, which may relate to interactions of prechondrogenic and chondrogenic tissues with surrounding connective tissues or epithelial structures; (3) remodelling processes, if present, for already chondrified components.

Homology: assumptions The concept of homology has been extensively discussed from systematic and phylogenetic points of view for many decades (see the summary in Patterson, 1982). However, as expressed by de Beer (197 1), with frustration, there has been little consensus concerning the definition, conceptual framework, or criteria for identifying homologous morphological structures. Recent trends of discussions on this subject have shifted to the realization and conceptualization of hierarchy and complex interactions of biological entities and have introduced the concept of biological homology as opposed to historical homology (Roth, 1984, 1988; Wagner, 1989). In addition, studies on development of morphological structures have been revitalized for understanding and elucidation of morphological evolution over the last decade (i.e. Bonner, 1982; Goodwin et al., 1983). The trend of incorporation of developmental data into understanding evolution of morphology is not new and had been intensively practised before the upsurge of the neo-Darwinian theory in the 1940s. During the course of our present studies, we have been impressed by insights of those who have contributed to the craniofacial morphology of fishes, i.e. Allis, de Beer, Edgeworth, Holmgren and recently, Bertmar, Bjerring, Compagno, Jarvik, Jollie, Maisey, Patterson and Schaeffer (see their references in Miyake, 1988). The introduction of the concept of biological homology may be a landmark for re-unification of these areas of biology. For this reason, we present our conceptual and empirical discussions on homology of rostral cartilages and ROSTRAL CARTILAGES IN BATOID FISHES 281 hopefully provide a research framework for studies of vertebrate morphology in general. Although our understanding of development of morphological structures has been based only on groups of vertebrates which have embryologically interested researchers or have been easily accessible experimentally, a body of earlier developmental data combined with recent developmental studies allows us to make six assumptions regarding the homology of rostral cartilages. These assumptions are put forward based on the data gleaned from development, morphology and evolution of several vertebrate structure (skeleton, teeth and adenohypophysis), which will be discussed in detail in Miyake & Hall (unpublished data): (1) morphological structures are defined as any structures which are produced by development of an individual in a given species; (2) a set of morphological variations, defined as the occurrence of discontinuous or continuous alternative forms of the structure in populations of a given species, reflect a norm of developmental expressions for the structure in development of individuals within populations of a given species; (3) development is thus considered as a causal process under a control of developmental mechanisms which produces collectively a set of morphological variations within populations of the species; (4) evolutionary forces may act on these variations and shift populations to another evolutionary regime where alternative forms of the structure may be fixed in populations (which may or may not coincide with speciation events); (5) assuming populations in a given species or species themselves an evolutionary unit, changes in a morphological structure during evolution of these evolutionary units, leading to the fixation of the alternative form, represent either changes in some constituted components of the structure or changes in most or all aspects of the structure; (6) these forms of the structure between or among the species may be considered homologous, not because of the causal process of development but because of both our recognition of similarities of the forms of the structure (with or without some modifications) and evolutionary or phylogenetic relationships of the species. There is a paucity of detailed knowledge of biological aspects of rostral cartilages, especially epigenesis and variations of rostral cartilages. However, it is our strong belief that our discussions of homology of rostral cartilage will bring clearer insights to the understanding and elucidation of biological and evolutionary significance of the rostral cartilage in batoid fishes. Our conceptual and empirical discussions of homology under the above assumptions, therefore, centre on: ( 1) recognition of complex interactions of biological structural entities in development and evolution; (2) elucidation of a set of interacting biological and evolutionary factors to define a given morphological structure(s) (biological homology); (3) determining the homologous structure(s) by elucidating genetic, epigenetic and evolutionary factors that bound the structure(s) to a given taxon (historical homology).

Homology: historical approach As discussed above, the rostral cartilage of elasmobranchs originates embryologically from the trabecula and lamina orbitonasalis. The final size and shape of the rostral cartilage depend on how these two elements behave developmentally in relation to other structural components in the ethmo-orbital 282 T. MIYAKE ElAL. area. The cells associated with the medial area of the trabecula appear to play a key role in determining the fates of the adult structure of the rostral cartilage. If the area chondrifies and grows, it gives rise to the main structural component of the rostral cartilage. On the other hand, if it remains blastematic and does not chondrify, either the lamina orbitonasalis is the contributor to the formation of the rostral cartilage or no rostral cartilaginous components are formed. Thus, the rostral cartilage of elasmobranchs is a cartilaginous outgrowth of the trabecula and lamina orbitonasalis ventro-medially anterior to the internasal septum and ventro-laterally beyond the anterior limit of the nasal capsules. Dorsally, the rostral cartilage is continuous with the anterior limit of the neurocranium, yet the definite boundary between two structures remains to be elucidated. The size and shape of the precerebral fontanelle appear to depend on how the rostral cartilage and the nasal capsules come about in adults (Miyake, 1988). This definition, however, is not satisfactory if we take into consideration evolutionary or taxonomic unit(s) and the entire spectrum of rostral cartilages in other vertebrate groups. The rostral cartilage must be defined in terms of biological and historical homology to elucidate its evolution among the vertebrates. This leaves us potentially two alternative or combined approaches by correlating the definition of different types of rostral cartilages to particular evolutionary or taxonomic unit(s) examined. The first approach is the phylogenetic reconstruction by cladistic methodology using shared derived characters (synapomorphies), a technique which has been extensively practised in the last decade (Wiley, 1981; Patterson, 1982, 1987). The analysis considers homology as evidence of common ancestry and non-homology (analogy) as evidence of convergent or parallel evolution. Homologies are considered as hypotheses “which are tested by other hypotheses of homology and their associated phylogenetic hypotheses” (Wiley, 1981). The major criterion for determining the character polarity and homologue is the out-group comparison. Once cladograms are produced by the comparison, the most parsimonious cladogram is chosen to represent a working hypothesis. The characters other than synapomorphies are considered either autapomorphic or plesiomorphic, and thus homologies of these characters are interpreted based on parsimony arguments. This interpretation may contradict homologies which are based on developmental information or other criteria for homologies. Rostra1 cartilages are found not only in holocephalans (de Beer & Moy-Thomas, 1935; de Beer, 1937; Holmgren, 1943; Patterson, 1965; Lund, 1986; Maisey, 1986), which have been proposed to be the sister group of elasmobranchs ( Maisey, 1986), and some actinopterygian fishes, Polypterus, Amia, Lepidosteus, Anguilla and Syngnathus which have an embryonic rostral projection (de Beer, 1937), but also in aquatic mammals, cetaceans and sirenians (de Beer, 1937; Carroll, 1988). All these cartilages embryologically arise from the medial area of the trabecula, possibly without any contribution from the lamina orbitonasalis. Of peculiar interest is the de nouo formation of the rostral cartilage dorsal to the trabecula in the euteleostean Gadus merlangus (de Beer, 1937). Several groups of larval euteleostean fishes, i.e. myctophid Lampanyctus achirus (Moser et al., 1984) and holocentrid fishes (Keene & Tighe, 1984), possess a rostral projection, yet which of the underlying cartilages are involved in the elongation of the rostrum remains unclear. One scombroid group, including ROSTRAL CARTILAGES IN BATOID FISHES 283 Sardini and Thunnini, Scomberomorus, Acanthocybium, Istiophoridae and Xiphias, possesses a larval beak (Collette et al., 1984;Johnson, 1986) which is composed of a laterally expanded rostral cartilage proximally embraced by the premaxillar bone and distally abutted by the ethmoid cartilage (Johnson, 1986). Therefore, Johnson (1986) proposed that this complex is one of the synapomorphies uniting these scombroid fishes within the family Scombridae, but he left the issue on homology of the modified break of the barracuda Sphyraena and most gempylids unanswered. In the medial area of the trabecula gives rise to a peculiar nasal septum accompanied by the growth of the cornu trabecula which morphologically resembles rostral cartilage (de Beer, 1937). In Lepidosiren, a small “rostral cartilage” is formed in front of the fan-shaped nasal septum (de Beer, 1937). In urodeles, there is a small cartilaginous process arising from the medial area of the trabecular cartilage (de Beer, 1937; Duellman & Trueb, 1986). In tadpoles of anurans, there is the suprarostral cartilage, which supports the upper horny break and functions as the upper jaw (de Beer, 1937; Duellman & Trueb, 1986; Wassersug & Pyburn, 1987; De Sa, 1988; Rocek & Vesely, 1989; Wiens, 1989). After metamorphosis, the cartilage disappears and the dermal , maxilla and premaxilla, take over support of the upper jaw (Trueb, 1985; Hanken & Summers, 1988; Davies, 1989). Although Sokol (1981) expressed uncertainty of the embryonic origin and homology of the suprarostral cartilage, a recent study on the differentiation of cranial neural crest cells in Xenopus laevis indicates that the suprarostral cartilage originates from neural crest cells, and that the suprarostral cartilage forms a continuous sheet of cartilage with the trabecula (Seufert, 1988; Seufert & Hall, 1990). Whether the anlage of the cartilage forms de nouo or as a continuous sheet of tissues with that of the cornu trabecula may depend on the species examined (Sokol, 1981; Rocek & Vesely, 1989). Recent cladistic analyses indicate that the rostral cartilage is not homologous between holocephalans and elasmobranchs, but each type of rostral cartilage may be unique to the subgroups. For instance, Lund (1986) described an elongate rostral cartilage as autapomorphic for the extinct Squaloraja but also stated that the cartilage is unique to both the extinct myriacanthids and Squaloraja. Maisey (1986) considered the rostral cartilage one of the synapomorphies uniting the extinct myriacanthids, Squaloraja, Ischyodus and extant holocephalans. Maisey (1984a) cast doubts on the phylogenetic significance of rostral cartilages because “ontogenetic studies of Squalus and some batoid fishes have revealed rostral elements that may be homologous to those forming the tripodal rostrum in galeomorphs.” In summarizing current opinion on homology of rostral cartilages, Compagno (1988), however, set the tone that spoon-shaped rostral cartilages in most squaloid sharks may not be homologous to tripodal cartilages in some carcharhinoid and lamnoid sharks. As described in this study, an analogous tripodal type of rostral cartilage is present in the electric ray Narke. In addition, as seen in development of rostral cartilages in Squalus and Etmopterus, similar patternings of the trabecula and lamina orbitonasalis may be responsible for the formation of a wide variety of rostral cartilages in skates. These conflicting observations and opinions raise the question of the levels or levels of vertebrate phylogeny at which the rostral cartilage can be considered a 284 T. MIYAKE ElAL. homologous structure. What has been meant by rostral cartilages appear to represent either all kinds of rostral cartilages, with unique size and/or shape being considered autapomorphic, i.e. an elongate rostral cartilage of the extinct holocephalans, or any kind of rostral cartilages regardless of the size and/or shape being considered synapomorphic at some taxonomic level. Considering the existence of the rostral cartilage in some actinopterygians, sarcopterygians and mammals, the following scenarios of rostral cartilage evolution in higher taxa are considered. (1) The rostral cartilage is a synapomorphy for at least the level of gnathostomes (sensu Maisey, 1986) and thus different forms have arisen in many different groups. Because lampreys have recently been shown to have a trabecula (Langille & Hall, 1988), the rostral cartilage may be a synapomorphy for the group including cyclostomes and gnathostomes. If the rostral cartilage is a synapomorphy for gnathostomes or for the vertebrates the definition of the rostral cartilage must be expanded because the whole spectrum of development of rostral cartilages in other vertebrates clearly involves different morphogenesis in the ethmo-orbital area. For instance, the cornu trabecula (as the trabecular horn) arises from the paired trabecular cranii in most groups of vertebrates, whereas it originates from the medial area of the trabecula in monotreme and placental mammals; the latter groups do not develop the trabecular cranii (de Beer, 1937). The rostral cartilage of actinopterygians originates from the ethmoidal plate which develops from the medial area, and we, therefore, assume that the ethmoidal plate and anterior growth of the medial area, i.e. as the medial plate and possibly rostral plate, are developmentally equivalent forms and/or homologous structures under this scenario. (2) The rostral cartilages arose separately in fishes (elasmobranchs, holocephalans (if it originates from the trabecular and lamina orbitonasalis), actinopterygians, sarcopterygians and possibly in placoderms) and in some mammals, and thus can be considered a synapomorphy each for fishes and for monotremes and placental mammals. The status of the suprarostral cartilage in tadpoles of anurans may fit well in this scenario. This scenario, however, poses a question; how do we account for the uniquely formed rostral cartilage originating from the trabecula and lamina orbitonasalis in elasmobranchs when the rostral cartilage of the other groups arises only from the trabecula? There are two possible explanations: (a): ancestor(s) of these fishes and some mammals independently acquired the rostral cartilage which embryologically originates from the medial area of the trabecula. Elasmobranchs (plus possibly holocephalans) then uniquely acquired the rostral cartilage which embryologically arises from the medial area of the trabecula and lamina orbitonasalis. (b): ancestor(s) of these fishes developed the rostral cartilage which embryo- logically arises from the medial area of the trabecula and/or lamina orbitonasalis, and then in the fishes excluding elasmobranchs (plus possibly holocephalans) the cartilage develops only from the trabecula. (3) all rostral cartilages are independently evolved in chondrichthyans, actinopterygians, sarcopterygians and some mammals, and thus they represent analogous characters. This may explain different embryonic origins of rostral cartilages between elasmobranchs and actinopterygians. The status of the suprarostral cartilage in anuran’s tadpoles may fit well in this scenario, too. ROSTRAL CARTILAGES IN BATOID FISHES 285 Because the rostral cartilage is formed and grows anteriorly between the nasal capsules, this topographic relationship, one of the classical criteria for homology, seem to favour the argument for the first and in part the second scenario but it is against the third. The third scenario, independent origin of the rostral cartilage in these groups of vertebrates, may account for why most vertebrates do not have any rostral cartilage. However, parsimony arguments minimizing possible convergent or parallel evolution of characters do not help test two possible alternatives in the second scenario without any reference to developmental data. The question still remains regarding the status of the suprarostral cartilage in tadpoles of anurans. Its functional specialization and morphology (Wassersug, 1980; Wassersug & Pyburn, 1987) lead us to consider it non-homologous with the rostral cartilage in fishes, but the anlage of the suprarostral cartilage may be developmentally equivalent to and/or homologous with that of the cornu trabecula if the latter is a synapomorphy for at least gnathostomes. Regardless of the validity of these scenarios given above, at least extant elasmobranchs (plus possibly holocephalans) possess a unique rostral cartilage which originates from the medial area of the trabecula and lamina orbitonasalis. Considering recent extensive attempts for phylogenetic analyses of this group (Compagno, 1973, 1977, 1988; Maisey, 1980, 1984a, b, 1985), it seems clear that batoid fishes and squaloid sharks may form a within the elasmobranchs (Compagno, 1977; Maisey, 1984a, 1985). Maisey (1980) introduced a clade orbitostylic group including sequentially Squatina, Chlamydoselachus, Hexanchoidea and squaloid sharks. Then he discussed in 1985, in describing a late Cretaceous Synechodus dubrisiensis, eight alternative hypotheses on phylogenetic relationships of galeomorphs, Synechodus, orbitostylic sharks and batoid fishes. Together with his previous work (Maisey, 1984a), his work gives the impression that batoid fishes are closely allied to squaloid sharks, which supports Compagno’s earlier work on phylogenetic relationships of batoid fishes (Compagno, 1973, 1977). Furthermore, as discussed by Maisey (1984a), galeomorphs are proposed to be monophyletic, but hypotheses of many previous studies on relationships of their contained taxa are questioned. Probable paraphyletic groups are ‘scyliorhinids’, ‘triakids’ and ‘carcharhinids’. Maisey (1984a) thus proposed that his galeomorphs form a sister group with orectoloboid sharks and that Lamna and Scapanorhynchus with fossil two genera are defined as a sister relationship with sequentially Cephaloscyllium, Scyliorhinus and Carcharhinus. He also showed a clear concordance of his galeomorph relationships with palaeontological data by transforming the galeomorph cladogram to the phyiogenetic tree. Suppose that Maisey’s galeomorphs, squaloid sharks (plus possibly Squatina, Chlamydoselachus and hexanchoids as the orbitostylic group) and batoid fishes are simply taken as a three-taxon relationship by assuming the latter two groups to be more closely related than the former, based on this state of flux in phylogenetic studies on elasmobranchs and on our current knowledge of development of rostral cartilages. Do we have a clearer picture of evolution of rostral cartilages within these groups? The answer, it seems to us, is ‘no’. The spoon-shaped and superficially similar rostral cartilages occur in most squaloids, hexanchoids, narcinidid electric rays, guitarfishes and some skates, yet other electric rays, Torpedo and narkinidids, have different forms of rostral cartilages. Even within skates, there are a variety of forms of rostral cartilages ornamented 286 T. MIYAKE ElAL. by the rostral appendix. The similarity of rostral cartilages between the Pristiophorus and sawfish Pristis may be convergent partly because they may not form the sister group relationship (Miyake & McEachran, unpublished data), although the former was suggested to be allied most closely to batoid fishes (Compagno, 1973). Maisey (1984a) proposed the tripodal rostral cartilage to be the synapomorphy for his galeomorphs including Lamna and Carcharhinus, but he suggested more a widespread distribution of this type of the rostral cartilage within elasmobranchs. As discussed above, the rostral cartilage of the squaloid shark Deania is analogous to the tripodal cartilage, and the electric ray .Narke possesses similar type of rostral cartilage. Orectoloboids, a sister group of Maisey’s galeomorphs, possess a simple rod-like rostral cartilage (Dingerkus, 1986). However, the latter type of rostral cartilage is found in the squaloid genera Centroscyltium, Etmopterus, Isistius and Squaliolus (Holmgren, 1941; Y ano, 1985) and embryonic stages of some stingrays. Even, an analogous simple rod- like rostral cartilage may represent the rostral cartilage formed only by the medial area of the trabecula in osteichthyans and the rest of vertebrates, as discussed above. It is more probable, therefore, that rostral cartilages may be of phylogenetic significance in each subgroup or subgroups of these putative monophyletic assemblages, but may not serve as a basis to unite these higher monophyletic assemblages into much higher assemblages. Furthermore, the formation of the cartilage may rely on its own development programme within each subgroup or subgroups with or without modifications of development in their ancestral groups. The elucidation of these accounts should await more detailed studies on taxonomic limits and relationships of these subgroups and development studies discussed above. Unfortunately, none of the scenarios and assumed distributions of different types of ‘‘rostra] cartilages” assures us that we are actually comparing homologous structures among the different groups of vertebrates because previous studies on rostral cartilages do not allow us to define rostral cartilages in the different groups of vertebrates. The discussion thus leads to circular arguments that to construct phylogeny, homologous characters are elucidated at the first place, but to find homologous characters, phylogenetic relationships have to be known. We also argue that topographic relationships are considered as developmental evidence because developmental process determines topographic relationships among morphological structures.

Homology: biological approach What we need to circumvent the circularity just discussed is to independently elucidate or define characters by other means. The definition given for the rostral cartilage in elasmobranchs reflects how different developmental pathways of two embryonic cartilages bring a variety of rostral cartilages in elasmobranchs. Therefore, regardless of the components involved, developmental patterns of the two cartilages are, in turn, key elements to define the rostral cartilage and the components within elasmobranchs as well as among vertebrate groups. The argument raised here may be comparable to what Wagner (1989) described as biological homology. He maintained that biological homology is applied only to “developmentally constrained morphological ROSTRAL CARTILAGES IN BATOID FISHES 287 pattern” and proposed that developmental contraints are internalized to create biologically homologous structures. The question is how do we specify the constraints imposed on the formation of rostral cartilage by developmentally self-regulating mechanisms. We do not need to reiterate recent discussions and arguments of genetic or developmental constraints (see Alberch, 1982; Dawid et al., 1982; Gerhart et al., 1982; Horn et al., 1982; Maderson et al., 1982; Kauffman, 1983; Maynard Smith et al., 1985; Loeschcke, 1987; Oster & Murray, 1989), but our discussion on homology of rostral cartilages should stimulate a search for underlying developmental mechanisms and their ‘constraints’ imposed on morphogenesis of rostral cartilages and may evaluate Wagner’s approach to biological homology. Tasks required are to specify the constraints: elucidation of developmentally self- regulating mechanisms and empirical presentation of developmental constraints imposed by these mechanisms. A set of developmental parameters, i.e. morphogenetic movements involving the anterior cranial areas; a series of earlier inductions to specify embryonic areas such as epidermal epithelium, neural epithelium, neural crest cell population, chordomesoderm etc.; size, timing and shape of prechondrogenic condensation and chondrogenesis, tissue interactions, including inhibitory effects for condensation and chondrogenesis; growth of embryonic cartilages, interactions of growing cartilages with other structural components, would all provide answers regarding self-regulating developmental mechanisms. However, this body of knowledge would not be incorporated into the evolutionary term unless we know how and to what extent these different developmental parameters contribute to phenotypic variability. Recent studies on development and genetics of the mandibular arch skeleton in mice took a combined approach of development and quantitative population genetics to understanding contributions of genetic and developmental factors to evolutionary changes in morphology (Atchley, Plummer & Riska, 1985a’ b; Atchley, Newman & Cowley, 1988). Developmental constraints have been defined as a “bias on the production of variant phenotypes or a limitation on phenotypic variability” (Maynard Smith et al., 19851, but it has been recognized that it is difficult to quantitatively or qualitatively assess constraints. The major difficulty is to distinguish the constraints caused by selection from those imposed by developmental mechanisms, from a mixture of both (Maynard Smith et al., 1985). Maynard Smith et al. (1985) devised several methods to assess and distinguish two different types of constraints, the results of which would have profound effects on discussions of evolution of morphology. They also emphasized that the interplay of developmental and selective constraints may be operating at various levels. The question asked is: how could we then describe developmental constraints as testable hypotheses? We present one potential approach to parameterize quantitatively developmental constraints. It is assumed that ( 1) development is the pathway(s) of differential genetic expressions through epigenesis, complex interactions of embryonic entities including cells, tissues and organs during development and with physical environments; (2) development is a genetic and epigenetic presentation creating a variation of developing morphological phenotype(s). That is, variations of phenotype(s), in turn, would reflect how much development would impose constraints of genetic and epigenetic factors interacting with physical environments on their end products. Of course, the 288 T. MIYAKE ElAL.

I

.o.: .:. 8...._/ Figure 17. Summary of development of rostral cartilages. The diagram does not indicate any actual shapes and sizes of components involved in the formation of rostral cartilages. The rostral process of urodeles, upper jaw complex of tadpoles and the contribution of the cornu trabecula to the formation of rostral cartilages are not included in this figure. Black: rostral cartilage; small dot: trabecular cranii; large circle: medial area of trabecula; cross: areas including the nasal capsules and rostral cartilage which originate from the lamina orbitonasalis. Given an example of the development of the rostral cartilage in narcinidid electric rays (NARC), the diagram will be read as follows: initial chondrification of the trabecular cranii (small dot); formation and contribution of the medial area of the trabecula between the trabecular cranii to the initial formation of the rostral cartilage (large circle) and formation of the nasal capsules (paired boxes without ventral wall of the nasal capsules in chondrichthyans) by the lamina orbitonasalis (cross); the formation of the trough- shaped rostral cartilage by the growth of the medial area of the trabecula (large circle); adult trough-shaped rostral cartilage (black). MAM: some mammals. ACTN: some actinopterygian fishes. RHINP: Rhinopteridae; MOB: Mobulidae; MYL: Myliobatidae; N-PEL STIG: non-pelagic stingrays (all belong to the order ). RAJI: rajoids (skates in the order ). RHINB: rhinobatoids (guitarfishes in the order Rhinobatiformes). PRIS: pristoids (sawfishes in the order Pristiformes). NARK: narcinidid electric rays; HYP: Hypnos;TORP: Torpedo (all belong to the order of Torpediniformes). PRISTO: pristiophorids (sawsharks); SQUA: Squalus acanthias (possibly including most of squaloid sharks); SCYLI: Scyliorhinus canicula (including carcharhinoid and lamnoid sharks except orectoloboid sharks); SHARK: other sharks. HOLC: holocephalans.

term ‘variations’ has to be specified in terms of variances of heritable developmental factors with/without interacting environments (Falconer, 1981) . We then may ask how much genetic variability generated during development would account for phenotypic variability and how much non-heritable factors account for phenotypic variability, an approach which has been explored by Atchley and his colleagues to understand genetic aspects of development in mandibular arch of mice in an evolutionary context (Atchley et al., 1985a, b; Atchley et al., 1988). Then, developmental constraints may be quantitatively measured and qualitatively visualized by comparisons of the magnitudes of relative contribution of heritable or non-heritable developmental components and of their interactions during development. The greater the additive genetic ROSTRAL CARTILAGES IN BATOID FISHES 289 variability, the more readily components of development would respond to selection, indicating the release of the constraints previously developed or less constraints developed at the present state of development (Atchley et al., 1985a, b; Atchley et al., 1988). As discussed by Maynard Smith et al. (1985), we have to devise means of distinguishing or assessing the magnitude of relative contributions of developmental and selective constraints, for example by using classical genetic selection experiments on structure (s) such as mandibular arches of mice, developmental aspects of which are fairly well known. Our ultimate task is then to elucidate what genetic and/or developmental factors or their interplays restricts or relax the constraints to create the variability of phenotype(s) .

Homology: conclusions The arguments given above lead us to several conclusions which should be carefully scrutinized with more empirical data on development, morphology and evolution of rostral cartilages in the future. Because we deal with three or potentially more types of hierarchies-development representing an individual of a given species and evolution representing either a group of these individuals in a given species or several species or higher taxa-we must specify which levels of hierarchies are presently concerned. This leads us to consider the following conclusions: ( 1) development may provide only causal explanations for similarities or differences in a homologous structure, and the hypothesis of homology is tested by “other hypotheses of homology and their associated phylogenetic hypotheses” (Wiley, 1981); (2) determination of homology of morphological structures between or among taxa higher than the specific category totally relies on the knowledge of the structures possessed by the constituting species in the taxa; (3) therefore, developmental and morphological information gleaned from several species in higher taxa should be carefully interpreted and utilized as supporting evidence to define or determine homology of the structure at other levels of categories; (4)it is concluded that there are no unified criteria or schemes to identity homologous structures; all rely on the knowledge of the structures and phylogenetic relationships of associated taxa and on how well we present the hypothesis of homology with supporting causal explanations and descriptions of the structures. Although Wagner’s approach to biological homology is still preliminary, his conceptual framework would give us a vision of how complex biological entities come about through development and of how we assess homology with our increasing knowledge of morphological structures from cell biology, and even molecular biology. The approach will strengthen, compensate for, or negate, arguments of homology based on the historical approach. In addition, the biological approach appeals to the fact that developmental and anatomical data gleaned experimentally and descriptively from a limited number of vertebrates may not reflect actual pictures of developmental and morphological structures in a wide variety of other vertebrate groups.

Homology of rostral cartilages We tentatively consider homology of rostral cartilages in the different groups of vertebrates as follows (Fig. 17) : 290 T. MIYAKE ET AL. (1) The entire rostral cartilage is defined as the structure which is formed by outgrowths of either the trabecula and/or lamina orbitonasalis. The cartilage is uniquely formed by the derivatives of two embryonic cartilages and grows anteriorly between the nasal capsules. Different patterns of morphogenesis of the two embryonic cartilages through interactions with other cranial structures result in a uniquely defined rostral cartilage in different groups of vertebrates. Thus it seems probable that the groups of vertebrates discussed each may have evolved a uniquely defined rostral cartilage from these embryonic cartilages. Search for the homology of these defined rostral cartilages clearly depends on how well we define each rostral cartilage in a given taxon and present supporting biological evidence based on our knowledge of the biology of rostral cartilages. Unfortunately, this study still does not permit us to elucidate homology of rostral cartilages at higher taxonomic categories. Because rostral cartilages may be transient features at earlier development of the crania in most vertebrate groups and disappear in adults, a broad ontogenetic survey of rostral cartilages throughout vertebrate groups will be needed. (2) The rostral cartilages of elasmobranchs may have evolved uniquely because the trabecula and lamina orbitonasalis both contribute to the formation. This may be causally related to the fact that the topographic relation of rostral cartilages and the nasal capsules in elasmobranchs may be developmentally predisposed to form this unique association. The development of the rostral cartilages in sharks other than squaloids and scyliorhinids is needed to test our hypothesis of the definition of rostral cartilages in elasmobranchs. The states of components forming rostral cartilages between elasmobranchs and holocephalans still remain unclear. However, it seems apparent that the rostral cartilage composed of one dorso-medial rod and paired ventro-lateral rods is unique in form, at least among extant holocephalans (de Beer & Moy-Thomas, 1935; Patterson, 1965). (3) The tripodal rostral cartilage may be unique in form in the group including some carcharhinoid and lamnoid sharks, but a possible exclusion of extant orectoloboids from the former two groups remains to be elucidated (Maisey, 1984a; Compagno, 1988). The developmental pattern of the cornu trabecula as seen in Scyliorhinus may represent a unique feature of the rostral cartilage in these groups. Unfused tripodal cartilage is found in the electric ray Narke and scyliorhinid sharks Scyliorhinus retifer and Cephaloscyllium ventriosum. Although we do not know the extent of variations in formation of the tripodal rostral cartilage within and among these groups, we conclude tentatively that unfused tripodal cartilage is developmentally equivalent but not homologous between the electric ray and sharks. The status of the analogous tripodal rostral cartilage in some squaloids remains uncertain. The answer should await more detailed studies on both development of rostral cartilages and phylogeny of subgroups of galeomorphs and squaloids. (4) Recent studies on phylogenetic interrelationships of several higher taxa, i.e. galeomorphs, squaloids (plus possibly Squatina, Chlamyhdoselachus and hexanchoids as the orbitostylic group) and batoid fishes, indicate that batoid fishes may form a clade with squaloids within the elasmobranchs (Compagno, 1973; 1977; Maisey, 1980, 1984a, 1985). However, our current knowledge of the development, variations and distribution of a variety of forms of rostral cartilages in the subgroups of these assemblages does not allow us to bring a clearer picture ROSTRAL CARTILAGES IN BATOID FISHES 29 1 of evolution of rostral cartilages. Our conclusion is, therefore, that rostral cartilages may be of phylogenetic significance in each subgroup or subgroups of these putative monophyletic assemblages, but may not serve as a basis to unite these assemblages into much higher assemblages. In addition, the formation of the cartilage may depend on its own developmental programme within each subgroup or subgroups with or without modifications of development in their ancestral groups. (5) The rostral cartilage of the electric ray Torpedo is uniquely formed by the ventro-medial area of the lamina orbitonasalis, but it is recognized as homologous with rostral cartilages of other elasmobranchs because of its structural similarities based on our definition of rostral cartilages and because of phylogenetic relationships of Torpedo with the rest of the electric rays which possess rostral cartilages. The absence of the contribution of the medial plate of the trabecula to the formation of the rostral cartilage poses interesting questions regarding morphogenesis of the medial area. (6) Narcinidid electric rays possess a rostral cartilage unique in forming dorso- ventrally flattened plate with lateral and/or ventral fenestrae. The unique nature of the chondrification in the medial plate of the trabecula in the group (putative cornu trabecula), as described above, may be related to the formation of the wide internasal septum (thus causally related to the formation of widely separated nasal capsules). Although both the groups form a trough-shaped rostral cartilage, this developmental pattern may distinguish the rostral cartilage between narcinidid electric rays and skates (possibly plus guitarfishes). The development of the trough-shaped cartilage in the carcharhinoid shark ~i~su~urin~(Compagno, 1977) remains uncertain. (7) The rostral cartilage, forming a trough-shaped cartilage, varies considerably within the subgroups of skates, yet the formation of a rostral appendix accompanied by the rostral shaft may be considered a synapomorphy for the group. The cartilage resembling the rostral appendix in shape was reported in several sharks such as Centrophorus (Yano, 1985) and in stingrays (Holmgren, 1940; Miyake, 1988). We tentatively conclude that the analogous cartilage in these sharks may be neither developmentally equivalent to nor homologous with the rostral appendix. As discussed above, the “rostral appendix’’ of Torpedo (Holmgren, 1940) is not developmentally equivalent to the appendix of skates but represents the rostral cartilage proper. Because of a wide variety of forms of rostral cartilages and because of well-elucidated phylogenetic interrelationships, skates would be one of the groups to be studied developmentally and evolutionarily. (8) Structural organization of the rostral cartilage differs between the sawshark Pristiophorus and sawfish Pristis. Although the development of both types of the rostral cartilage is unknown, it would be interesting to know the morphogenesis of rostral teeth and how much two embryonic cartilages discussed in this study would contribute to the formation of the rostral cartilage. (9) The rostral cartilage is vestigial in benthic stingrays and pelagic myliobatidid stingrays. The unique form of the lamina orbitonasalis displays an interesting morphological feature of the rostral cartilage in the pelagic stingray families Rhinopteridae and Mobulidae. (10) The absence or loss of the rostral cartilage may not reflect the same phenomenon in different groups, because it may involve quite different 292 T. MIYAKE ET AL developmental mechanisms, as exemplified by the difference in the absence of the chondrification of the medial plate between the electric ray Torpedo and benthic stingrays: non-differentiation of the blastema in the medial area (for instance, probably due to inhibitory effects of the forebrain epithelium on chondrogenic differentiation) vs cessation of growth accompanied by a remodelling of already formed cartilaginous structures. (1 1) The rostral cartilage of actinopterygians arises only from the medial area of the trabecula (ethmoidal plate) and has no contribution from the lamina orbitonasalis, although they develop the trabecula cranii. The medial and rostral plates of elasmobranchs and ethmoidal plate of actinopterygians appear to be developmentally equivalent, but the latter structure is rather transient at embryonic stages and, for the most part, is replaced by bony structures. Differential patterns of would lead to a variety of forms in the ethmo- orbital complex of actinopterygians. Because there have been no agreements as to phylogenetic relationships of chondrichthyans and actinopterygians (Gardiner, 1984; Maisey, 1986) and because there is little information on the development of the rostral cartilage in holocephalans, the homology of the medial and rostral plates in elasmobranchs and ethmoidal plate in actinopterygians remains uncertain. As Johnson ( 1986) showed, one scombroid group, including Sardini and Thunnini, Scomberomorus, Acunthocybium, Istiophoridae and Xiphius, possesses a unique larval beak which is composed of the rostral cartilage, ethmoid cartilage and premaxillar bone. Whether the peculiar nasal septum of the lungfishes is developmentally equivalent to the derivatives of the ethmoidal plate or rostral cartilages, remains to be elucidated. Data are needed concerning which groups of osteichthyan fishes possess the rostral cartilage and how they develop. (12) Monotreme and placental mammals are unique in having only the medial area of the trabecula developed, and thus the rostral cartilage of cetaceans and sirenians originates only from the medial area. No contribution of the lamina orbitonasalis to the rostral cartilage has been discerned. (1 3) The medial process of urodeles is probably developmentally equivalent to the medial process of the rostral cartilage in some batoid fishes, yet the occurrence in urodeles is either atavistic or unique (autapomorphic). (14) The morphological complex composed of the surprarostral cartilage is unique in tadpoles of anurans. Thus the complex is considered non-homologous with the rostral cartilage, but the anlage of the suprarostral cartilage is probably developmentally equivalent to the outgrowth of the cornu trabecula in batoid fishes.

ACKNOWLEDGEMENTS This study was part of a dissertation prepared in partial fulfilment of the requirement for the degree of Doctor of Philosophy at Texas A&M University, Texas, U.S.A., by the senior author. We are greatly indebted to the following individuals for the loan of specimens for the anatomical study: P. J. P. Whitehead and M. L. Holloway (BMNH); J. E. Randall and A. Suzumoto (BPBM); T. Iwamoto, A. E. Anderson and D. Catania (CAS); I. Nakamura (FRSKU); R.K. Johnson (currently Grice Biological Laboratory, Charleston, North Carolina, U.S.A.) and D. J. Stewart (currently Center for Limnology, ROSTRAL CARTILAGES IN BATOID FISHES 293 University of Wisconsin, Madison, U.S.A.) (FMNH); H. Hatanaka and S. Kawahara (FSFL); C. E. Dawson and S. G. Poss (GCRL); R. I. Lavenberg, C. C. Swift and J. A. Seigel (LACM); K. L. Liem, M. L. J. Stiassny (currently American Museum of Natural History, New York, U.S.A.) and K. E. Hartel (MCZ); R. Rosenblatt, H. J. Walker, Jr. and J. Haugsness (SIO); T. B. Thorson (TBT); M. Retzer (currently University of Illinois, Urbana, Illinois, U.S.A.) (TCWC); R. S. Rosa (UFPB); V. G. Springer, S. L. Jewett and L. P. Norrod (USNM); Gerald R. Allen (WAM). Special thanks goes to Ernst Mayr, K. L. Liem, M. L. J. Stiassny and K. E. Hartel for their hospitality and assistance during visits to the Museum of Comparative Zoology, Harvard University, as a recipient of the Ernst Mayr Grant (TM).Thanks also goes to K. Amaoka, K. Nakaya and K. Nishida for their hospitality and discussions with us on morphology and systematics of batoid fishes during visits to the Laboratory of Marine Zoology, Faculty of Fisheries, Hokkaido University, Japan. We acknowledge Gulf Specimen Marine Laboratories, Inc., Florida, for their donation of embryos and adult specimens of . The senior author has had great benefits from W. R. Atchley and the people who have been associated with B. K. Hall’s laboratory with stimulating discussions on development, genetics and evolution. Finally, J. G. Maisey is greatly acknowledged for reviewing our manuscript and giving us valuable comments. This study was supported in part by the NSF grants (DEB 82-04661, INT 84-05423 and BSR 87-00292) to JDM and by the Ernst Mayr Grant of Museum of Comparative Zoology, Harvard University, to TM. The manuscript was completed with support from an Izzac Walton Killam Postdoctoral Fellowship to TM and Natural Sciences and Engineering Research Council of Canada Operating Grant to BKH.

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ABBREVIATIONS USED IN FIGURES acf anterior cerebral vein foramen anb antorbital cartilage at-bcpc anterior basicapsular commissure ROSTRAL CARTILAGES IN BATOID FISHES 297 bcf basicranial fenestra ctr cornu trabecula endfo endolymphatic fossa esf foramen for efferent pseudobranchial artey est stalk ePP epiphysial bar f I1 n. opticus foramen f 111 n. oculomotorius foramen f hyml2 facio-hyomandibular lateralis foramen 2 fP1 fronto-parietal fontanelle ftl trigeminal lateralis foramen fVIIpal facio-palatine foramen hPf hypophysial fenestra hYm hyomandibular cartilage hyml 1 facio-hyomandibular lateralis foramen 1 icf foramen for internal carotid artery ins internasal septum lbc labial cartilage lc lateral commissure Imb lamina orbitonasalis lmhy lamina hypotica 1. rost lateral rostral lamella of rostral appendix Mc Meckel’s cartilage m-tr medial plate of trabecula ns nasal capsules nt oc otic capsule ocn orbitonasal canal orbc orbital cartilage Palq palatoquadrate cartilage Par parachordal plate PfC prefacial commissure precerebral fontanelle pila antotica pila metoptica P‘ polar cartilage Pm preoptic root pre-spr prespiracular cartilage pt-bcpc posterior basicapsular commissure ra rostral appendix rc rostral cartilage rs rostral shaft mP rostral plate spec spheno-ethmoidal commissure supob supraorbital cartilage tam taenia marginalis tr trabecula tri trabecular cranii

APPENDIX

Acronoms of museums or institutions through which specimens were examined follow Levinton cf a1 (1985); BMNH (British Museum of Natural History, London, U.K.); BPBM (Bernice P. Bishop Museum, Honolulu, Hawaii, U.S.A.); CAS (California Academy of Sciences, San Francisco, California, U.S.A.); FMNH (Field Museum of Natural History, Chicago, Illinois, U.S.A.); FRSKU (Fisheries Research Station, Faculty of Agriculture, Kyoto University, Maizuru, Japan); FSFL (Far Seas Fisheries Research Laboratory, Distance-water Trawl Resources Section, Japanese Fisheries Agency, Shimizu, Japan); LACM (Los Angeles County Museum of Natural History, Los Angeles, California, U.S.A.); MCZ (Museum of Comparative Zoology, Harvard University, Cambridge, Massachusetts, U.S.A.); NMNZ (National Museum of , Wellington, New Zealand); SIO (Scripps Institute of Oceanography, Collection, University of California, La Jolla, California, U.S.A.); TBT (Dr T. B. Thorson’s Collection, University of Nebraska, Lincoln, Nebraska, U.S.A.); TCWC (Texas Cooperative Wildlife Collection, Texas A&M University, College Station, Texas, U.S.A.); UFPB (Universidade Federal de Paraiba, Departmento de Sistematica e Ecologia, Joao Pessoa, Brasil); USNM (National Museum of Natural History, Smithsonian 298 T. MIYAKE ET AL. Institution, Washington, D.C., U.S.A.); WAM (Western Australia Museum, Perth, Australia). The specimens which were examined and used to glean information on rostra1 cartilages in this study are as follows: Batoidei Pristiformes Pristidae Pristis pectinatus Latham (MCZ 36960, 1028 mm TL) Rajiformes Rhinoba toidea Rhinobatidae Rhinobatosproductus Girard (TCWC 6183.1, 554 mm TL); Rhynchobatus djiddensis (Forsskal) (MCZ 806, 490 mm TL) Rajoidea Raj (Dipturus) bafis Linnaeus (TCWC 2819.5); Raja (Dipturus) johannisdavisi Alcock (BMNH 1939.5.24.19); Raja (Leucoraja) erinacea Mitchill (TCWC uncat., 53 mm and 55 mm TL); Raja (Leucoraja) garmani Whitely (TCWC 1888. I, 283 mm TL); Ragu (Leucoraja) ocellata Mitchill (TCWC, uncat.); Breuiraja colesi Bigelow & Schroeder (MCZ 41998); Bathyraju peruanu McEachran & Miyake (USNM 267045); Arhynchobatis asperrimus Waite (NMNZ 5200); Psammobatis scobina (Philippi) (TCWC uncat.); Irolita wadi (McCulloch, 191 1) (WAM P702); Pseudoraja Jischeri Bigelow & Schroeder (TCWC uncat.); Gergesiella furuescens de Buen (TCWC uncat.); Notoraja tobitukai (Hiyama) (MCZ 40284); Pavoraja nitidu (Giinther) (WAM B1.5275); Pauoraja (Subgenus) asperulu (Garrick & Paul) (NMNZ 55697) Torpediniformes Torpedinoidea Torpedinidae Torpedo califarnica Ayres (MCZ 43, 334 mm TL) Hypnidae Hypnos monoptevgium (Shaw & Nodder) (MCZ 38602, 282 mm TL) Narcinoidea Narkinididae Narke japonica (Schlegel) (MCZ 1339, 270 mm TL); Typhlonarke aysoni (Hamilton) (FRSKU 46477, 317 mm TL) Narcinididae Narcine brasiliensis (Olfers) (TCWC uncat., 253 mm , 34 mm, 36 mm TL); DzscopTEe tschudiz (Heckel) (FRSKU 105043, 394 mm TL); Benthobatis marcida Bean & Weed (TCWC 1903.1, 137 mm TL); Diplobatispictus Palmer (TCWC 1900.1, 119 mm TL) Myliobatiformes Dasyatoidea Urolophidae Urotrygon daviesi Wallace (BPBM 24578 481 mm TL); Urotrygon microphthalmum Delsman (USNM 222692, 244 mm TL; UFPB uncat., 80 mm TL); Urotrygon ueneruelae Schultz (TBT 76-28, 28 mm TL); Urotrygon munda Gill (USNM 220612, 201 mm TL); Urotrygon nana Miyake & McEachran (SIO 65-167, 152 mm TL); Urotrygon reticulala Miyake & McEachran (USNM 222644, 188 mm TL); Urotrygon rogersi uordan & Starks) (LACM 50-57, 355 mm TL); Urotrygon chilensis (Giinther) (LACM 7013 352 mm TL); Urotrygon simulatrix Miyake & McEachran (USNM 13064, 267 mm TL); Urotrygon aspidura ('Jordan & Gilbert) (CAS 51834, 284 mm TL); Urolophus jamaicensis (Cuvier) (TCWC 0815.1, 285 mm TL); Urolophus halleri Cooper (SIO uncat., 381 mm TL); Urolophus concentricus (Osburn & Nichols) (LACM 3177 I, 278 mm TL); Urolophus pausimaculatw Dixon (FSFL ED189, 407 mm TL); Urolophus cruriatus Lacepede (TCWC uncat., 231 mm TL); Urolophus aurantiacus Muller & Henle (TCWC uncat., 251 mm TL); Urolophus bucculentus Macleay (FSFL EC361, 538 mm TL); Urolophw viridis McCulloch (USNM 205519, 122 mm TL) Dasyatididae Dayatis americana Hildebrand & Schroeder (TCWC 5820.1, 614 mm TL); Dayatis uiolaceu (Bonaparte) (MCZ 57667, 958 mm TL); Taeniura lymma (Forsskal) (TCWC 5276.1, 442 mm TL) Potamotrygonidae Potamotrygon magdalenae (Valenciennes) (TBT 76-54, 419 mm TL) Gymnuridae Gymnura micrura (Blotch & Schneider) (TCWC uncat., 203 mm TL) Myliobatoidea Myliobatidae Myliobatis goodei Garman (TCWC 3699, 725 mm TL); Myliobatis califomicus (Gill) (MCZ 395, 500 mm TL); narznari (Euphrasen) MCZ, 1400, 950 mrn TL); Rhinopteridae Rhinoptera bonasus (Mitchill) (TCWC 4423.1, 604 mm TL) Mobulidae Mobula hypostoma (Bancroft) (MCZ 36406, 490 mm in Disc Width)