JOURNAL OF MORPHOLOGY 235:183–237 (1998)

Ontogeny of the Osteocranium in the African Catfish, Clarias gariepinus Burchell (1822) (Siluriformes: Clariidae): Ossification Sequence as a Response to Functional Demands

DOMINIQUE ADRIAENS* AND WALTER VERRAES Institute of Zoology, University of Ghent, Ghent, Belgium

ABSTRACT The ontogeny of the bony skull of the African catfish, Clarias gariepinus, is studied from initial ossification until a complete skull is formed. The ossification sequence in C. gariepinus seems to be related to the func- tional demands that arise in a developing larva. Early ossification of the opercular bone coincides with the initiation of opercular skin movements. Early ossifications involve several dentulous bones, formed shortly before the transition phase from endogenous to exogenous feeding. The enlarging bran- chiostegal membrane becomes supported by the gradual adding of branchio- stegal rays. Parasphenoid ossification may be related to protection of the brain during prey transport, whereas the several hyoid bones, including the parurohyal, are formed in relation to the increasing loads exerted onto the tendons of the sternohyoideus and consequently onto the hyoid bar. Overall skull reinforcement occurs almost simultaneously, with a whole set of perichon- dral bones arising especially at places of high mechanical load. The suspenso- rium becomes protected against dislocation in an anteroposterior direction through a ligamentous connection, which even becomes partially ossified, forming the sesamoid entopterygoid. Later, the cranial lateral-line system becomes enclosed by a set of gutters, which close, frequently becoming plate-like later in ontogeny. The brain also becomes covered dorsally. Addi- tional dentition (prevomeral tooth plates) formation seems to coincide with formation of the opercular four-bar system, as well as with the time the digestive system becomes completely functional. Eventually, unossified re- gions between the bones become closed off, fortifying and completely covering the skull. J. Morphol. 235:183–237, 1998. ௠ 1998 Wiley-Liss, Inc.

The osteology of catfishes has been a basis hito, ’91; Teugels et al., ’91; Kobayakawa, for several studies, especially for taxonomy ’92; Chen and Lundberg, ’95; de Pinna and and phylogeny. In the present study, the Vari, ’95; Ferraris, ’96). Special attention taxonomy follows that of Fink and Fink (’96), has been paid to some clariid species (Nawar, in which the Siluriformes, or catfish, repre- ’54; Greenwood, ’56; Poll, ’57, ’77; Tilak, ’63c). sent the sister group of the Gymnotiformes, Much confusion concerning homologies is or knife-fish. Both are grouped within the being cleared up, as data from several stud- Siluriphysi, which together with the Char- ies and several catfish groups are put to- aciphysi and Cypriniphysi comprise the oto- gether (Fink and Fink, ’81, ’96; Arratia and physan group of the Ostariophysi. An exten- Gayet, ’95; Arratia and Huaquin, ’95). For a sive literature has been produced on the general review of bone nomenclatural syn- descriptive cranial osteology of siluriform onymies, we also refer to Harrington (’55). fish, mostly on adult forms (e.g., McMurrich, 1884; Bhimachar, ’33; Merriman, ’40; Hubbs and Miller, ’60; Tilak, ’61, ’63a,b, ’64, ’65a,b, Contract grant sponsor: Institute for Science and Technology ’67, ’71; Rastogi, ’63; Gauba, ’66; ’70; Taverne (D.A.); contract grant sponsor: National Funds for Scientific and Aloulou-Triki, ’74; Lundberg, ’75, ’82; Research (W.V.). *Correspondence to: Dominique Adriaens, University of Srinivasa Rao and Lakshmi, ’84; Vignes and Ghent, Zoological Institute, K.L. Ledeganckstraat 35, B-9000 Garcia, ’87; de Pinna, ’88; Howes and Fumi- Gent, Belgium. E-mail: [email protected]

௠ 1998 WILEY-LISS, INC. 184 D. ADRIAENS AND W. VERRAES The main purposes of the present work MATERIALS AND METHODS are, first, to describe the ontogeny of the Observations were made on 30 different cranial bones in order to observe the pres- ontogenetic stages of the African catfish Clar- ence or absence of cranial bones. Addition- ias gariepinus, originally described by ally, ontogenetic stages provide information Burchell (1822) and synonymized by Teugels concerning fusions or reductions of certain (’82). Specimens ranged from 4.1 mm SL skeletal elements, of which no trace can be (standard length) (ϭ1 day posthatching) to seen in adults. Further, we attempt to con- 174.5 mm SL (age unknown) (Table 1). Eggs sider the ontogeny of skeletal elements from a functional morphological point of view. Why were obtained from the Laboratory of Ecol- do certain bones develop at a certain mo- ogy and Aquaculture (Catholic University of ment, and why do some develop simulta- Leuven) and raised at a temperature of 25°C. neously while others do not? Such attempts Most juveniles (100 days posthatching) were (Weisel, ’67; Verraes, ’74, ’75, ’77; Verraes commercially raised and obtained from W. and Ismail, ’80; Hunt von Herbing et al., Fleure (Someren, The Netherlands). At dif- ’96b; Mabee and Trendler, 1996) can yield ferent time intervals, the developing larvae important information on the adaptations of were sedated in MS-222 and fixed in 4% structures present at a certain stage. Addi- buffered formaldehyde or paraformaldehyde- tionally, as indicated by Arratia (’87), ‘‘de- glutaraldehyde. Clearing and staining fol- tailed ontogenetic studies of different struc- lowed Hanken and Wassersug (’81) with tryp- tures in different groups are needed as base sin being replaced by a 1% KOH solution. for a future phylogenetic interpretation of These specimens were studied using a stereo- the relationships of the families of Siluroi- scopic microscope (WILD M5). Eight speci- dei.’’ mens were used for serial sectioning after

TABLE 1. Specimens used for present study nr SL1 (mm) TL2 (mm) PAL 3 (mm) Age4 Method Staining5 Used for 1 4.1 4.4 2.3 1 clearing AB ϩ ARS drawing 2 5.9 6.4 3.3 2 serial sections T 3D reconstructions 3 6.0 6.4 2.7 1 clearing AB ϩ ARS drawing 4 6.6 7.0 3.2 2 clearing AB ϩ ARS drawing 56 6.8 7.4 3.1 3 clearing AB ϩ ARS observations 6 7.2 7.8 3.8 7 serial sections T 3D reconstructions 7 7.3 7.9 3.3 7 clearing ARS observations 8 7.7 8.7 3.7 9 clearing AB ϩ ARS drawing 96 8.2 8.6 4.1 17 clearing AB ϩ ARS observations 10 8.3 9.2 4.4 10 clearing ARS observations 116 8.4 9.1 4.2 13 serial sections T observations 126 9.3 10.7 4.9 21 clearing AB ϩ ARS observations 136 10.0 11.3 5.0 21 clearing AB ϩ ARS drawing 146 11.1 12.4 5.5 21 serial sections T observations 156 11.6 13.0 5.0 26 clearing AB ϩ ARS drawing 166 12.0 13.5 6.2 26 clearing ARS drawing 176 12.7 15.0 6.1 26 clearing AB ϩ ARS drawing 186 13.0 14.7 6.4 31 serial sections T observations 196 14.8 16.4 8.1 31 clearing ARS drawing 206 15.2 16.9 8.0 26 serial sections T observations 216 15.4 16.7 7.7 31 clearing ARS observations 226 15.5 17.8 8.3 31 clearing AB ϩ ARS observations 236 18.7 20.9 9.2 31 serial sections T observations 246 19.0 21.1 11.1 31 clearing AB ϩ ARS drawing 256 21.5 24.7 11.7 31 clearing AB ϩ ARS drawing 26 46.8 50.2 23.9 119 serial sections T 3D reconstructions 27 125.5 144.9 67.1 100 clearing AB ϩ ARS drawing 28 127.0 143.6 65.5 100 clearing AB ϩ ARS drawing 29 140.1 158.4 72.4 100 clearing ARS drawing 30 174.5 187.7 90.9 ? clearing AB ϩ ARS drawing

IT ϭ improved trichromous staining, T ϭ T. 1Standard length. 2Total length. 3Preanal length. 4Number of days posthatching. 5AB ϭ alcian blue; ARS ϭ alizarine reds. 6Gynogenetic specimens. OSTEOCRANIUM ONTOGENY IN CLARIAS 185 embedding using Epon and Paraplast. The make a clear distinction between certain obtained 2 µm and 5 µm thick sections, bones and their margins. Serial microscopic respectively, were stained with toluidine and sections, however, revealed clearly the pres- an improved trichrome staining according to ence or absence of bones. The drawings were Mangakis et al. (’64). Sections were studied based on cleared material, so drawings of using a Leitz Diaplan light microscope. certain stages may not show the presence of Drawings of both cleared and sectioned ma- a certain bone, although that bone was al- terial were created using a camera lucida. ready observed in serial sections of a smaller Three-dimensional reconstructions of serial specimen. Despite this, the drawings do give sections were done using a commercial soft- ware package (PC3D, Jandel Scientific). a good representation of the morphology, as The examined specimens were obtained well as the chronology of ossification. from different egg batches. However, 17 of For anatomical references of the chondro- them belong to one single batch of gynoge- cranium, we refer to Adriaens and Verraes netical eggs, obtained by and according to (’97c), where the relation of the cranial bones Volckaert et al., (’94) (Table 1). with the cranial lateral-line system is dealt The present study uses body length as an with separately (Adriaens et al., ’97). The indicator of developmental stage. As several initiation of ossification of the cranial bones, factors such as temperature, prey size, prey grouped per region, is given in Table 2. The type, or water quality, may influence growth ontogeny of the Weberian apparatus is given rate, independent from age, growth itself is in the figures, but is not discussed here. For a better indication of maturity.Also, coupled that, we refer to Radermaker et al. (’89). to body size is the fluctuation in spatial constraints in a developing larva. Arratia The 4.1–6.0 mm SL stage (Fig. 1) and Schultze (’90) noted that ‘‘the sequence Neurocranium of appearance of bones is maintained intra- specifically and interspecifically within ex- No signs of any ossification. tant neopterygians despite age differences.’’ An epigenetic control of bone formation has Splanchnocranium been demonstrated by several experiments, from which it could be derived that once a The first signs of bony elements involve certain bone is formed (genetic differentia- the opercular bone. At 4.1 mm SL, where the tion), its growth is regulated through the chondrocranium is still in a primordial phase mechanical load (and other factors as hor- (Adriaens and Verraes, ’97c), a condensation mones and metabolic factors) (epigenetic dif- of what appears to be a bony matrix (al- ferentiation) (Herring, ’93). The rate of devel- though still unstained in the specimen), at opment of other structures like the the level of the opercular process of the hyo- suprabranchial organ, and consequently the symplectic could be observed. In the 5.9 mm coupled behavior of air breathing, depends SL serial sections, a condensation of cells on the water temperature during ontogeny could be observed at that spot, although no (Haylor and Oyegunwa, ’93). The fact that bony matrix could be distinguished. Presum- the embryological and early larval period of ably, this condensation concerns the initia- Clarias gariepinus occurs extremely fast, in tion of opercular bone formation. At 5.9 mm which the uptake of yolk resources occurs in SL, dentition is present in the lower jaw, a very efficient way (Kamler et al., ’94), borne by a dentary. The latter is a dermal implies that skull formation also must be plate covering the lateral face of Meckel’s fast. However, a developing larva has to deal cartilage. The rostral tips of that cartilage, with a varying set of demands in order to however, are still unossified at that stage. survive, with most of these demands related They will ossify perichondrally, forming the to body size. Consequently, skull formation mentomeckelian bone (somewhere between will be coupled to the developmental needs that arise, where those apparatuses, which 5.9 and 6.0 mm SL). This ossification seems can satisfy those needs, have to develop prior to initiate at the anterior tip of the dermal to their corresponding needs. dentary, which is fused to it from the mo- ment it arises. This bony lower jaw at this RESULTS stage is referred to as the os dento-ment- In some cleared specimens, especially the omeckelium (Fig. 2B). No signs of other ossi- smallest ones, staining was insufficient to fications could be discerned. 186 TABLE 2. Ossification sequence and type of the cranial bones in Clarias gariepinus, grouped by region Initiation Bone Initiation Bone Region Bone (mm SL) type1 Region Bone (mm SL) type1 Neurocranium Splanchnocranium Ethmoid region supraethmoid 11.1 mm PC Premandibular arch autopalatine 11.6 mm PC hypoethmoid 11.1 mm PC laterodermethmoid 11.1 mm D Mandibular arch dentary 5.9 mm D nasal 12.7 mm DC mentomeckelium 6.0 mm PC lateral ethmoid 12.7 mm PC ϩ M splenial (dentary) 8.4 mm DC prevomeral tooth plate 11.6 mm D angular 8.4 mm D prevomeral bone 18.7 mm D articular 8.4 mm PC retroarticular 8.4 mm PC Orbito-temporal region frontal 10.0 mm D splenial (angular) 11.1 mm DC orbitosphenoid 18.7 mm PC splenials (inter-articular) 46.8 mm DC pterosphenoid 12.7 mm PC quadrate 7.2 mm PC

parasphenoid 6.6 mm D metapterygoid 11.6 mm PC VERRAES W. AND ADRIAENS D. antorbital 11.6 mm DC entopterygoid 8.4 mm S lacrimal 11.6 mm DC coronomeckelium 12.0 mm S infraorbitale II 12.7 mm DC infraorbitale III 18.7 mm DC Hyoid arch ventral hypohyal 7.2 mm PC infraorbitale IV 18.7 mm DC dorsal hypohyal 46.8 mm PC anterior ceratohyal 7.2 mm PC Otic region dermosphenoticum 10.0 mm DC posterior certohyal 8.4 mm PC autosphenoticum 11.6 mm PC interhyal — — prooticum 10.0 mm PC hyomandibula 7.2 mm PC dermopteroticum 10.0 mm DC symplecticum — — autopteroticum 11.1 mm PC parurohyal2 6.6 mm S ϩ PC posttemporo-supracleithrum 7.7 mm DC ϩ D Branchial arches basibranchialia II–III 11.6 mm PC Occipital region parieto-supraoccipital 11.1 mm D ϩ PC hypobranchialia I–II 11.6 mm PC epioticum ?140.1 mm PC hypobranchialia III–IV — — exoccipital 7.2 mm PC ceratobranchialia I–V 7.2 mm PC basioccipital 7.2 mm PC epibranchialia I–IV 7.2 mm PC infrapharyngobranchialia III 18.7 mm PC Maxillary bones premaxillary 6.0 mm D infrapharyngobranchialia IV 46.8 mm PC maxillary 6.0 mm D lower pharyngeal tooth plate 8.4 mm D upper pharyngeal tooth plate 6.0 mm D Opercular bones opercular 4.1 mm D preopercular 10.0 mm DC interopercular 11.6 mm D suprapreopercular 18.7 mm DC branchiostegal rays 7–10 6.0 mm D branchiostegal rays 5–6 6.6 mm D branchiostegal rays 2–4 7.7 mm D branchiostegal rays 1 21.5 mm D

1PC ϭ perichondral bone; D ϭ dermal anamestic bone; DC ϭ dermal canal bone; M ϭ membrane bone; S ϭ sesamoid bone. 2Although the parurohyal is not part of the hyoid arch, it is noted here because of its close relation to the hyoid bar. OSTEOCRANIUM ONTOGENY IN CLARIAS 187 The 6.0–6.6 mm SL stage (Fig. 2) tions of the inner side of cranial walls sur- Neurocranium rounding the lagena and the sacculus. Several bones are present at this stage. Rostrally, a paired, rudimentary premaxil- Splanchnocranium lary bone lies at the ventral surface of the At 6.6 mm SL, the dento-mentomeckelian ethmoid plate. Although rudimentary, they complex reaches up to the coronoid process already bear several teeth (Fig. 2B). The two of Meckel’s cartilage. In the middle, the left premaxillaries do not yet meet in the mid- and right mentomeckelian parts of the com- line (Fig. 2A). At the rostral tip of the pala- plexes are separated from each other by the tine, a maxillary bone is formed, already fused tips of the Meckelian cartilages. At the enclosing the base of the maxillary barbel. caudal face of the hypohyals, two ossicles The articulation of the maxillary bone with have formed, separated from each other by the palatine is improved by an additional the anterior copula (Figs. 3C, 4A). These cartilaginous element, the submaxillary car- paired elements correspond to the sesamoid, tilage. unfused parts of the future median paruro- hyal bone. Two more branchiostegal rays Splanchnocranium have been added, as six of them border the The opercular bone has clearly ossified ceratohyal. At 7.2 mm SL, most of the now. It consists of a horizontal rod bearing a splanchnocranial perichondral bones have ventral, triangular membranous plate. The formed. The hyoid bar bears an anterior articular facet is more differentiated. Along ceratohyal bone, as well as a ventral hypo- the caudoventral margin of the posterior hyal one. The cartilaginous hyosymplectic- part of the ceratohyal, four branchiostegal pterygoquadrate plate bears an ossification rays have developed. Based on their position at its articular facets with the mandibula and the further development of these rays, it and the opercular bone, i.e., the quadrate is clear that those four involve the posterior and opercular process of the hyomandibular four branchiostegal rays of adults (radii bran- bones, respectively (the latter was over- chiostegi VII–X). During further ontogeny looked in a previous study by Adriaens and they become enlarged, whereas other bran- Verraes, ’97a). The ventral plate of the oper- chiostegal rays are added in front of them. cular bone now reaches the level of the inter- At the ventral face of the fourth infrapharyn- hyal, whereas the horizontal rod is extended gobranchial element, a tooth plate is formed. posteriorly. The bone is still triangularly This plate corresponds to the tooth bearing shaped. Serial sections of a 7.2 mm SL speci- part of the upper pharyngeal jaws. men indicate the differentiation of a dorsal process at the lateral face of the articulatory The 6.6–7.7 mm SL stage (Figs. 3, 4A) facet of the opercular bone with the hyosym- Neurocranium plecticum. At this stage, a very indistinct Whereas in the previous stages the neuro- tendon of the dilatator operculi inserts onto cranial floor is reinforced by cartilaginous this process. The branchial basket bears most elements only, a bony support is present at of its ossifications at this 7.2 mm SL stage. 6.6 mm SL. The parasphenoid arises as what All five ceratobranchials and all four epibran- appears to be a U-shape bone, following the chials are present. Ossification seems to start curvature of the trabecular bars and the at the anterior half of the cartilaginous ele- acrochordal cartilage. Although the hypophy- ments. The ceratobranchials V, however, do sial fenestra is open almost completely, the not seem to possess any teeth yet. foramen for the internal carotid artery is already cut off (Fig. 3A). The serial sections The 7.7–10.0 mm SL stage (Figs. 5, 6A, 7A) of the 5.9 mm SL stage reveal no sign of a Neurocranium parasphenoid, whereas the 6.8 mm SL stage At 7.7 mm SL, the parasphenoid now com- already has a parasphenoid closing off the pletely covers the hypophyseal fenestra, leav- hypophysial fenestra almost completely. At ing only a paired foramen for the entrance of that stage, the parasphenoid is no longer the internal carotid artery (Figs. 5A, 6A). U-shaped. The premaxillary bones have be- Posteriorly it becomes extended and almost come more elongated. At 7.2 mm SL, ossifica- contacts with the basioccipital, which now tion at the anterior tip of the notochord has has an extracranial ossified part also (Fig. started, corresponding to the basioccipital. 5C). The exoccipital bones cover the bases of Exoccipitals arise as perichondral ossifica- the pilae occipitales (Fig. 5A). Posterior to Figure 1 OSTEOCRANIUM ONTOGENY IN CLARIAS 189 the chondrocranium, two small ossicles are eral stages indicate that the perichondral formed, which lie between the caudal mar- articular and retroarticular bones and the gin of the otic capsules and the parapophysis dermal angular bone arise at about the same of the fourth vertebra (Figs. 5B, 6A). Based moment (Adriaens et al., ’97). The retroar- on its position, in relation to the cleithral ticular bone is formed as an ossification of bone, as well as evidence from further ontog- the ventral face of the cartilaginous retroar- eny, this bone must correspond to the dorsal ticular process of Meckel’s cartilage. The process of the posttemporal part of the fu- angular bone is plate-like, bordering the pos- ture posttemporo-supracleithral bone com- terior part of Meckel’s cartilage ventrolater- plex. A discussion on the true nature of this ally. At the ventromedial face of the, still bone complex in siluriform fishes is given by cartilaginous, palatine a thin, plate-like bone Arratia and Gayet (’95). The premaxillaries is formed. At this stage (8.4 mm SL), this now almost meet in the midline. bone seems to be surrounded by ligamen- tous tissue. Based on its morphology, posi- Splanchnocranium tion, and data from further ontogeny, this Both processes, i.e., the lateral and ven- bone must correspond to the sesamoid ‘‘ent- tral processes, of the future dentosplenio- opterygoid’’ type 4 (Arratia, ’92). The hyoid mentomeckelian complex can already be dis- bar now bears a posterior ceratohyal ossifica- tinguished at 7.7 mm SL. A total of nine tion (frequently referred to as the epihyal). branchiostegal rays is present. The left and The paired, sesamoid part of the parurohyal right parurohyal parts have become forked bone has become fused to what seems to be a at their caudal tip, whereas both are con- perichondral ossification of the ventral face nected with their rostral tip to the ventral of the anterior basibranchial element. The hypohyals through a ligament (Fig. 4B). The complex nature of this bone, which has the upper pharyngeal tooth plate is enlarged position and function of the pure dermal and heavily toothed, but is still supported by urohyal bone of teleosts, led to the designa- the fourth infrapharyngobranchial element tion of the name ‘‘parurohyal’’ for that bone only (Figs. 5C, 7A). At 8.4 mm SL, a gutter in Siluriformes (see Discussion) (Arratia and has formed, covering the posterior part of Schultze, ’90). Teeth were observed at this the dental bone laterally. This gutter corre- stage on the dorsal face of the fifth cerato- sponds to the dental splenial, but whether or branchial bone. Both pharyngeal jaws are not one or more of these splenial bones are thus present from 8.4 mm SL. involved could not be discerned. As ex- The 10.0–11.6 mm SL stage (Figs. 6B, 7B, 8) pected, the initiation of this gutter forma- Neurocranium tion is at the level of a superficial neuro- mast, which has started to sink in. More The ossification of the skull roof has anteriorly, the neuromasts also have started started. The ossification centers of the fron- to invaginate, although not so deeply, and tal bones are situated at the branching point are not yet supported by a gutter. At this of the taenia marginalis posterior in the stage, a perichondral ossification surround- epiphysial bridge, the lamina orbito-nasalis, ing the articulation between Meckel’s carti- and the commissura spheno-septalis. Appar- lage and the pars quadrata of the pterygo- ently, this ossification initiates by the forma- quadratum indicates the presence of the tion of the neurodermal component of the articular bone (Fig. 8B). Observations of sev- frontal bone, as a gutter-like bone follows the taenia marginalis posterior and the com- missura spheno-septalis. Apparently, this os- sification initiates by the formation of the Fig. 1. Skull of Clarias gariepinus (4.1 mm SL stage). neurodermal component of the frontal bone, A, dorsal view; B, lateral view; C, ventral view. (Grey as a gutter-like bone follows the taenia mar- indicates bone, shaded areas indicate cartilage.) bb-I, ginalis posterior and the commissura spheno- basibranchiale I; c-ac, cartilago acrochordalis; c-Meck, septalis. At the level of the epiphysial bridge, cartilago Meckeli; c-ot-a, cartilago oticalis anterior; c- ot-p, cartilago oticalis posterior; c-pc, cartilago para- a medial opening of that gutter is present, chordalis; cb-I, ceratobranchiale I; cd, chorda dorsalis; corresponding to the future epiphysial ch, ceratohyale; cm-bc-a, commissura basicapsularis an- branch of the supraorbital canal (Adriaens terior; fr-tr-hm, foramen truncus hyomandibularis ner- et al., ’97). Posterior to the frontal bone, two vus facialis; hs, hyosymplecticum; ih, interhyale; o-op, os operculare; p-q, pars quadrata of the palatoqua- paired and consecutive gutter-like bones bor- dratum; prc-op, processus opercularis; tr-cr, trabecula der the otic capsules laterally (Fig. 8A). The cranii. anterior one represents the neurodermal Figure 2 OSTEOCRANIUM ONTOGENY IN CLARIAS 191 component of the dermosphenotic bone, ventral and dorsal surface of the cartilagi- whereas the posterior one corresponds to the nous floor (Figs. 6B, 8A). At this stage the neurodermal component of the dermopter- bone is rather slender and broadens at its otic bone. Apparently the supraorbital canal posterior half. The basioccipital now covers is already elongated into the otic and tempo- the exposed notochord completely. The exoc- ral canals at this stage. Both neurodermal cipital expands along the dorsal face of the components are still separated from each pila occipitalis (Fig. 6B). At 11.1 mm SL, other, although the dermopterotic bone al- additional skull roof bones are formed. In ready reaches the posttemporo-supraclei- the ethmoid region, two dermal plates come thral complex. to cover the pre-ethmoid cornua and the Based on its morphology, the latter com- ethmoid cartilage, which have already plex is represented by both the posttemporal started to become ossified perichondrally, and supracleithral parts at this stage. Three both ventrally and dorsally. At the dorsal processes can be distinguished: (1) a dorsal face, however, this bone bears a distinct lat- one, which reaches the dorsocaudal margin eral, plate-like extension, which indicates a of the neurocranium, medial to the pterotic possible dermal origin. The ventral and dor- bone, (2) a ventral one, which runs to the sal perichondral ossification must corre- lateroventral face of the pterotic bone, and spond to the hypo- and supraethmoid bones, (3) a ventromedial process, which runs to- respectively, whereas the dermal component ward the parapophysis of the fourth verte- probably represents the laterodermethmoid bra. Most probably, the two anteriorly di- bones (Taverne, pers. comm.). Consequently, rected processes represent the dorsal and this bony complex is referred to as the mes- ventral processes of the posttemporal bone, ethmoid (Adriaens et al., ’97). The posterior and the ventromedial one corresponds to the part of the skull roof has also started to transscapular ligament of the supracleithral ossify. As is the case for the ethmoid region, bone, which has become ossified (see Discus- the roof of the occipital region seems to con- sion). In the skull floor, the parasphenoid sist of two dermal plates covering an un- has reached the basioccipital, and even over- paired, perichondral bone. This perichon- laps it (Fig. 6B). The foramina for the inter- dral supraoccipital covers the tectum nal carotid arteries have shifted laterally, posterius, as well as the roof of the posterior coupled to the ongoing excavation of the part of the otic capsules. The dermal plates trabecular bars. At that level the parasphe- overlap with the whole supraoccipital bone, noid has formed two conspicuous lateral but are extended anteriorly, up to the ante- wings that reach up to the posterior margin rior part of the otic capsule. Apparently, of the sphenoid fenestra (Fig. 6B). At the what seems to correspond to the parietals anteromedial face of the otic capsules, the becomes fused to the supraoccipital at the initiation of the prootic bone covers both the moment they are formed, as has been ob- served in several siluriform fishes (see Dis- cussion). This complex is further referred to as the parieto-supraoccipital bone. However, Fig. 2. Skull of Clarias gariepinus (6.0 mm SL stage). what part of this complex exactly corre- A, dorsal view; B, lateral view; C, ventral view. (Grey sponds to the supraoccipital would require indicates bone, shaded areas indicate cartilage.) c-eth, some more detailed research. At the level of cartilago ethmoideum; c-Meck, cartilago Meckeli; cb-II, ceratobranchiale II; cb-IV, ceratobranchiale IV; cb-V, the dermopterotic bone, the otic cartilage ceratobranchiale V; cd, chorda dorsalis; ch, ceratohyale; has begun to ossify perichondrally, corre- cm-sphsep, commissura spheno-septalis; cp-a, copula sponding to the autopterotic bone. A corre- anterior; eb-IV, epibranchiale IV; fn-hyp, fenestra hy- sponding ossification of the autosphenotic pophysea; fn-sph, fenestra sphenoidea; hb-II, hypobran- chiale II; hh, hypohyale; hs, hyosymplecticum; ih, inter- bone, however, is still lacking. hyale; ipb-IV, infrapharyngobranchiale IV; mnd-b, mandibular barbel; mx-b, maxillary barbel; ns-b, nasal Splanchnocranium barbel; o-den-mm, os dento-mentomeckelium; o-mx, os Suspensorial ossifications, like the quad- maxillare; o-op, os operculare; o-prmx, os praemaxillare; ot-cap, otic capsule; p-q, pars quadrata of the palatoqua- rate and hyomandibular bone, can be dis- dratum; pal, palatinum; pc-pl, parachordal plate; pl-oc, cerned on the cleared specimens of 10.0 mm pila occipitalis; pns-ep, pons epiphysialis; prc-op, proces- SL, whereas no sign of a symplectic bone is sus opercularis; prc-pc, processus praecerebralis; prc-ra, present (Fig. 8B). The latter bone does not processus retroarticularis; r-br, radius branchiostegus; sol-n, solum nasi; su-ph-tpl, superior pharyngeal tooth develop at all in Clarias gariepinus, which is plate; tn-m-p, taenia marginalis posterior; tr-cr, tra- a general feature for Siluriformes (Fink and becula cranii. Fink, ’81, ’96). The hyomandibular bone en- Figure 3 OSTEOCRANIUM ONTOGENY IN CLARIAS 193 closes the foramen for the truncus hyoman- now be clearly distinguished from the neuro- dibularis of the nervus facialis (VII), as well dermal, gutter-like one, as it has started to as the base of the opercular process (Fig. cover the postpineal foramen and, conse- 8B). Between the hyomandibular and quad- quently, the brain. Posterolaterally, the fron- rate bones, a gutter-like preopercular bone tal bone gutter has reached that of the der- follows the ventrolateral border of the sus- mosphenotic bone. The latter bone, as well pensorium, thereby partially covering the as the dermopterotic bone, has started to cartilaginous interhyal. The anterior cerato- form the membranodermal component as hyal bone is now apparent in the cleared well. Additionally, a perichondral ossifica- specimen as well. The forked branches of the tion covers the taenia marginalis posterior parurohyal bones have become extended, at the level of these dermosphenotics, espe- both in an anterior and posterior direction. cially at the ventral side (Fig. 6C). Appar- Their medial tips now have fused with each ently, the autosphenotic has also formed. It other posteriorly (Fig. 4C). Ceratobranchial is directly fused to the dermal counterpart, ossifications are observed in the cleared as is the case for the dermopterotics, and specimen. Both their articular facets, with autopterotic bones. This fused bony complex the hypobranchials and epibranchials, re- of double origin is consequently referred to main unossified. Apart from an increase in as the sphenotic bone (ϭdermosphenotic and number of teeth, no striking difference can autosphenotic bones) and the pterotic bone be observed in the upper pharyngeal tooth (dermopterotic and autopterotic bones). plate (Fig. 7B). No more branchiostegal rays At the posterior corners of the chondrocra- have been added since the previous stage; nium, contacting the pterotics, lies the post- thus nine are present. At 11.1 mm SL, temporo-supracleithral bone, bearing a fora- the angular bone bears a lateral gutter, men for the temporal canal (Fig. 9B). The which indicates the fusion with at least one latter feature is an additional argument for splenial bone. Consequently, a complex an- stating that the posttemporal is present and gulo-splenio-articulo-retroarticular bone is formed. part of the bony complex. From this stage on, the parasphenoid plays an important The 11.6–12.7 mm SL stage (Figs. 6C, 7C, 9) role in the reinforcement of the skull floor, as Neurocranium the trabecular bars have become split in two Ventral to the ethmoid plate, the premax- by the continued lateral expansion of the illaries become more and more plate-like, as foramen for the internal carotid artery (Fig. a posterior extension is noted. The membra- 9A). At this stage, the parasphenoid conse- nodermal component of the frontal bone can quently establishes the ventral connection between the anterior part of the neurocra- nium (anterior to the sphenoid fenestra) and the posterior part (posterior to the fenestra). Fig. 3. Skull of Clarias gariepinus (6.6 mm SL stage). The previously slender, lateral wings have A, dorsal view, B, lateral view, C, ventral view. (Grey consequently become broader. The basioccipi- indicates bone, shaded areas indicate cartilage.) c-eth, cartilago ethmoideum; c-Meck, cartilago Meckeli; c- tal has formed two longitudinal ridges be- sbmx, cartilago submaxillaris; cb-I, ceratobranchiale I; tween which the posterior extension of the cb-V, ceratobranchiale V; cd, chorda dorsalis; ch, cerato- parasphenoid comes to lie (Fig. 6C). At this hyale; cm-sphsep, commissura spheno-septalis; cp-a, stage, the basioccipital supports the otoliths copula anterior; eb-I, epibranchiale I; eb-II, epibran- chiale II; eb-III, epibranchiale III; fn-hyp, fenestra hy- of the lagena and the sacculus, i.e., the as- pophysea; fn-sph, fenestra sphenoidea; fr-car-i, foramen teriscus and the sagitta, respectively. Lat- arteria carotis interna; fr-I, foramen fila olfactoria; fr-X, eral to the basioccipital, the exoccipitals have foramen nervus vagus; hb-I, hypobranciale I; hh, hypo- enclosed the foramen of the nervus vagus hyale; hs, hyosymplecticum; ih, interhyale; ipb-IV, in- frapharyngobranchiale IV; Im-on, lamina orbitonasalis, (X). The latter bones form the lateral mar- sensu latu; o-den-mm, os dento-mentomeckelium; o-mx, gins of the foramen magnum, thus enclosing os maxillare; o-op, os operculare; o-para, os parasphenoi- the pilae occipitales, until they contact the deum; o-prmx, os praemaxillare; o-puh, os parurohyale; parieto-supraoccipital bone complex dor- ot-cap, otic capsule; p-q, pars quadrata of the palatoqua- dratum; pal, palatinum; pc-pl, parachordal plate; pl-oc, sally. The prootics have become enlarged pila occipitalis; pns-ep, pons epiphysialis; prc-op, proces- and ovally shaped and take part in the ossi- sus opercularis; prc-pt, processus pterygoideus; prc-ra, fication of the border of the sphenoid fenes- processus retroarticularis; r-br, radius branchiostegus; sb, swimbladder; sol-n, solum nasi; su-ph-tpl, superior tra (Fig. 6C). They support the otolith of the pharyngeal tooth plate; tn-m-p, taenia marginalis poste- utriculus, i.e., the lapillus. Anteriorly, be- rior; tr-cr, trabecula cranii. hind the premaxillary bones, two small, Fig. 4. Ontogeny of the parurohyale in Clarias gari- puh, fissura parurohyalis; hb-I, hypobranchiale I; hb-II, epinus (ventral view). A, 6.6 mm SL stage; B, 7.7 mm SL hypobranchiale II; hh, hypohyale; I-puh-hh, ligamen- stage; C, 10.0 mm SL stage; D, 11.6 mm SL stage; E, tum parurohyalo-hypohyale; o-bb-II, os basibranchiale 12.7 mm SL stage; F, 21.5 mm SL stage; G, 127.0 mm SL II; o-bb-III, os basibranchiale III; o-cb-I, os ceratobran- stage. (Black indicates parurohyal bone, grey indicates chiale I; o-cb-II, os ceratobranchiale II; o-ch-a, os cerato- ligaments, shaded areas indicate cartilage.) bb-I, basi- hyale anterior; o-hb-I, os hypobranchiale I; o-hb-II, os branchiale I; bb-II, basibranchiale II; cb-I, ceratobran- hypobranchiale II; o-hh-v, os hypohyale ventrale; o-puh, chiale I; cb-II, ceratobranchiale II; ch, ceratohyale; fr-hy, os parurohyale. foramen hyoideum; fr-puh, foramen parurohyalis; fs- OSTEOCRANIUM ONTOGENY IN CLARIAS 195 splint-like bones arise on the ventral face of ceratohyal in the middle, and the newly the ethmoid plate (Fig. 6C). As can be de- formed posterior ceratohyal and ventral hy- rived from further ontogeny, these bones pohyal (Fig. 7C). The latter bone is pen- correspond to the prevomeral tooth plates etrated by a foramen, through which passes that are formed prior to the median pre- the hyoid artery (Fig. 4D). The articulation vomeral bone itself. Dentition, however, of the nine branchiostegal rays occurs with could not yet be discerned. At this stage, the the anterior ceratohyal bone and the carti- first signs of the dermal bones bordering the laginous part separating the latter from the rigid skull laterally are present. At the level posterior ceratohyal bone. The parurohyal of the palatine, two small ossicles have bone now clearly forms one single unit, as a formed, enclosing the anterior part of the horizontal bony lamella has formed between infraorbital canal. These ossicles correspond the median and two lateral processes (Fig. to the neurodermal component of the antor- 4D). The ligamentous connection of the paru- bital bone and lacrimal bone (ϭos infraorbit- rohyal with the hyoid bar occurs at the level ale I) (Fig. 9B). of the ventral hypohyals. The anterior copula Splanchnocranium bears two ossified rings at the position of the second and third basibranchial bones. At The previously cartilaginous lower jaw has their anterolateral tips, the hypobranchials now become almost completely enclosed with I and II have started to ossify as well (Fig. bone. An anterolateral process of the angulo- 7C). All the epibranchial bones are observed splenio-retroarticular complex fits between in the cleared specimen of 11.6 mm SL. The the ventral and lateral processes of the dento- upper pharyngeal tooth plate has enlarged splenio-mentomeckelian complex. At this in- rather isometrically, but is still supported terdigitating surface, the coronoid process, mainly by the unossified, fourth infrapharyn- as well as almost the complete medial face of gobranchial element. At 12.0 mm SL, all Meckel’s cartilage is still exposed (Fig. 7C). mandibular ossifications are present. The At the level of its articulation with the orbito- last to develop is the coronomeckelian bone, nasal lamina, the palatine has become ossi- which is formed at the medial face of the fied perichondrally, thus forming the auto- coronoid process, between the dento-splenio- palatine bone (Fig. 6B). The articular facet mentomeckelian and the angulo-splenio- of the autopalatine itself is still cartilagi- articulo-retroarticular bone complexes (Fig. nous, although surrounded by bone. The ent- 10A). At this stage, the splenial gutter of the opterygoid is now a small, triangular bone, dental bone is still open anteriorly, but gradu- bordering the autopalatine ventrally (Figs. ally becomes closed off and incorporated in 6C, 7C, 9B). The anterior tip of the pterygoid the bone complex (Fig. 10). process of the suspensorium has also started to ossify, forming the metapterygoid bone. The 12.7–21.5 mm SL stage This perichondral bone, however, is still sepa- (Figs. 4E, 11, 12A) rated from the quadrate bone. The latter has Neurocranium become expanded dorsally and caudally, com- The bones of the skull roof have started to pared with the previous stage. Dorsally, the close up the unossified regions in the skull initiation of the membranous outgrowth of as they make contact with the surrounding the quadrate has started. Although still sepa- bones. The laterodermethmoids follow the rated from the quadrate, the hyomandibular commissura spheno-septalis as they grow bone has formed the corresponding membra- posteriorly. Serial sections of a 13.0 mm SL nous outgrowths as well, at its anterior bor- specimen show that the laterodermethmoids der. The preopercular bone has become tube- are fused to the perichondral supraethmoid, like now, as the gutter has closed. This bone up to the level of the precerebral lamina. still follows the ventrocaudal margin of the Posterior to that level, the supraethmoid is hyosymplecticum, with its anterior part ly- missing, whereas the dermal bones have ing horizontally and its posterior part verti- extended above the posterior part of this cally.At the level of the articulation with the lamina and the sphenoseptal commissures, neurocranium, the hyomandibular bone separated from it by connective tissue. This bears a cartilaginous rim. Compared to the configuration supports a possible dermal ori- previous stage, the opercular bone has ex- gin of these bones. The plate-like part of the tended even more ventrally. In this cleared frontal bone becomes extended anteriorly, and stained specimen, three ossifications of medially and posteriorly. Posteriorly, the the hyoid bar are discernable: the anterior frontals have contacted the parietals (thus Figure 5 OSTEOCRANIUM ONTOGENY IN CLARIAS 197 fused to the supraoccipital bone), and the covered the ventral part of the preorbital initiation of an interdigitation can be ob- base. At the anterodorsal border, the pteros- served. Medially, the frontals have contin- phenoid bones have appeared at the ventral ued to cover the pre- and postpineal fenes- part of the taenia marginalis posterior (Fig. tra, as well as enclosing the cartilaginous 12A). The prevomeral tooth plates have ex- epiphysial bridge (Fig. 11A). Anterior to the panded, and the first teeth can be distin- entrance of the supraorbital canal into the guished. The parasphenoid becomes ex- frontal bone, a small, gutter-like nasal bone tended anteriorly, touching the prevomeral has differentiated. Laterocaudally, the bony tooth plates. Posteriorly, the parasphenoid skull margin is formed by the sphenotic and narrows and terminates in a slender process pterotic bone, covering the posterior part of lying between the longitudinal ridges of the the taenia marginalis posterior and the otic basioccipital. This kind of overlap allows the capsule. The sphenotic bone is restricted to long and slender interdigitation, which can the taenia marginalis and the anterior part be observed in later stages. The basioccipital of the otic capsule, whereas the pterotic bone has enlarged, especially in an anterior direc- covers about two thirds of that capsule. The tion, as it now forms the complete floor for perichondral part of both these bones has the sacculus, enclosing the sagitta (Fig. 12A). expanded over the otic capsules, ventrally The exoccipitals now border the foramen for and dorsally. Posteriorly, the two anterior the glossopharyngeus nerve posteriorly. At processes of the posttemporo-supracleithral the lateral face of the skull, a second infraor- bone have expanded anteriorly, as the fork bital bone has been added, posterior to the between them fits onto the posterior margin lacrimal bone. of the pterotic bone (Fig. 11B). The ossified As is still the case for the anterior two transscapular ligament is a stout process, canal bones, this infraorbital bone is tubu- contacting the laterodorsal margin of the lar. It borders the eye ball anteroventrally parapophysis of the fourth vertebra. In the (Fig. 11B). Ventral to the preopercular bone, skull floor, two more bones have been added. a triangular interopercular bone is formed, Ossification of the lateral ethmoid has close to the anterior margin of the opercular started at the level of the articular facet of ventral tip. Serial sections of a 15.2 mm SL the lamina orbito-nasalis, for the articula- specimen indicate that the lateral ethmoid tion with the autopalatine (Fig. 12A). It bor- consists of a perichondral ossification, bear- ders the articular facet laterally, and has ing a plate-like extension. The lamina orbito- nasalis ossifies perichondrally and becomes laterally and dorsally enlarged by a bony plate. However, a separate ossification of the Fig. 5. Skull of Clarias gariepinus (7.7 mm SL stage). plate-like part could not be discerned. At A, dorsal view; B, lateral view; C, ventral view. (Grey 18.7 mm SL, the prevomeral tooth plates are indicates bone, shaded areas indicate cartilage.) ast, interconnected and have fused to the me- asteriscus (lagenar otolith); c-eth, cartilago ethmoid- dian prevomeral bone. This bone already eum; c-Meck, cartilago Meckeli; cb-I, ceratobranchiale I; cd, chorda dorsalis; ch, ceratohyale; cm-sphsep, commis- interdigitates with the parasphenoid. Three sura spheno-septalis; cp-a, copula anterior; cp-p, copula more dermal canal bones are added at this posterior; eb-I, epibranchiale I; fn-sph, fenestra sphenoi- stage, covering the skull laterally: the third dea; fr-car-i, foramen arteria carotis interna; fr-I, fora- and fourth infraorbital bones, and the supra- men fila olfactoria; fr-X, foramen nervus vagus; hb-I, hypobranchiale I; hh, hypohyale; hs, hyosymplecticum; preopercular one. All of them are still tubu- ih, interhyale; ipb-III, infrapharyngobranchiale III; lap, lar or gutter-like. The infraorbital series is lapillus (utricular otolith); Im-on, lamina orbitonasalis, completed in this stage. The onset of peri- sensu latu; o-boc, os basioccipitale; o-den-mm, os dento- chondral ossification of the orbitosphenoid mentomeckelium; o-eoc, os exoccipitale; o-mx, os maxil- lare; o-op, os operculare; o-para, os parasphenoideum; could also be discerned, covering the carti- o-prmx, os praemaxillare; o-pt-scl, os posttemporo- laginous floor of the ethmo-orbital region. supracleithrum; o-puh, os parurohyale; ot-cap, otic cap- sule; p-q, pars quadrata of the palatoquadratum; pal, Splanchnocranium palatinum; pns-ep, pons epiphysialis; pp-v4, parapophy- sis of vertebra 4; prc-co, processus coronoideus; prc-op, The bones of the hyoid arch have ex- processus opercularis; prc-pt, processus pterygoideus; panded, especially the anterior ceratohyal. prc-ra, processus retroarticularis; r-br, radius branchio- It is, however, still separated from both the stegus; sag, sagitta (saccular otolith); sb, swimbladder; ventral hypohyal and posterior ceratohyal sn, supraneurale; su-ph-tpl, superior pharyngeal tooth plate; tn-m-p, taenia marginalis posterior; tr-cr, tra- by cartilage (Fig. 11B). The interhyal is still becula cranii; trip, tripus; tt-p, tectum posterius; v2, cartilaginous and is continuous with both vertebra 2; v6, vertebra 6. the hyoid arch and the hyosymplectic carti- Figure 6 OSTEOCRANIUM ONTOGENY IN CLARIAS 199 lage. As is the case for the previous stage, all men, the lateral ethmoid ossification is now branchiostegal rays articulate with the ante- observed on the dorsal face of the skull (Fig. rior ceratohyal and the cartilaginous part 14A). The frontal bones have expanded sub- between the latter and the posterior cerato- stantially in all directions. Anterolaterally, hyal. All suspensorial bones have expanded, they interdigitate with the lateral ethmoid, closing in the cartilaginous parts between anteriorly with the mesethmoid, posterolat- them. The membranous outgrowths of the erally with the sphenotics and the pterotics, quadrate and hyomandibular are more pro- caudally with the parieto-supraoccipital com- nounced, and a distinct articular ridge be- plex, and medially the two frontals meet at tween the latter and the neurocranium is the level of the epiphysial bridge (Fig. 14A). present (Fig. 11B). The articulation between The parieto-supraoccipital bone has started the suspensorium and the neurocranium is to close off the postpineal fenestra, as have situated at the level of the posterior part of the frontals. Posteriorly, the former bone the sphenotic and the anterior part of the complex bears a distinct, pointed supraoccipi- pterotic. Evidence of a paired origin of the tal process, which reaches up to the fourth parurohyal is now lost, as the anterior tips vertebra. Rudimentary contact between the also have fused, leaving a foramen for the parieto-supraoccipital bone and the pterotic passage of the parurohyal artery, as can be bone is already established. Anterior expan- derived from serial sections of a 46.8 mm SL sion of the pterotic bone has deprived the specimen (Figs. 4E, 13). At 18.7 mm SL, the sphenotic bone from any contact with the third infrapharyngobranchial also becomes parieto-supraoccipital bone complex. Medial ossified. expansion of the sphenotic bone, at the ven- The 21.5–127.0 mm SL stage tral side of the skull, has resulted in the (Figs. 4F, 12B, 14) ossification of the lateral border of the sphe- Neurocranium noid fenestra. At its posterior margin, the posttemporo-supracleithral bone has be- All bones are present. Most of them have started to close off the unossified parts al- come plate-like and borders the pterotic bone most completely. In the 21.5 mm SL speci- both laterocaudally and mediocaudally. The men, the premaxillaries have expanded pos- lateral-line canal, which exits this bone com- teriorly, thus supporting the nasal sacs. The plex, is enclosed by a pair of small ossicles. ethmoid plate and precerebral lamina are In the skull floor, the orbitosphenoid is almost completely ossified, as the contralat- clearly visible (Fig. 12B). It arises as a paired eral laterodermethmoid bones have fused ossification of the ethmoid plate and the medially. The nasal bones have become tubu- preorbital base, lateral to the anterior part lar, in which the branching of the supraor- of the parasphenoid. The orbitosphenoids bital canal can already be distinguished are still separated from the surrounding (Adriaens et al., ’97). In the cleared speci- bones and border the sphenoid fenestra ante- riorly. The lateral wings of the parasphenoid have expanded extensively, as they contact the ventral extension of the pterosphenoid Fig. 6. Neurocranium of Clarias gariepinus (ventral bones. Consequently, this connection subdi- view). A, 7.7 mm SL stage; B, 10.0 mm SL stage; C, 11.6 vides the sphenoid fenestra into an anterior mm SL stage. (Grey indicates bone, shaded areas indi- foramen for the optic nerve and a posterior cate cartilage.) fn-sph, fenestra sphenoidea; fr-car-i, fora- men arteria carotis interna; fr-IX, foramen nervus glos- trigemino-facial foramen. The prootics have sopharyngeus (ϭfenestra basicapsularis posterior); fr-X, come into close contact with the parasphe- foramen nervus vagus; fr-m, foramen magnum; I-trsc, noid, sphenotics, pterotics, basioccipital, and ligamentum transcapularis; o-ant, os antorbitale; o- exoccipital bones, but no true interdigitation apal, os autopalatinium; o-apt, os autopteroticum; o- asph, os autosphenoticum; o-boc, os basioccipitale; o- has occurred (Fig. 12B). Basioccipital and dpt, os dermopteroticum; o-dsph, os dermosphenoticum; parasphenoid, however, already strongly in- o-enp4, sesamoid ‘‘os entopterygoideum’’ type 4; o-eoc, os terdigitate. The second infraorbital bone has exoccipitale; o-lac, os lacrimale (ϭos infraorbitale I); extended caudally, as it also now borders the o-mx, os maxillare; o-para, os parasphenoideum; o- prmx, os praemaxillare; o-prot, os prooticum; o-pt-scl, os eye ventrally. The third infraorbital bone posttemporo-supracleithrum; o-susp, os suspensorium; lies at the posterior margin of the eye. A gap ot-cap, otic capsule; pal, palatinum; pp-v4, parapophysis is still present between the third and the of vertebra 4; pvm-tpl, prevomeral tooth plate; sb, swim- bladder; tn-m-p, taenia marginalis posterior; tr-cr, tra- second infraorbital bone. All these infraor- becula cranii; trip, tripus; v1, vertebra 1; v2, vertebra 2; bital bones are still tubular, apart from the v6, vertebra 6. fourth one, which lies at the lateral margin Figure 7 OSTEOCRANIUM ONTOGENY IN CLARIAS 201 of the skull, between the frontal and sphen- The autopalatine is almost completely ossi- otic bones. Apparently, the fourth infraor- fied, except for its rostral and caudal tips bital bone arises last, although it subse- (Fig. 12B). Anteriorly, the tip establishes an quently becomes the largest one of the series, articular facet for the double-headed maxil- as can be derived from the later stages. The lary bone, enclosing the maxillary barbel. suprapreopercular bone has become plate- Although no true articulation is present with like, where the neurodermal and membrano- the caudal tip of the autopalatine, an ossifi- dermal components already can be clearly cation is lacking. The sesamoid entoptery- distinguished. In the 46.8 mm SL specimen, goid bone connects to the metapterygoid bone a plate-like, nasal, triangular in cross sec- and the prevomeral bone through a ligamen- tion, could be observed, indicating the pres- tous strap. The metapterygoid bone has ence of the membranodermal component. An started to form its membranous outgrowths, articulation is present between the small as was already the case for the quadrate and antorbital bone and a small cartilaginous the hyomandibular bone. Although more ex- protuberance of the anterior articular facet tensively ossified, the latter two bones are of the autopalatine (Fig. 15A). The antor- still separated from each other by cartilage bital bone partially encloses the base of the (Fig. 14B). The preopercular bone has initi- nasal barbel. ated the formation of the membranodermal Splanchnocranium component, and the branching of the preoper- cular canal can be recognized in the neuro- The mandibula has become more heavily dermal part. The dorsal opening of that ca- ossified, as the dento-splenio-mentomeck- nal in the preopercular bone is positioned elian complex becomes connected more sol- exactly ventral to the entrance of the canal idly to the angulo-splenio-articulo-retroar- into the suprapreopercular bone. The opercu- ticular complex through an extensive lar bone is still triangular, being ligamen- interdigitation (Fig. 10B,C). The splenial tously connected to the interopercular bone bones have closed their gutters completely, leaving only the pores where the mandibu- at its ventral tip. The latter bone has become lar canal exits the bone (Adriaens et al., ’97). broader compared to the previous stage. At its medial face, the coronoid process The anterior ceratohyal bone now reaches gradually becomes enclosed as well (Fig. the posterior one. 10C). A well-developed articular facet for Interdigitation between these two bones articulation with the quadrate is present, has started only at the dorsal side of the bearing a substantial retroarticular process. hyoid bar, whereas cartilage separates them ventrally (Fig. 14B). Anteriorly, the anterior ceratohyal bone has reached the ventral hy- pohyal bone, where the first signs of inter- digitation are apparent (Fig. 4F). The articu- Fig. 7. Splanchnocranium of Clarias gariepinus (dor- lation between the hyoid bar and the sal view). A, 7.7 mm SL stage; B, 10.0 mm SL stage; C, branchiostegal rays is still comparable to 11.6 mm SL stage. (Grey indicates bone, shaded areas indicate cartilage.) c-Meck, cartilago Meckeli; cb-I, cera- that in the previous stage, i.e., at the level of tobranchiale I; ch, ceratohyale; cp-a, copula anterior; the anterior ceratohyal bone and the carti- cp-p, copula posterior; eb-I, epibranchiale I; eb-IV, epi- laginous region posterior to it. At this stage, branchiale IV; hb-I, hypobranchiale I; hh, hypohyale; hs, one branchiostegal ray is added, comprising hyosymplecticum; in-ph-tpl, inferior pharyngeal tooth- plate; ipb-III, infrapharyngobranchiale III; ipb-IV, in- a total of 10. The parurohyal bone bears a frapharyngobranchiale IV; o-ang-c, os angulo-splenio- small but distinct foramen, and the three articulo-retroarticulare complex; o-art, os articulare; o- caudal processes have become elongated (Fig. bb-II, os basibranchiale II; o-bb-III, os basibranchiale 4F). Anteriorly, the double ligamentous con- III; o-cb-I, os ceratobranchiale I; o-cb-III, os ceratobran- chiale III; o-cb-IV, os ceratobranchiale IV; o-cb-V, os nection to the ventral hypohyals is still pre- ceratobranchiale V; o-ch-a, os ceratohyale anterior; o- sent. The bones of the branchial basket have den-c, os dento-splenio-mentomeckelium complex; o-den- not changed substantially compared to the mm, os dento-mentomeckelium; o-eb-I, os epibranchiale previous stage. Basibranchials of the second I; o-enp4, sesamoid ‘‘os entopterygoideum’’ type 4; o-hb-I, os hypobranchiale I; o-hh-v, os hypohyale ventrale; o- and third branchial arches are separated by hm, os hyomandibulare; o-mp, os metapterygoideum; cartilage on the anterior copula. Very small o-op, os operculare; o-pop, os praeoperculare; o-puh, os hypobranchials are present on the first and parurohyale; o-q, os quadratum; prc-op, processus oper- cularis; prc-pt, processus pterygoideus; prc-un, proces- second branchial arch; all ceratobranchials sus uncinatus; r-br, radius branchiostegus; su-ph-tpl, are well ossified. Their articular facets, with superior pharyngeal tooth plate. the hypo- and epibranchials, however, re- Figure 8 OSTEOCRANIUM ONTOGENY IN CLARIAS 203 mains unossified. Although no fifth epibran- preethmoid processes, which correspond to chial has developed, the corresponding cera- the well-ossified preethmoid cornua (Adriaens tobranchial element bears an unossified head and Verraes, ’97c). Interdigitation occurs with at both ends. The third epibranchial ele- the lateral ethmoids, laterally, and the fron- ment bears a well-developed, medially di- tals, posteriorly. Together with the lateral rected uncinate process at its caudal mar- ethmoid, the laterally curved preethmoid gin. At 46.8 mm SL, the splanchnocranium processes enclose the nasal bone. The lateral is complete. A small, gutter-like splenial bone ethmoid has become plate-like, enclosing the is present lateral to the articulation between cartilaginous orbito-nasal lamina. At its ven- mandibular and suspensorium. This sple- tral face, the initial articular facet between nial embraces the sensory canal, at the this lamina and the palatine can still be transition from the mandibular to the pre- distinguished, as an ossification is lacking opercular canal. Dorsomedially, the angulo- (Fig. 17A). Medially the lateral ethmoid bears a splenio-articulo-retroarticulare complex has funnel through which the olfactory lobes pass. become extended rostrally, coming into con- Laterally, it bears a distinct process for the tact with the dento-splenio-mentomeckelian articulation with the dorsal process of the bone. As a result, the cartilaginous coronoid second infraorbital bone (Fig. 15A). Posteri- process is enclosed by bone laterally and orly, the lateral ethmoid interdigitates with medially. At its dorsomedial face, the hypo- the frontals, but no overlapping could be hyal cartilage bears a small, but distinct observed. At its lateral margin, the lateral process, which becomes ossified at this stage. ethmoid is connected through a connective Lying at the dorsal face of the ventral hypo- tissue sheet to the dorsal margin of the ante- hyal, this bone corresponds to the dorsal rior part of the fourth infraorbital bone. The hypohyal. The fourth infrapharyngobran- frontals are the largest, paired skull bones, chial element also starts to ossify, bordering which border the anterior fontanella. Com- the cartilage dorsally, medially and ven- pared to the previous stage, both frontals trally. are sutured to each other posteriorly, thus The 127.0 mm SL stage subdividing the postpineal fenestra into a (Figs. 4G, 10D, 12C, 15–23) postepiphysial part of the anterior fonta- Neurocranium nella and the posterior fontanella (Fig. 15A). This juvenile stage is to some degree a In small specimens, the anterior fontanella good copy of the adult configuration, al- is bordered anteriorly by the mesethmoid. though reduced in size. The mesethmoid is A second region of interdigitation between relatively broad, bearing two well-developed contralateral frontals occurs at the level of the epiphysial bridge, which has become com- pletely enclosed by a tubular outgrowth of Fig. 8. Skull of Clarias gariepinus (10.0 mm SL the dermal frontals (Fig. 17B). In the skull stage). A, dorsal view; B, lateral view; C, ventral view. roof, the frontals connect through sutures to (Gray indicates bone, shaded areas indicate cartilage.) the sphenotics (posterolaterally), the pterot- c-Meck, cartilago Meckeli; fn-sph, fenestra sphenoidea; fr-car-i, foramen arteria carotis interna; fr-I, foramen ics (posteriorly) and the parieto-supraoccipi- fila olfactoria; fr-IX, foramen nervus glossopharyngeus tal bone complex (posteromedially). Later- (ϭfenestra basicapsularis posterior); fr-ot, foramen ra- ally, they connect through connective tissue mus oticus nervus facialis; I-trsc, ligamentum transcapu- to the central part of the fourth infraorbital laris; lm-on, lamina orbitonasalis, sensu latu; lm-pc, lamina praecerebralis; o-art, os articulare; o-boc, os basi- bone. Ventrally, the frontals connect rigidly occipitale; o-cb-I, os ceratobranchiale I; o-cb-II, os cerato- to the orbitosphenoids and the pterosphe- branchiale II; o-cb-V, os ceratobranchiale V; o-ch-a, os noids (Fig. 12C). The sphenotics have be- ceratohyale anterior; o-den-c, os dento-splenio-ment- come plate-like as well, enclosing the antero- omeckelium complex; o-dpt, os dermopteroticum; o- dsph, os dermosphenoticum; o-eoc, os exoccipitale; o-fr, lateral part of the otic capsule (Fig. 12C). os frontale; o-hm, os hyomandibulare; o-mx, os maxil- They are bordered by the frontals and pterot- lare; o-op, os operculare; o-para, os parasphenoideum; ics, to which they are strongly sutured. They o-pop, os praeoperculare; o-prmx, os praemaxillare; o- have lost contact with the parieto-supraoc- prot, os prooticum; o-pt-scl, os posttemporo-supraclei- thrum; o-puh, os parurohyale; o-q, os quadratum; o- cipital bone (Fig. 15A). Ventrally, they con- susp, os suspensorium; ot-cap, otic capsule; pal, nect to the prootic and pterosphenoid bones. palatinum; pns-ep, pons epiphysialis; pp-v4, parapophy- At their ventrolateral margin, they enclose sis of vertebra 4; prc-co, processus coronoideus; prc-un, processus uncinatus; sb, swimbladder; su-ph-tpl, supe- the anterior part of the articular facet for rior pharyngeal tooth plate; tn-m-p, taenia marginalis the suspensorium, which is preceded by a posterior; trip, tripus; v6, vertebra 6. stout lateroventral process (Figs. 17C, 21E). Figure 9 OSTEOCRANIUM ONTOGENY IN CLARIAS 205 The pterotics are large bones, enclosing posterior part of the frontals, as no signs of a the posterolateral part of the otic capsule, suture can be observed anymore (Fig. 17E). and bearing large, lateral, plate-like exten- The left-right fusion is not complete, as a sions. Medially, they are sutured to the pari- posterior fontanella remains. Observations eto-supraoccipital bone. The connection with of larger adults indicate a slow, but progres- the posttemporo-supracleithral bone is re- sive closure of both the anterior and poste- stricted to the posterolateral part. Conse- rior fontanella. At its ventral face, the com- quently, the pterotics form part of the poste- plex bears a median ridge, which is formed rior margin of the juvenile skull (Fig. 15A). in the mediosagittal septum. Caudolater- Ventrally, they connect to the prootics and ally, the bone interdigitates with the exoccipi- exoccipitals (Figs. 12C, 17D) and form the tals (Fig. 17E). The posttemporo-supraclei- posterior part of the articular facet for articu- thral bone is a plate-like complex, bordering lation with the suspensorium (Fig. 21E). In the pterotics laterally and laterocaudally. a specimen of 140.1 mm SL, however, the Ventrally, it bears a mediocaudally directed pterotic seemed to be separated partially process for articulation with the parapophy- from the parieto-supraoccipital and exoccipi- sis of the fourth vertebra, as well as an tal bones (which may be due to insufficient articular facet for articulation with the clei- staining), where a very small ossicle was thral bone (Fig. 17F). It does not bear any found covering that region (Fig. 16). Based connection with the basioccipital, which is on its position, as well as the fact that it the case in many siluriform fishes (Fig. 12C). seems to be a perichondral bone, at the level Most presumably, this process corresponds of the posterior semicircular canal, it must to the ossification of the transcapular liga- correspond to the epiotic, which could not be ment (see Discussion). observed in any previous stage. It is, how- Anteriorly, two plate-like premaxillaries ever, possible that because of its reduced articulate with the ventral face of the pre- size and insufficient staining, it has been ethmoid processes of the mesethmoid. Pre- overlooked in the other specimens, as was also the case for Nawar (’54). The parieto- maxillaries, with the exception of the poste- supraoccipital bone complex is large, with rior margin, are completely covered with its previously paired anterior part now fused teeth. They lack any differentiations like an to each other medially. Apparently, a true ascending process or maxillary process (Fig. fusion has occurred, in contrast with the 18A). No true articular facet, with second- ary cartilage, is present between the premax- illary bone and the mesethmoid. Both con- nect only through connective tissue. An articulation of the premaxillary with the Fig. 9. Skull of Clarias gariepinus (11.6 mm SL stage). A, dorsal view. B, lateral view; C, ventral view. maxillary bone is also absent. Only a dis- (Grey indicates bone, shaded areas indicate cartilage.) tinct string of ligamentous tissue connects fn-sph, fenestra sphenoidea; fr-I, foramen fila olfactoria; the two. The maxilla is typically advanced o-ang-c, os angulo-splenio-articulo-retroarticulare com- siluriform, as it is socket-like, enclosing the plex; o-ant, os antorbitale; o-apal, os autopalatinum; o-bb-II, os basibranchiale II; o-bb-III, os basibranchiale base of the maxillary barbel. It bears a III; o-boc, os basioccipitale; o-cb-I, os ceratobranchiale I; double-headed articular facet for articula- o-cb-IV, os ceratobranchiale IV; o-ch-a, os ceratohyale tion with the rostral tip of the palatine (Fig. anterior; o-ch-p, os ceratohyale posterior; o-den-c, os 18B). As in most siluriform fish, the maxil- dento-splenio-mentomeckelium complex; o-dpt, os der- mopteroticum; o-dsph, os dermosphenoticum; o-eb-IV, os lary bone has been modified to take part in epibranchiale IV; o-enp4, sesamoid ‘‘os entopterygoi- the palatine-maxillary mechanism (Adri- deum’’ type 4; o-eoc, os exoccipitale; o-fr, os frontale; aens and Verraes, ’97a). The antorbital bone o-hh-v, os hypohyale ventrale; o-hm, os hyomandibulare; is still small and overlies the rostral tip of o-iop, os interoperculare; o-lac, os lacrimale (ϭos infraor- bitale I); o-ldeth, os latero-dermethmoideum; o-mp, os the palatine. Compared to the previous stage, metapterygoideum; o-mx, os maxillare; o-op, os opercu- most infraorbital bones have undergone a lare; o-par-soc, os parieto-supraoccipitale; o-para, os major transformation in shape, as they are parasphenoideum; o-pop, os praeoperculare; o-prmx, os no longer tubular. The lacrimal bone bears a praemaxillare; o-prot, os prooticum; o-pt-scl, os posttem- poro-supracleithrum; o-puh, os parurohyale; o-q, os qua- serrated ventral edge, whereas the second dratum; o-susp, os suspensorium; pp-v4, parapophysis of infraorbital bone possesses a massive part vertebra 4; pp-v5, parapophysis of vertebra 5; pvm-tpl, for the articulation with the lateral process prevomeral tooth plate; r-br, radius branchiostegus; scaph, scaphium; su-ph-tpl, superior pharyngeal tooth of the lateral ethmoid (Fig. 15A). Conse- plate; trip, tripus; v1, vertebra 1; v6, vertebra 6; v7, quently, both these bones form the anterior vertebra 7. border of the orbita, whereas the remaining Figure 10 OSTEOCRANIUM ONTOGENY IN CLARIAS 207 part of the second infraorbital bone borders but now interdigitates with the complex of the orbita anteroventrally. The posteroven- vertebrae, formed in relation to the Webe- tral and posterior border is demarcated by rian apparatus (Figs. 16, 19C). Lateroven- the third infraorbital bone (Fig. 15B). Dor- trally, the basioccipital connects to the exoc- sally, the slender supraorbital process of the cipitals, posteriorly, and to the prootics, fourth infraorbital bone forms the dorsal anteriorly, both through short sutures. At its margin, but is elongated and much broader dorsal face, the basioccipital bears a bowl- prosterior to the eye. In Clarias gariepinus, like structure housing the sinus impar peri- the fourth infraorbital bone reaches up to lymphaticus of the Weberian complex (Fig. the suprapreopercular bone. The latter bone 16) (Chardon, ’67; Radermaker et al., ’89). has become plate-like as well, filling up the The orbitosphenoids connect to the lateral gap between the fourth infraorbital bone ethmoids anteriorly, the frontals dorsally, and the posttemporo-supracleithral bone and the parasphenoid posteriorly. They are (Fig. 15B). separated from the pterosphenoids by the The skull floor consists of a central, longi- foramen for the optic nerve (Fig. 12C). Al- tudinal and narrow bridge, which becomes though the orbitosphenoids are formed as broader posteriorly, as it forms the base of paired ossifications, they have fused to a the braincase. Anteriorly, the hypoethmoid single, gutter-like bone in the juvenile stage part of the mesethmoid interdigitates with (Fig. 19D). The pterosphenoids, however, re- the prevomeral bone. The latter has become main paired ossifications, which connect to arrow-like, bearing two well-developed tooth the orbitosphenoids, the parasphenoid, and plates, to which it is fused. Posteriorly, the the sphenotics (Fig. 12C). The prootics form prevomeral bone interdigitates through a the anterolateral floor of the brain cavity very slender, forked suture with the paras- and enclose the utriculus with its otolith phenoid (Fig. 19A). Dorsally, the prevomeral (the lapillus) (Fig. 19F). The perichondral bone is connected to the lateral ethmoid, prootics are sutured to a whole set of bones: more exactly to the part enclosing the olfac- the pterosphenoid, the parasphenoid, the tory lobes (Fig. 12C). The parasphenoid con- sphenotics, the pterotics, the exoccipitals, sists of a small anterior part, which is ex- the basioccipital, and the parieto-supraoc- panded laterally at the level of the earlier cipital. It takes part in the bordering of the formed lateral wings, which contact the trigemino-facial foramen (Fig. 12C). Finally, pterosphenoid bones. Posterior to these the exoccipitals form the posterolateral ossi- wings, the parasphenoid becomes narrow fications of the brain cavity floor and also again, finally interdigitating posteriorly with enclose the otoliths of the sacculus and the the basioccipitals. The interdigitation with lagena (i.e., the sagitta and asteriscus respec- the latter occurs through many more su- tively) (Fig. 19G). These bones interdigitate tures than is the case for the prevomeral with the basioccipitals, prootics, and the pa- bone (Fig. 19B). At its dorsal face, the paras- rieto-supraoccipital bone, whereas a syn- phenoid connects rigidly to the orbitosphe- chondrosis seems to be present with the noid (in front of the lateral wings), the pteros- pterotics. The exoccipital bone encloses the phenoids (at the lateral wings), and the foramina for the glossopharyngeus, vagus, prootics (posterior to the lateral wings) (Figs. and hypoglossus nerves (Figs. 12C, 16, 19G). 12C, 19B). The basioccipital no longer forms Splanchnocranium an articular surface with the first vertebra, The mandibula is fully ossified, although Meckel’s cartilage remains partially exposed (Fig. 10D). The coronoid process is well pro- Fig. 10. Ontogeny of the lower jaw in Clarias gariepi- tected by bone, with only its dorsal tip re- nus (medial view). A, 12.0 mm SL stage; B, 14.8 mm SL maining uncovered. Caudally, the retroar- stage; C, 19.0 mm SL stage; D, 127.0 mm SL stage. ticular process is rather short and bears two (Grey indicates Meckel’s cartilage, shaded areas indi- processes, both for the attachments of liga- cate bone.) af-III, articulatory facet of the os angulo- splenio-articulo-retroarticulate with the os quadratum; ments: a ligament running to the interoper- c-Meck, cartilago Meckeli; con-t, conical teeth; mnd- cular bone and one to the hyoid bar (Fig. sym, mandibular symphysis; o-ang, os angulare; o-art, 20A). The dento-splenio-mentomeckelian os articulare; o-com, os coronomeckelium; o-den, os den- bone complex is provided with a large patch tale; o-den-spl, os spleniale (dentale); o-den-vp, ventral process of the os dento-splenio-mentomeckelium com- of teeth. Two types of teeth are observed: plex; o-mm, os mentomeckelium; o-rart, os retroarticu- villiform teeth at the outer margin, and coni- lare; prc-co, processus coronoideus; vil-t, villiform teeth. cal teeth at the inner margin (Fig. 20B). The Figure 11 OSTEOCRANIUM ONTOGENY IN CLARIAS 209 surface for dentition seems to be expanded, plate, which connects to a similar outgrowth as a kind of rostral extension of the bone has of the quadrate (Fig. 21A). Dorsally, a carti- occurred. At its medial face, this bone com- laginous strip is present for articulation with plex bears a socket that encloses the ante- the neurocranium. Anterior to this strip, the rior part of Meckel’s cartilage. Caudally, a bone has a distinct dorsal process, which fits well-developed ventral and lateral process into a cavity in the sphenotic bone, addition- can be distinguished, between which fits the ally, a ventral process of the sphenotic fits lateroventral process of the angulo-splenio- into a cavity posterior to the hyomandibular articulo-retroarticular complex. At the level process (Fig. 21E). Due to such a connection, of the coronoid process, a distinct cor- the suspensorium and neurocranium are in- onomeckelian bone covers Meckel’s cartilage terlocked to a certain degree (see Discus- dorsally, anterior to the coronoid process (Fig. sion). At its posterior margin, the hyoman- 10D). The autopalatine is a rod-shape bone, dibula bears a substantial opercular process bearing three unossified regions: (1) the an- with a cartilaginous articular facet (Fig. terior tip that enables the articulation with the maxillary bone, (2) the slender and elon- 21B). At its medial face, the hyomandibula gated articular facet for the articulation with bears two foramina: (1) a small foramen just the lateral ethmoid, and (3) the posterior tip, above the insertion of the ligament running which does not articulate with any bone to the hyoid bar, and (2) a larger one poste- (Fig. 20C). Between the mandibula and the rior and dorsal to it. This larger foramen is suspensorium, the number of isolated sple- penetrated by the truncus hyomandibularis, nial bones has increased to two, whereas in whereas through the smaller foramen a blood larger specimens three of them were present vessel enters. (Fig. 15B) (Adriaens et al., ’97). Compared to the situation in previous The quadrate bone bears a solid articular stages, the foramen through which the trun- facet with the mandibula and is provided cus leaves has shifted ventrally, due to the with a substantial dorsal, membranous out- fact that a bony crest of the hyomandibular growth (Fig. 21C). The quadrate connects bone has grown ventrally, thereby covering both synchondrally and through sutures with the foramen of the hyosymplectic cartilage. the metapterygoid bone, anteriorly, and with At this stage, the latter foramen is bordered the hyomandibula, posteriorly (Fig. 21A). by both the hyomandibula and the quadrate. The hyomandibula also has a membranous Medially, the hyomandibula forms a ridge for the attachment of a stout ligament that runs to the hyoid bar (Fig. 21A) (Adriaens and Verraes, ’94). The unossified part, sepa- Fig. 11. Skull of Clarias gariepinus (12.7 mm SL rating the hyomandibula from the quadrate, stage). A, dorsal view; B, lateral view; C, dorsal view of corresponds to the part that in previous the splanchnocranium. (Grey indicates bone, shaded areas indicate cartilage.) fr-I, foramen fila olfactoria; stages was connected to the interhyale and I-trsc, ligamentum transcapularis; o-ang-c, os angulo- may be considered as an unossified symplec- splenio-articulo-retroarticulare complex; o-ant, os antor- tic. At its ventral margin, the hyomandibula bitale; o-apal, os autopalatinum; o-bb-II, os basibran- interdigitates with the preopercular bone chiale II; o-bb-III, os basibranchiale III; o-boc, os basioccipitale; o-cb-II, os ceratobranchiale II; o-ch-a, os (Figs. 15B, 21A). This bone does not bear a ceratohyale anterior; o-ch-p, os ceratohyale posterior; distinct vertical and horizontal limb, as is o-den-c, os dento-splenio-mentomeckelium complex; o-eb- the case in many catfish (Fig. 24A). Its cau- III, os epibranchiale III; o-eoc, os exoccipitale; o-fr, os frontale; o-hb-I, os hypobranchiale I; o-hh-v, os hypohy- dal margin consists of the neurodermal part ale ventale; o-hm, os hyomandibulare; o-io-II, os infraor- enclosing the preopercular canal with two of bitale II; o-iop, os interoperculare; o-lac, os lacrimale its branches (Adriaens et al., ’97). At its (ϭos infraorbitale I); o-ideth, os latero-dermethmoid- anterodorsal side, it bears a plate-like exten- eum; o-leth, os latero-ethmoideum; o-mp, os metaptery- goideum; o-mx, os maxillare; o-ns, os nasale; o-op, os sion for the interdigitation with the hyoman- operculare; o-par-soc, os parieto-supraoccipitale; o-para, dibula and the quadrate (Fig. 24A). The os parasphenoideum; o-pop, os praeoperculare; o-prmx, plate-like expansion has persisted in the os praemaxillare; o-prot, os prooticum; o-psph, os pteros- phenoideum; o-pt, os pteroticum; o-pt-scl, os posttemporo- metapterygoid and entopterygoid bone as supracleithrum; o-puh, os parurohyale; o-q, os qua- well (Fig. 21D). As can be derived from the dratum; o-sph, os sphenoticum; o-susp, os suspensorium; previous stages, the ossification of the pp-v4, parapophysis of vertebra 4; pp-v5, parapophysis of metapterygoid initiates from a perichondral vertebra 5; r-br, radius branchiostegus; scaph, scaphium; su-ph-tpl, superior pharyngeal tooth plate; trip, tripus; ossification of the pterygoid process, which v6, vertebra 6. can still be distinguished in this stage. In Figure 12 OSTEOCRANIUM ONTOGENY IN CLARIAS 211 this stage, both ventrally and dorsally, this pohyal is much larger than the dorsal. Ven- central rod bears plate-like extensions. The tromedially, the latter bone forms an inter- ventral extension corresponds to the digitation with the anterior ceratohyal bone, ectopterygoid process of Arratia (’92). Consid- whereas the rest is interconnected through a erable discussion concerns the homology of synchondrosis. Mediolateral to these su- the pterygoid bones in catfish and even in tures, it is attached through a stout liga- fish in general (Howes and Teugels, ’89; Arra- ment that is connected to the anterior tips of tia, ’90, ’92). The metapterygoid is strongly the parurohyal bone (Fig. 4G). A basihyal connected to the quadrate through the ven- bone is absent. The anterior ceratohyal bone tral synchondrosis, but more strongly is the largest of the hyoid bar (Fig. 22C). At through the extensive suturation along the its medial face, it bears a shelf against which dorsal plates (Fig. 21A). Consequently, the the first six branchiostegal rays articulate metapterygoid no longer contacts the hyo- (Fig. 22A). The following two rays articulate mandibula. Rostrally, the metapterygoid has with the cartilaginous strip, which sepa- a straight margin, connected to the entop- rates the anterior ceratohyal bone from the terygoid through a ligamentous strip. The posterior one ventrally. The posterior two entopterygoid in Clarias gariepinus is liga- branchiostegal rays articulate with the ven- mentously connected to a whole set of sur- tral margin of the posterior ceratohyal bone. rounding bones: (1) the metapterygoid, pos- The posterior two branchiostegal rays articu- teriorly, (2) the autopalatine, dorsally, (3) late with the ventral margin of the posterior the prevomeral bone, anteriorly, and (4) the ceratohyal bone. The posterior ceratohyal lateral ethmoid, anterolaterally. Caudally, bone tapers posteriorly and terminates as a the entopterygoid bone bears a distinct pro- solid cone (Fig. 22D). At the dorsal face of cess (Fig. 21D). this cone is a stout ligament that connects The hyoid bar is almost completely ossi- the hyoid bar to the hyomandibula (Fig. fied, although cartilaginous strips seem to 21A). Another large ligament connects the persist between all bones (Figs. 15C, 22A). At the medial face of the ventral hypohyal, lateral face with the medial process of the the dorsal hypohyal remains separated by mandibular retroarticular process (Fig. 20A). cartilage from both the ventral hypohyal In this stage, the interhyal is completely and the anterior ceratohyal. The ventral hy- reduced (Adriaens and Verraes, ’94). The parurohyal bone has become more solidly ossified, with three distinct, caudal pro- cesses. Although the median process is the result of a fusion of two processes, it is more Fig. 12. Neurocranium of Clarias gariepinus (ven- slender than the lateral processes (Fig. 4G). tral view). A, 12.7 mm SL stage; B, 21.5 mm SL stage; C, 127.0 mm SL stage. (Grey indicates bone, shaded areas The two heads of the paired sternohyoideus indicate cartilage.) af-V, articulatory facet of the os lat- muscle fit nicely into the fork of the paruro- eroethmoideum with the os infraorbitale II; fn-sph, hyal bone (Adriaens and Verraes, ’97d). The fenestra sphenoidea; fr-II, foramen fasciculus opticus; length of the ligaments, in relation to the fr-IX, foramen nervus glossopharyngeus (ϭfenestra ba- sicapsularis posterior); fr-X, foramen nervus vagus; fr-V- size of the parurohyal, seems to have become VII, foramen of the trigemino-facial nerve complex; I- reduced substantially, as the rostral tips of trsc, ligamentum transcapularis; o-ant, os antorbitale; the parurohyal now partially fit into a small o-apal, os autopalatinum; o-apt, os autopteroticum; gap at the medial face of the ventral hypohy- o-asph, os autosphenoticum; o-boc, os basioccipitale; o-dpt, os dermopteroticum; o-enp4, sesamoid ‘‘os entoptery- als. The main body of the parurohyal still goideum’’ type 4; o-eoc, os exoccipitale; o-fr, os frontale; bears a foramen, penetrated by a ventral o-hm, os hyomandibulare; o-io-II, os infraorbitale II; branch of the ventral aorta (Fig. 13). The o-io-III, os infraorbitale III; o-io-IV, os infraorbitale IV; latter artery is here referred to as the paru- o-lac, os lacrimale (ϭos infraorbitale I); o-leth, os latero- ethmoideum; o-II, lateral line ossicle; o-mp, os metaptery- rohyal artery. It enters the parurohyal dor- goideum; o-mx, os maxillare; o-ns, os nasale; o-op, os sally, where it splits into two arteries before operculare; o-osph, os orbitosphenoideum; o-par-soc, os it leaves the bone ventrally. Between the two parieto-supraoccipitale; o-para, os parasphenoideum; rostral processes of the parurohyal, a vein o-prmx, os praemaxillare; o-prot, os prooticum; o-psph, os pterosphenoideum; o-pt, os pteroticum; o-pt-scl, os (here referred to as the vena parurohyalis) posttemporo-supracleithrum; o-pvm, os prevomerale; o-q, branches off of the vena jugularis inferior os quadratum; o-sph, os sphenoticum; o-spop, os supra- and curves caudally, against the ventral side praeoperculare; o-susp, os suspensorium; pp-v4, par- of the bone and between the two branches of apophysis of vertebra 4; pp-v5, parapophysis of vertebra 5; pvm-tpl, prevomeral tooth plate; r-v7, rib of vertebra the artery (Fig. 13B). The branchiostegal 7; trip, tripus; v6, vertebra 6. rays differ slightly in shape, depending on Figure 13 OSTEOCRANIUM ONTOGENY IN CLARIAS 213 their position. The anterior ray is a slender (Adriaens and Verraes, ’97c). This cartilage rod with a broader articular part, whereas connects these epibranchials to the anterior the posterior ray has a distinct articular tip of the third infrapharyngobranchial bone facet, penetrated by a foramen (Fig. 22E), (Fig. 15C). Epibranchiale III has a well- with a distal, elongated, plate-like part (Fig. developed uncinate process at its caudal mar- 22A). gin (Fig. 23C). Medially, this epibranchial Ossification of the branchial basket seems articulates with the posterior tip of the third to have completed, although several carti- infrapharyngobranchial and the anterior laginous elements lack any bone (Fig. 15C). margin of the fourth one (Fig. 15C). The The anterior copula still bears two ossifica- fourth epibranchial bone bears a horizontal, tions, corresponding to the second and third plate-like extension at its middle portion, for basibranchial bones (Fig. 23B–C). No signs the support of the uncinate process (Fig. of the first basibranchial could be discerned 23D). Medially, this epibranchial articulates at this stage (Fig. 23A). The posterior copula with the posterolateral margin of the fourth also lacks ossifications of the fourth and fifth infrapharyngobranchial element. As men- basibranchial (Fig. 23D–E). Hypobranchial tioned, only the third and fourth infrapha- bones I and II of the previous stage have ryngobranchials ossify in Clarias gariepinus expanded, as they now cover almost half of (Fig. 15C). The third is rod-like, with both the surface of the corresponding cartilagi- its articular tips nonossified (Fig. 23C). The nous elements (Figs. 15C, 23A,B). The third fourth one is rather triangular, with its lat- hypobranchial element is still unossified. The eral margin nonossified (Fig. 23D). The up- cartilaginous fourth hypobranchial is be- per pharyngeal tooth plate is enlarged and is lieved to remain fused to the ceratobran- supported dorsally by the fourth infrapha- chial cartilage and is unossified as well (Fig. ryngobranchial bone, as well as the medial 23D) (Adriaens and Verraes, ’97c). part of epibranchials III and IV (Fig. 15C). All five ceratobranchials are well ossified. The opercular series in juvenile Clarias They consist of the central, perichondral rod, gariepinus consists of the opercular bone, which bears two caudal ridges for attach- the preopercular, and interopercular bones ment of the gill rods, as well as for the (Fig. 15B). The subopercular bone is miss- housing of blood vessels and nerves. The ing, as is the case in most siluriform fish (see tooth plate, covering the dorsal face of the Discussion). The preopercular bone attaches fifth ceratobranchial bone, has become to the suspensorium, as mentioned above. broader, especially at its anterior half (Fig. The opercular bone is triangular and articu- 23E). The epibranchials are more elongated, lates with the opercular process of the hyo- compared with the previous stage, espe- mandibula (Fig. 15B). Consequently, at its cially epibranchials I and II (Fig. 15C). This anterior margin, the opercular bone bears a elongation seems to be coupled to the elonga- well-developed articular facet, which is in tion of the third infrapharyngobranchial contact with a medial crest for the insertion bone. Medially, these bones converge and of the levator operculi muscle and the oper- attach to each other through cartilage, which cular part of the hyohyoidei adductor muscles most probably corresponds to the unossified (Fig. 24B) (Adriaens and Verraes, ’97b). The remains of infrapharyngobranchials I and II ventral tip of the opercular bone attaches to the interopercular bone through a ligamen- tous strip (Fig. 15B). This interopercular bone remains triangular, as it tapers ros- Fig. 13. Graphical, 3-D reconstruction of the paruro- trally to become elongated in a stout liga- hyal bone and related bloodvessels in Clarias gariepinus ment that is attached to the lateral process (46.8 mm SL stage). A, frontal view; B, frontal view with vena jugularis inferior and arteria hyoidea removed; C, of the mandibular retroarticular process caudal view; D, caudal view with vena jugularis inferior (Figs. 20A, 24C). The interopercular bone and arteria hyoidea removed. (Grey indicates bone.) also attaches through connective tissue to a-br-aff-I, arteria branchialis afferens I; a-br-aff-II, arte- the lateral face of the posterior ceratohyal ria branchialis afferens II; a-hy, arteria hyoidea; a- puh-c, arteria parurohyalis communis; a-puh-I, arteria bone. parurohyalis lateralis; an-v-j-inf, anastomosis between DISCUSSION left and right venae jugulares inferiores; ao-v, aorta ventralis; cp-a, copula anterior; o-bb-II, os basibran- Bone in Clarias gariepinus is cellular, al- chiale II; o-puh-lp, lateral process of the os parurohyale; o-puh-mp, medial process of the os parurohyale; v-j-inf, though the number of osteocytes is low. Os- vena jugularis inferior; v-j-inf-br, branch of the vena tariophysans, in general, together with some jugularis inferior; v-puh, vena parurohyalis. other primitive teleostean lineages (Meu- Figure 14 OSTEOCRANIUM ONTOGENY IN CLARIAS 215 nier and Huysseune, ’92), are characterized Siluriform relationships of the by the presence of cellular bone. According osteocranium of Clarias gariepinus to the terminology of Patterson (’77), all Although a comparative osteology is be- three bone types in fish are formed in C. yond the scope of this report, some of the gariepinus: cartilage bones, dermal bones, and membrane bones. In C. gariepinus, all features used to describe the cranial osteology cartilage bones ossify perichondrally. True of Clarias gariepinus, are briefly discussed. enchondral ossification was not observed but The paired origin of the prevomeral bone may occur in larger specimens. Bony ele- has been observed in most teleost fish (De ments are compact, lamellar ossifications Beer, ’37; Verraes, ’73). This bone is consid- early in ontogeny, whereas most of them ered nonhomologous with the vomeral bone become spongious in the juvenile stage. The of tetrapods, although it is frequently re- cartilage bones, lining the cartilaginous ferred to as ‘‘vomeral bone’’ in teleosts (de structures, are surrounded with membra- Beer, ’37; Harrington, ’55; Daget, ’64). In nous apolamellae, which frequently are Clarias gariepinus, it appears that the pre- formed in a radiating manner. Most dermal vomeral bone itself is unpaired (whether or bones are extremely trabecular, with the not a fusion occurs between paired blaste- exception of the prevomeral bone and paras- mata or not could not be discerned) and phenoid bone. Dermal tooth plates become becomes fused to paired tooth plates. In the spongy also. At least one type of membrane closely related heteropneustid species, Het- bone is also present: the sesamoid bones eropneustes fossilis, an unpaired prevomeral (coronomeckelian and entopterygoid bones). bone arises, prior to its dentition (Srinivasa- The fully developed skull of C. gariepinus char, ’58). Researchers seem to come to a consists of about 150 bones (both sides in- consensus that the toothless prevomeral cluded), which can be subdivided into five bone is an unpaired, dermal bone that be- categories: (1) 22 anamestic dermal bones, comes fused to paired, autogenous tooth- (2) 14 dermal canal bones, (3) 37 perichon- plates, whereas the latter may be homolo- dral bones, (4) two sesamoid bones and (5) gous with pharyngeal tooth plates of the three bones of a compound nature. premandibular arch (Daget, ’64; Srinivasa Rao and Lakshmi, ’84; Arratia, ’87). The prevomeral bone in siluriform fish is gener- ally T-shaped, bearing two patches of teeth at its lateral wings and interdigitating with Fig. 14. Skull of Clarias gariepinus (21.5 mm SL the parasphenoid through very long sutures stage). A, dorsal view; B, lateral view; C, dorsal view of splanchnocranium. (Grey indicates bone, shaded areas (Bhimachar, ’33; Merriman, ’40; Tilak, ’63b, indicate cartilage.) fr-I, foramen fila olfactoria; ipb-IV, ’65a; Lundberg, ’82; Srinivasa Rao and Lak- infrapharyngobranchiale IV; o-ang-c, os angulo-splenio- shmi, ’84; Howes and Fumihito, ’91). Both articulo-retroarticulare complex; o-ant, os antorbitale; o-bb-II, os basibranchiale II; o-boc, os basioccipitale; tooth patches may be fused to each other o-cb-IV, os ceratobranchiale IV; o-ch-a, os ceratohyale (Merriman, ’40; Tilak, ’61, ’63b,c; Rastogi, anterior; o-cl, os cleithrum; o-com, os coronmeckelium; ’63; Arratia, ’87; Howes and Fumihito, ’91) or o-den-c, os dento-splenio-mentomeckelium complex; may even be absent (Tilak, ’65a, ’67; Gauba, o-eb-I, os epibranchiale I; o-eb-IV, os epibranchiale IV; o-enp4, sesamoid ‘‘os entopterygoideum’’ type 4; o-eoc, os ’70; Lundberg, ’82; Vignes and Garcia, ’87; exoccipitale; o-fr, os frontale; o-hb-II, os hypobranchiale Ferraris, ’96). Fusion between both tooth II; o-hh-v, os hypohyale ventrale; o-hm, os hyomandibu- plates in adult Clariidae seems to be the lare; o-io-II, os infraorbitale II; o-io-III, os infraorbitale general trend (Tilak, ’63c; Teugels, ’82). In III; o-io-IV, os infraorbitale IV; o-iop, os interoperculare; o-ipb-III, os infrapharyngobranchiale III; o-lac, os lacri- case dentition is lacking, an unpaired origin male (ϭos infraorbitale I); o-leth, os latero-ethmoideum; of the prevomeral bone has been observed o-II, lateral line ossicle; o-meth, os mesethmoideum; (Kindred, ’19), with its lateral wings some- o-mp, os metapterygoideum; o-mx, os maxillare; o-ns, os nasale; o-op, os operculare; o-osph, os orbitosphenoi- times being absent (Lundberg, ’82; Howes, deum; o-par-soc, os parieto-supraoccipitale; o-para, os ’83). Heavily dentate prevomeral tooth plates parasphenoideum; o-pop, os praeoperculare; o-prmx, are present in Pangasiidae and Siluridae, os praemaxillare; o-prot, os prooticum; o-psph, os ptero- where these plates become closely aligned sphenoideum; o-pt, os pteroticum; o-pt-scl, os post- temporo-supracleithrum; o-q, os quadratum; o-sph, os with autogenous palatine tooth plates, or sphenoticum; o-spop, os suprapraeoperculare; o-susp, os may even fuse to them (Tilak, ’61; Roberts suspensorium; pp-v4, parapophysis of vertebra 4; pp-v5, and Vidthayanon, ’91). In Chacidae, the pre- parapophysis of vertebra 5; prc-op, processus opercu- vomeral bone is absent as an independent laris; r-br, radius branchiostegus; r-v7, rib of vertebra 7; v6, vertebra 6; vc, vertebral complex of the Weberian ossification (Tilak, ’71; Brown and Ferraris, apparatus. ’88). Figure 15 OSTEOCRANIUM ONTOGENY IN CLARIAS 217

Fig. 16. Skull of Clarias gariepinus (140.1 mm SL impar perilymphaticus; I-trsc, ligamentum transcapu- stage). Caudal view. (Small circles indicate cartilage.) laris; o-boc, os basioccipitale; o-eoc, os exoccipitale; o- cd, chorda dorsalis; fr-IX, foramen nervus glossopharyn- epot, os epioticum; o-par-soc, os parieto-supraoccipitale; geus (ϭfenestra basicapsularis posterior); fr-m, foramen o-prot, os prooticum; o-pt, os pteroticum; o-pt-scl, os magnum; fr-X, foramen nervus vagus; fr-XII, foramen supracleithrum. nervus hypoglossus; Gr sin-imp, groove for the sinus

For a discussion and in- and outgroup presence of a separate parietal ossification, comparison of the parietal and supraoccipi- which has been observed in several siluri- tal bone complex, we refer to Fink and Fink form taxa, is now generally accepted to ex- (’81, ’96) and Arratia and Gayet (’95). The plain the compound nature of this bone (Ar- ratia, ’87; Fink and Fink, ’96). For a discussion on the homology of the Fig. 15. Skull of Clarias gariepinus (127.0 mm SL posttemporo-supracleithral bone in Silurifor- stage). A, dorsal view; B, lateral view; C, dorsal view of mes, we refer to Lundberg (’75), Arratia and the splanchnocranium. (Grey indicates bone, shaded Gayet (’95), and Fink and Fink (’81, ’96). areas indicate cartilage.) af, anterior fontanella; af-V, Concerning the transscapular ligament, articulatory facet of the os lateroethmoideum with the os infraorbitale II; c-Meck, cartilago Meckeli; I-trsc, several constructions seem to be present in ligamentum transcapularis; o-ang-c, os angulo-splenio- Siluriformes. In many catfish, the posttem- articulo-retroarticulare complex; o-ant, os antorbitale; poro-supracleithral bone connects to the ba- o-apal, os autopalatinum; o-bb-III, os basibranchiale III; o-cb-I, os ceratobranchiale I; o-cb-III, os ceratobran- sioccipital through a bony limb, the ossified chiale III; o-cb-V, os ceratobranchiale V; o-ch-a, os cerato- transscapular ligament, which attaches hyale anterior; o-ch-p, os ceratohyale posterior; o-com, through a ligament to the lateral face of the os coronomeckelium; o-den-c, os dento-splenio-ment- basioccipital (Bhimachar, ’33; Merriman, ’40; omeckelium complex; o-eb-I, os epibranchiale I; o-enp4, sesamoid ‘‘os entopterygoideum’’ type 4; o-ep-br, ossifica- Hubbs and Miller, ’60; Tilak, ’61, ’63b, ’65a, tion of the os frontale around the epiphyseal bridge; o-fr, ’67, ’71; Rastogi, ’63; Lundberg, ’75; Poll, ’77; os frontale; o-hb-I, os hypobranchiale I; o-hb-II, os hypo- Srinivasa Rao and Lakshmi, ’84; Howes and branchiale II; o-hh-d, os hypohyale dorsale; o-hh-v, os hypohyale ventrale; o-hm, os hyomandibulare; o-io-II, Fumihito, ’91). In others, no such a connec- os infraorbitale II; o-io-III, os infraorbital III; o-io-IV, os tion is observed. Then, mostly, a medial limb infraorbitale IV; o-iop, os interoperculare; o-ipb-III, os connects the anterior vertebrae, at the level infrapharyngobranchiale III; o-ipb-IV, os infrapharyn- of the vertebral center (Tilak, ’63b), or con- gobranchiale IV; o-lac, os lacrimale (ϭos infraorbitale I); o-leth, os latero-ethmodeum; o-II, lateral line ossicle; tacts the parapophyses (Srinivasacher, ’58; o-meth, os mesethmoideum; o-mp, os metapterygoi- Nawar, ’54; Tilak, ’63c; Gauba, ’70; Howes, deum; o-mx, os maxillare; o-ns, os nasale; o-op, os opercu- ’83; present study). Regan (’11) mentioned lare; o-par-soc, os parieto-supraoccipitale; o-pop, os praeo- perculare; o-prmx, os praemaxillare; o-pt, os pteroticum; that a lower limb of the posttemporal (post- o-pt-scl, os posttemporo-supracleithrum; o-puh, os paru- temporo-supracleithral bone of present rohyale; o-q, os quadratum; o-sph, os sphenoticum; o-spl, study) to the basioccipital is absent in Clari- ossa splenialia; o-spop, os suprapraeoperculare; pf, poste- idae, Loricariidae, and Callichthyidae. This rior fontanella; prc-co,processus processus coronoideus; prc- un, processus uncinatus; r-br, radius branchiostegus; su-ph- author also mentioned that the posttempo- tpl, superior pharyngeal tooth plate; TC, temporal canal. ral is absent in Clariidae, whereas the su- Figure 17 OSTEOCRANIUM ONTOGENY IN CLARIAS 219

Fig. 18. Maxillary bones of juvenile Clarias gariepi- prmx, dorsal crest of the os praemaxillare, for the nus. A, premaxillary, dorsal view (125.5 mm SL stage); articulation with the cornua prae-ethmoidea; I-mx-pal, B, maxillary, caudal view (174.5 mm SL stage). (Orienta- ligamentum maxillo-palatinum; s-mx-b, socket of the tion cross: D, dorsal, V: Ventral.) af-X, articulatory fac- maxillary barbel. ets of the os maxillare with the os autopalatinum; cr- pracleithral bone is connected ‘‘with a poste- temporo-supracleithral bone, which is bifur- rior process firmly united to the air-bladder cated and attached to the cranium (at the capsule.’’ In Diplomystidae, Arratia (’87) ob- level of the basioccipital) and to the par- served a ventromedial process of the post- apophyses through ligaments. The question raised here is whether the process, attaching to the basioccipital in most catfish corresponds to the one connecting Fig. 17. Skull roof bones of juvenile Clarias gariepi- the Weberian apparatus in others. The con- nus (125.5 mm SL stage). A, lateral ethmoid, ventral nection to the basioccipital is believed to be view; B, frontal, ventral view; C, sphenotic, ventral view. D, pterotic, ventral view; E, Parieto-suproccipital, the ossified Baudelot’s ligament, which gen- ventral view; F, posttemporo-supracleithral, ventral view. erally runs from the supracleithral bone to (Orientation cross: A: Anterior, P: Posterior, L: Lateral, the basioccipital in other teleosts. Lundberg M: Medial.) af-V, articulatory facet of the os lateroethmoi- deum with the os infraorbitale II; af-VIII, articulatory (’75) considered the connection to the par- facet of the os lateroethmoideum with the os autopal- apophyses of the vertebrae as a differentia- atinum; af-XI, articulatory facet of the os posttem- tion (ventral process) of the transscapular poro-supracleithrum with the os cleithrum; cr-par-soc, ligament of his supracleithrum (ϭposttem- medioventral crest of the posterior part of the os parieto- supraoccipital; fn-pop-a, anterior part of the postpineal poro-supracleithrum of present study). A foramen; fn-prp, fenestra praepinealis; fr-m-b, border of double connection of the posttemporo-su- the foramen magnum; fr-V-VII-b, border of the foramen pracleithral bone to the basioccipital and of the trigemino-facial nerve complex; Gr a-sc-c, groove for the anterior semicircular canal; Gr nerv-I, groove of Weberian apparatus, as observed by Arratia the fila olfactoria; intd-eoc, interdigitation with the os (’87) in Diplomystidae, is considered to be a exoccipitale; intd-fr, interdigitation with the os frontale; feature of the Siluriphysi (present Silurifor- intd-leth, interdigitation with the os lateroethmoideum; intd-meth, interdigitation with the os mesethmoideum; mes and Gymnotiformes) (Fink and Fink, intd-osph, interdigitation with the os orbitosphenoi- ’81: character 98). Although such a bifurca- deum; intd-par-soc, interdigitation with the os parieto- tion is absent in Clarias gariepinus, it could supraoccipitale; intd-prot, interdigitation with the os indicate the homology of the ventromedial prooticum; intd-psph, interdigitation with the os pteros- phenoideum; intd-pt, interdigitation with the os pteroti- process of the posttemporo-supracleithral cum; intd-pt-scl, interdigitation with the os posttemporo- bone with a part of the transscapular liga- supracleithrum; intd-sph, interdigitation with the os ment in other catfish groups. sphenoticum; IOC, infraorbital canal; I-trsc, ligamen- tum transcapularis; lap, lapillus (utricular otolith); o-ep- A dermopalatine is considered to be ab- br, ossification of the os frontale around the epiphyseal sent in ostariophysan fish. Tooth plates at bridge; o-sph-sp, sphenotic spine; OTC, otic canal; pf, the level of the autopalatine are then re- posterior fontanella; PMC, preoperculo-mandibular ca- garded as neomorphic, autogenous tooth nal; sch-eoc, synchondrosis with the os exoccipitale; SOC, supraorbital canal; TC, temporal canal; TC-pt, pterotic plates (Fink and Fink, ’81, ’96; Arratia and branch of the temporal canal. Schultze, ’91; Arratia, ’92). The dermopala- Fig. 19. Skull floor bones of juvenile Clarias gariepi- with the os basioccipitale; intd-eoc, interdigitation with nus. A, prevomer, ventral view (125.5 mm SL stage); B, the os exoccipitale; intd-fr, interdigitation with the os parasphenoid, dorsal view (174.5 mm SL stage); C, frontale; intd-leth, interdigitation with the os lateroeth- basioccipital, dorsal view (125.5 mm SL stage); D, Orbi- moideum; intd-meth, interdigitation with the os meseth- tosphenoid, dorsal view (125.5 mm SL stage); E, pteros- moideum; intd-osph, interdigitation with the os orbito- phenoid, lateral view (125.5 mm SL stage); F, prootic, sphenoideum; intd-par-soc, interdigitation with the os medial view (125.5 mm SL stage); G, exoccipital, lateral parieto-supraoccipitale; intd-para, interdigitation with (left) and medial (right) view (125.5 mm SL stage). ast, the os parasphenoideum; intd-prot, interdigitation with asteriscus (lagenar otolith); cd, chorda dorsalis; fr-II-b, the os prooticum; intd-psph, interdigitation with the os border of the foramen fasciculus opticus; fr-IX, foramen pterosphenoideum; intd-pt, interdigitation with the os nervus glossopharyngeus (ϭfenestra basicapsularis pos- pteroticum; intd-pvm, interdigitation with the os pre- terior); fr-m-b, border of the foramen magnum; fr-V- vomerole; intd-sph, interdigitation with the os sphenoti- VII-b, border of the foramen of the trigemino-facial cum; intd-vc, interdigitation with the vertebral complex; nerve complex; fr-X, foramen nervus vagus; fr-XII, fora- o-para-lp, lateral wing of the os parasphenoideum; pvm- men nervus hypoglossus; Gr sin-imp, groove for the tpl, prevomeral tooth plate; sch-pt, synchondrosis with sinus impair perilymphaticus; intd-boc, interdigitation the os pteroticum; tn-m-p, taenia marginalis posterior. OSTEOCRANIUM ONTOGENY IN CLARIAS 221

Fig. 20. Lower jaw and palatine of juvenile Clarias Meckeli; con-t, conical teeth; mx-b, maxillary barbel; gariepinus. A, angulo-splenio-articuloϭretroarticular o-ang-lp, lateral process of the os angulo-splenio-articulo- bone complex with Meckel’s cartilage, dorsal view (125.5 retroarticulare complex; o-apal, os autopalatinum; o-den- mm SL stage); B, dento-splenio-mentomeckelian bone fe, frontal extension of the os dento-splenio-mentomeck- complex, dorsal view (174.5 mm SL stage); C, autopala- elian complex; o-den-lp, lateral process of the os dento- tine and maxillary, dorsal view (125.5 mm SL stage). splenio-mentomeckelium; o-den-vp, ventral process of af-III, articulatory facet of the os angulo-splenio-arti- the os dento-splenio-mentomecklium complex; o-mx, os culoretroarticulare with the os quadratum; af-VII, maxillare; prc-ang-ch, processus angulo-ceratohyalis; articulatory facet of the os autopalatinum with the os prc-ang-iob, processus angulo-interopercularis; s-Meck, lateroethmoideum; af-IX, articulatory facet of the os socket for Meckel’s cartilage; t-re-t, tendon of the m. autopalatinum with the os maxillare; c-Meck, cartilago retractor tentaculi; vil-t, villiform teeth. Figure 21 OSTEOCRANIUM ONTOGENY IN CLARIAS 223 tine of Vandewalle et al., (’93), observed in also observed in the present study, in which Clarias gariepinus, corresponds to the sesa- development of the parurohyal bone is very moid entopterygoid bone of the present study. similar to that observed in Trichomycterus The ‘‘thin dermal plate’’ covering the ante- areolatus (Trichomycteridae) (Arratia and rior end of the cartilaginous pterygoid pro- Schultze, ’90). In Clarias gariepinus, a peri- cess probably may have been confused with chondral bone appeared at the ventral mar- the perichondral metapterygoid, which bears gin of the cartilaginous basibranchial I (8.4 a dorsal and ventral membranous apola- mm SL), after the paired elements were mella. The cartilaginous connection between observed (6.6 mm SL). At 8.4 mm SL, the the metapterygoid and the entopterygoid, as perichondral part was already fused to the mentioned by Vandewalle et al., (’93), could sesamoid one. The chondroid bone of some not be observed. The dermopalatine in Galei- catfish, however, could not be observed in C. chthys felis (Ariidae), reported by Bamford gariepinus (Arratia and Schultze, ’90). (’48: Fig. 7B), seems to correspond to the Three hypotheses can be put forward: (1) prevomeral tooth plates, as they attach to the lateral ethmoids at the same spot as is the perichondral part corresponds to the en- the case in most siluriforms. doskeletal urohyal of sarcopterygians, which The homology of the urohyal bone in ostei- becomes fused to dermal bones, the latter chthyan fishes has been extensively re- homologous with the sarcopterygian inter- viewed by Arratia and Schultze (’90). In most clavicles (Patterson, ’77), or (2) as Patterson teleost fishes the urohyal bone is an un- (’77) in a way predicted, the perichondral paired ossification of the aponeurosis, be- part corresponds to the ventral part of the tween the contralateral sternohyoideus anterior basibranchial bone, which becomes heads, bearing two rostral processes for the fused with the paired tendon bones. A final ligamentous connection with the ventral hy- possibility is (3) the secondary invasion of pohyals, and a ventral, horizontal plate (de the ossification centers of the tendons of the Beer, ’37; Patterson, ’77; Arratia and Sch- sternohyoideus muscle into the basibran- ultze, ’90). In siluriform fish, however, a chial cartilage, which thus become perichon- perichondral ossification participates in the dral, as already proposed by Arratia and formation of that bone, together with a paired Schulze (’90). However, they mentioned that bone of apparent sesamoid origin. This was ‘‘the parurohyal of siluroids is a novelty within teleosts in its combination of paired tendon bones with the addition of secondary cartilage or chondroid bone.’’ In Clarias gari- Fig. 21. Suspensorium of juvenile Clarias gariepi- epinus, no such secondary cartilage, nor nus. A, suspensorium and hyoid bar, medial view (125.5 chondroid bone could be observed. The carti- mm SL stage); B, hyomandibula, lateral view (125.5 mm lage involved was the basibranchial I. An SL stage). C, quadrate, lateral view (125.5 mm SL stage); D, metapterygoid and entopterygoid, lateral view ossified basibranchial I is absent in pres- (125.5 mm SL stage); E, Locking mechanism of suspen- ently studied siluriforms, but could be ob- sorium and neurocranium, medio-ventral view (127.0 served in some other ostariophysans, e.g., mm SL stage). af-I, articulatory facet of the os hyoman- Cyprinidae (Harrington, ’55; Cubbage and dibulare with the os sphenoticum; af-II, articulatory facet of the os sphenoticum with the os hyomandibulare; Mabee, ’96), Catostomiade (Cypriniformes) af-IV, articulatory facet of the os quadratum with the os (Weisel, ’60), Characidae (Weitzman, ’62), angulo-splenio-articulo-retroarticulare; af-XIII, articula- Hemiodontidae, Parodontidae (Characifor- tory facet of the os hyomandibulare with the os opercu- lare; fr-tr-hm, foramen truncus hyomandibularis nervus mes) (Roberts, ’74). Although the parurohyal facialis; intd-hm, interdigitation with the os hyomandibu- of Siluriformes is considered to be homolo- lare; intd-mp, interdigitation with the os metapterygoi- gous with the urohyal of other ostariophys- deum; intd-q, interdigitation with the os quadratum; ans and other teleosts (Arratia and Sch- I-hm-ch, ligamentum hyomandibulo-ceratohyale; memb- pl, membranous plate of the os hyomandibulare; o-ch-a, ultze, ’90), a detailed ontogenetic study, at os ceratohyale anterior; o-ch-p, os ceratohyale posterior; an ultrastructural level, of the parurohyal o-enp4, sesamoid ‘‘os entopterygoideum’’ type 4; o-hh-d, and basibranchial cartilage could yield con- os hypohyale dorsale; o-hh-v, os hypohyale ventrale; o-hm, os hyomandibulare; o-hm-sp, hyomandibular clusive result on the true nature of the paru- spine; o-mp, os metapterygoideum; o-pop, os praeopercu- rohyal in siluriform fish. lare; o-pt, os pteroticum; o-q, os quadratum; o-sph, os Another feature related the parurohyal sphenoticum; o-sph-sp, sphenotic spine; prc-ect, proces- bone, in contrast with the teleostean uro- sus ectopterygoideus; prc-op, processus opercularis; prc- pt, processus pterygoideus; tr-hm, truncus hyomandibu- hyal bone, is the penetration of it by an laris nervus facialis. artery. Arratia and Schultze (’90) noted the Fig. 22. Hyoid bar of juvenile Clarias gariepinus. A, articulation with the radii branchiostegi; fr-br, foramen hyoid bar with branchiostegal rays, lateral view (125.5 of the radii branchiostgi; intd-ch-a, interdigitation with mm SL stage). B, hypohyals, dorsal view (127.0 mm SL the os ceratohyale anterior; intd-ch-p, interdigitation stage); C, anterior ceratohyal, medial view (125.5 mm with the os ceratohyale posterior; intd-hh-v, interdigita- SL stage); D, posterior ceratohyal, medial view (125.5 tion with the os hypohyale ventrale; I-hm-ch, ligamen- mm SL stage); E, detail of articular facet of a branchio- tum hyomandibulo-ceratohyale; I-puh-hh, ligamentum stegal ray, frontal view (125.5 mm SL stage). af-XV, parurohyalo-hypohyale; o-ch-a, os ceratohyale anterior; articulatory facet of the os ceratohyale anterior with the o-ch-p, os ceratohyale posterior; o-hh-d, os hypohyale radii branchiostegi; af-XVI, articulatory facet of the ra- dorsale; o-hh-v, os hypohyale ventrale; r-br, radius bran- dius branchiostegus with the os ceratohyale anterior; chiostegus. cr-ch-a, crest of the os ceratohyale anterior, for the OSTEOCRANIUM ONTOGENY IN CLARIAS 225

Fig. 23. Branchial basket of juvenile Clarias gariepi- II, os basibranchiale II; o-bb-III, os basibranchiale III; nus (125.5 mm SL stage). A, branchial arch I, medial o-cb-I, os ceratobranchiale I; o-cb-II, os ceratobranchiale view; B, branchial arch II, medial view; C, branchial II; o-cb-III, os ceratobranchiale III; o-cb-IV,os ceratobran- arch III, medial view; D, Branchial arch IV, medial view; chiale IV; o-cb-V,os ceratobranchiale V; o-eb-I, os epibran- E, branchial arch V, medial view. brct, branchictenium; chiale I; o-eb-II, os epibranchiale II; o-eb-III, os epibran- c-hembr, cartilaginous supporting rods of the hemibran- chiale III; o-eb-IV, os epibranchiale IV; o-hb-I, os chia; cr-eb-IV, crest of the os epibranchiale IV, for the hypobranchiale I; o-hb-II, os hypobranchiale II; o-ipb- support of the processus uncinatus of the os epibran- III, os infrapharyngobranchiale III; o-ipb-IV, os infrapha- chiale III; hb-III, hypobranchiale III; hb-IV, hypobran- ryngobranchiale IV; prc-un, processus uncinatus; su-ph- chiale IV; in-ph-tpl, inferior pharyngeal toothplate; o-bb- tpl, superior pharyngeal tooth plate. 226 D. ADRIAENS AND W. VERRAES

Fig. 24. Opercular bones of juvenile Clarias gariepi- foramen truncus hyomandibularis nervus facialis; intd- nus. A, preopercular bone, lateral view (125.5 mm SL hm, interdigitation with the os hyomandibulare; intd-q, stage); B, opercular bone, medial view (174.5 mm SL interdigitation with the os quadratum; I-an-iop,ligamen- stage); C, interopercular bone, medial view (174.5 mm tum angulo-interoperculare; PMC, preoperculo-man- SL stage). af-XIV, articulatory facet of the os operculare dibulare canal; prc-do, processus dorsalis; r-op, insertion with the os hyomandibulare; fr-tr-hm-b, border of the ridge on the os operculare for the m. levator operculi. passage of the hypobranchial artery, which the dorsal face of the parurohyal bone and runs anteriorly and enters the parurohyal penetrates it through the foramen. Ven- bone dorsally, in Trichomycterus areolatus trally, it immediately splits off two branches, and Noturus flavus (Ictaluridae). It then pen- as in N. flavus, but they run posteriorly, etrates the bone, leaving it ventrally. In T. instead of anteriorly. The terminology ‘‘hypo- areolatus, the artery runs anteriorly, split- branchial artery’’ is not adopted in the pre- ting into three branches at the anterior mar- sent study, as it appears that the artery gin of the hypohyals. In N. flavus, however, penetrating the parurohyal is not homolo- the artery splits into two, immediately after gous with the hypobranchial artery ob- it leaves the parurohyal (Arratia and Sch- served in other teleosts. In teleosts, the hypo- ultze, ’90: Fig. 19). Each branch then gives branchial artery always splits off from an off a lateral branch at the anterior margin of efferent branchial artery, in most cases the the hypohyals. The situation in Clarias gari- second one (Bertin, ’58; Goodrich, ’58). In C. epinus seems to differ even more (Fig. 13): a gariepinus, the hypobranchial artery splits ventral branch of the ventral aorta runs to off from the fourth efferent branchial artery OSTEOCRANIUM ONTOGENY IN CLARIAS 227 (Nawar, ’55). As this is not the case for the tostomus commersoni (Cypriniformes, Catos- artery penetrating the parurohyal bone in tomidae) (McElman and Balon, ’80) and C. gariepinus, it cannot be referred to as the Gadus morhua (Gadiformes, Gadidae) (Hunt hypobranchial artery. Consequently, the term von Herbing et al., ’96a). In Poecilia reticu- ‘‘arteria parurohyalis’’ is applied. lata (Cyprinodontiformes, Poeciliidae) ossifi- The absence of the subopercular bone is a cation already starts while the larva still lies synapomorphic feature of Siluriformes (Gos- within the egg membrane. Although no true line, ’73; Fink and Fink, ’81, ’96). This is also active respiration, by means of gills, is per- the case for Clarias gariepinus, where none formed, respiratory movements were al- of the ontogenetic stages revealed the pres- ready observed (Weisel, ’67). It appears that ence of any subopercular primordium. Sur- the alternating mechanical load onto the prisingly, a ‘‘small, rudimentary subopercu- opercular skin fold, early during ontogeny, lum’’ was observed in Plotosius canius may be related to the early ossification of the (Plotosidae) and Osteogeniosus militaris (Ari- opercular bone, prior to mechanical load ex- idae) (Bhimachar, ’33). erted by muscular contraction of the opercu- Sequences in bone formation as response lar muscles (Herring, ’93). The formation of to functional demands a substantial, horizontal rod in the opercu- lar bone in C. gariepinus may well be related The sequences in bone formation appear to the insertion of the levator operculi, as in to be related to functional demands that the adult situation, this rod corresponds to arise in developing larvae and juveniles. These demands tend to vary to a large ex- the levator operculi crest (Fig. 24B) (Adri- tent during the transformation from larva to aens and Verraes, ’97b). adult, with several vulnerable moments at Other ossifications related to the onset of which the functional demands, and struc- respiratory movements are the branchioste- tural adaptations to support these demands, gal rays, as they support the branchiostegal differ only very little (Galis et al., ’94). membrane in a similar way as the opercular The initiation of cranial ossification dur- bone supports the opercular skin fold (Fig. ing ontogeny in teleost fish is coupled to 25). The initiation of branchiostegal ray respiratory and feeding requirements. In ossification generally follows that of the oper- Clarias gariepinus, the opercular bone is the cular bone rather rapidly, e.g., in Clarias first one to appear (4.1 mm SL), supporting gariepinus, Heterobranchus longifilis (Vande- the opercular skin fold (Fig. 25). Opercular walle et al., ’97), Galeichthys feliceps (Tilney ossification starts at the articular facet of and Hecht, ’93), Chrysichthys auratus the opercular process of the hyosymplecti- (Vandewalle et al., ’95), Barbus barbus cum, as could be observed in other teleosts (Vandewalle et al., ’92), Danio rerio (Cub- (Verraes, ’73; Huysseune, ’85). From there bage and Mabee, ’96), Catostomus macrochei- on, the bone becomes a posteriorly extended lus (Weisel, ’67), Oncorhynchus mykiss (Ver- rod, bearing a ventral plate (Fig. 2B). Myo- raes, ’73), Poecilia reticulata (Weisel, ’67), logical data indicate that some of the muscles Oryzias latipes (Langille and Hall, ’87). In acting on the opercular bone arise between Gadus morhua, however, a time lapse is 5.2 and 6.8 mm SL. Although the adductor present between the two ossifications (Hunt and levator operculi are present at that von Herbing et al., ’96a). The gradually add- stage, it is only the levator muscle that al- ing of branchiostegal rays consequently cov- ready inserts onto the opercular bone (Adri- ers the support of the gradually increasing aens and Verraes, ’97b). However, prior to a branchiostegal membrane. The early differ- possible muscular displacement of the oper- entiation of a dorsal process of the opercular cular bone, the opercular skin fold is dis- bone in C. gariepinus seems to be related to placed by active respiratory movements, the insertion of the dilatator operculi muscle. which already seem to occur at the 5.2 mm At 7.2 mm SL the insertion of a very indis- SL stage (Surlemont et al., ’89). At this stage, tinct tendon of the dilatator onto the opercu- opening and closing of the mouth is ob- lar bone was overlooked in a previous study served, coupled to ad- and abduction of the (Adriaens and Verraes, ’97b), as in the serial cheek region. A similar correlation between sections, the tendon appeared to be the ante- the onset of opercular ossification and the rior ossification of the opercular bone. initiation of active respiratory movements Early ossifications involve most, but not was observed in Galeichthys feliceps (Siluri- all, dentulous bones. In Clarias gariepinus formes, Ariidae) (Tilney and Hecht, ’93), Ca- the premaxilaries, dental bones, and upper 228 D. ADRIAENS AND W. VERRAES

Figure 25 OSTEOCRANIUM ONTOGENY IN CLARIAS 229 pharyngeal tooth plates are present from tion to accommodate the arising functional the 6.0 mm SL stage on, whereas the initia- demands for the active uptake and manipu- tion of tooth formation could already be ob- lation of food (Vandewalle et al., ’94). Addi- served in the 5.6 mm SL specimen. The tionally, it was observed in Lates calcarifer other toothed bones, the lower pharyngeal (Centropomidae, Perciformes) that dentate and prevomeral tooth plates, arise at the 8.4 bones appeared only after a whole set of mm and 11.6 mm SL stage, respectively toothless bones (both dermal and perichon- (Table 2, Fig. 25). Mandibular elevation oc- dral), which seemed to be related to a shift in curs from the 5.2 mm SL stage on, as a feeding method as a response to prey size well-developed adductor mandibulae be- alteration during growth. Initially, bones re- comes functional (Surlemont et al., ’89; Adri- lated to suction feeding appear, followed by aens and Verraes, ’96). At the transition dentulous bones at the moment prey grasp- from endogenous to exogenous feeding, at ing is performed (Kohno et al., ’96a). In 7 mm SL, most tooth-bearing bones are Chanos chanos (Chanidae, Gonorhynchifor- thus present. A rapid increase in food uptake mes) dentition was not observed, even 150 occurs in the first few days of exogenous hours after mouth opening, at which feeding feeding, whereas once the feeding process appeared not to be of the suction/grasping has stabilized, the gastric content remains type, but rather of the ‘‘straining’’ type fairly constant (close to 21% of the body (Kohno et al., ’96b). It is believed that the weight) (Haylor, ’93). Early ossification of morphogenesis of certain bones is, in a way, the dentate bones could also be found in the influenced by the presence or absence of closely related Heterobranchus longifilis teeth (Huysseune, ’89). In C. gariepinus, the (Vandewalle et al., ’97). In Chrysichthys au- mentomeckelian bone is the first perichon- ratus, the dentary appears prior to the pre- dral bone to appear (Fig. 25). Presumably, it maxillaries and pharyngeal tooth plates, al- may assist in reinforcing the dentary plate though its dentition arises synchronously to the cartilaginous lower jaw, in order to be (Vandewalle et al., ’95). In Galeichthys feli- able to resist pressure forces more ad- ceps the dentaries also appear earlier than equately. the premaxillaries (13.9 mm and 18.8 mm The transition from endogenous feeding to TL, respectively) (Tilney and Hecht, ’93). In exogenous feeding implies that solid food cypriniform fishes, which lack any dentition particles have to be taken in and conse- on either the dentary or premaxillary (homo- quently transported from the oro-branchial plastic feature of Gonorhynchiformes and cavity into the esophagus. Along their course, Cypriniformes [Fink and Fink, ’81, ’96; Tav- these particles pass along the hypophyseal erne, pers. comm.]) the lower pharyngeal fenestra, which is very large in platybasic jaw, which is dentate, appears as one of the skulls. It is thus not surprising that from first ossifications, e.g., Danio rerio (Cubbage that moment on, the overlying brain has to and Mabee, ’96), or at least prior to the be protected more strongly from objects pass- premaxillaries, e.g., Barbus barbus (Vande- ing below than by the epithelial lining alone. walle et al., ’92), Catostomus macrocheilus A parasphenoid is formed, preventing pos- (Weisel, ’67) and C. commersoni (McElman sible damage to the brain, a little prior to the and Balon, ’80). This shift in sequence is an transition phase (Fig. 25). The supportive additional support for the supposition that role of the parasphenoid, as observed in sal- the sequence of cranial ossification is a reac- monids (Verraes, ’74), does not seem to be as critical as could be supposed at this moment, as the trabecular bars are still intact. The resorption of the middle part of these tra- Fig. 25. Chronological initiation of cranial ossifica- becular bars occurs at 11.6 mm SL. At that tions and certain related items (grey bars) in Clarias stage, the parasphenoid has become broader gariepinus. Because of the doubtful and late initiation of at its lateral wings, thus interconnecting the the epiotic bone, it has not been added to the figure. A, interrupted bars. However, its role in sup- mouth and opercular movements; B, transition from endogenous to exogenous feeding; C, cannibalism type I porting the central axis of the skull, together starts; D, four bar-system and digestive system become with the basioccipital, may not be ruled out functional; E, onset of air-breathing (first marker indi- completely (Weisel, ’67). Early ossification of cates the stage at which no ossification is observed; the the parasphenoid is a general feature in second marker indicates the presence, as well as the type of ossification). D, dermal, anamestic bone; DC, teleost fish, but when comparing its appear- dermal canal bone; PC, perichondral bone; S, sesamoid ance in relation to the standard length, it is bone, compound, compound bone. present in clariid species much earlier than, 230 D. ADRIAENS AND W. VERRAES e.g., in some silurids (Kobayakawa, ’92) and the hyoid artery (Verraes, ’75). As men- cyprinids (Vandewalle et al., ’92). However, tioned, tension forces are already exerted in some cases the parasphenoid develops onto this ligament from the 6.8 mm SL on: a even earlier, at lower standard length val- reinforcement may thus be required. The ues (e.g., some Cyprinodontiformes) (Weisel, anterior ceratohyal ossification may have a ’67; Langille and Hall, ’87). Surprisingly, in supportive function for the elongated carti- the guppy, Poecilia reticulata, the parasphe- laginous hyoid bar, especially at the inser- noid and several other bones are present tion site of muscles, which are already pre- long before hatching starts, and consequently sent and inserting onto it (Adriaens and long before active food uptake (Weisel, ’67), Verraes, ’97d). It may also support the in- whereas in the cyprinodont Oryzias latipes, creasing number of articulating branchioste- the parasphenoid is formed a little prior to gal rays. A similar relation has been ob- hatching (Langille and Hall, ’87). served in other teleosts (Verraes, ’75). The Exogenous feeding also requires a func- same can be suggested for the long branchial tional apparatus, which enables active up- skeletal elements: the cerato- and epibran- take of food particles. The sternohyoideus chials. An asynchrony in epibranchial ossifi- muscle is found to insert onto the hyoid bar cation in Heterobranchus longifilis is related at the 6.8 mm SL stage (Surlemont and to the formation of a suprabranchial organ Vandewalle, ’91). The paired, sesamoid parts (Vandewalle et al., ’97). Such an asynchrony of the parurohyal bone already can be ob- does not occur in Clarias gariepinus, which served a little earlier (6.6 mm SL). This indicates that it may be absent or that it is ossification of the tendons of the sternohyoid spread over a much shorter time span. is an adaptation to mechanical stress resis- Apart from overall reinforcement, the peri- tance, indicating a possible activity of that chondral ossification of the above noted muscle. As this is the case (Surlemont and structures may also be coupled to a change Vandewalle, ’91), a functional mouth open- in feeding behavior. It has been observed ing mechanism is present from this stage on: that Clarias gariepinus performs cannibal- contraction of the sternohyoideus results in ism extensively. Two types of cannibalistic the depression of the hyoid bar, which is behavior can be distinguished, based on body coupled to the depression of the lower jaw length and the way prey items are swal- through the protractor hyoidei muscle and/or lowed (Hecht and Appelbaum, ’87). Type I the ligament between the lower jaw and the cannibalism starts at 8 mm total length hyoid bar (Osse, ’69; Winterbottom, ’74; Ver- (corresponds to about 7.5 mm SL) until 45 raes, ’77; Lauder, ’80; Lauder and Liem, ’80; mm TL, and is characterized by capturing Aerts, ’91). the prey by the tail, swallowing it up to the Once the primary functional demands are head, followed by biting off of the head. This dealt with, the overall skull must become implies that large forces have to be produced reinforced as it gradually becomes larger. by the adductor mandibulae complex, and Quite simultaneously, a whole set of peri- consequently that a high mechanical stress chondral bones are formed, which enable is exerted onto the jaws. A well-developed such a reinforcement (Fig. 25). These ossifi- adductor mandibulae complex is present at cations include both neurocranial elements, 7.2 mm SL (Adriaens and Verraes, ’96). The as well as splanchnocranial parts. The first ossification of the quadrate may thus be an neurocranial ones involve the reinforcement adaptation to resist large pressure forces of the attachment of the skull to the noto- during biting. chord (basi- and exoccipital bones). Suspen- Once exogenous feeding has become obliga- sorial ossifications appear at articular fac- tory and cannibalism has started, mouth ets, which already have become functional: opening also becomes more and more impor- the quadrate for the mandibular articula- tant. The mouth-opening mechanism, noted tion and the hyomandibula for neurocranial earlier, involves the retraction of the hyoid and the opercular articulation. All these ar- bar through the contraction of the sternohy- ticulations become increasingly important oideus. This muscle interconnects the hyoid in feeding and respiratory movements. The bar with the pectoral girdle, at the level of ossification of the ventral hypohyal may be the cleithral bone. During contraction of the related to the ligamentous insertion, which sternohyoideus, the cleithrum is held in po- connects the hyoid bar to the parurohyal, as sition, or even retracted through the hypaxi- well as the protective role of that bone for als (Osse, ’69; Lauder, ’80; Lauder and Liem, OSTEOCRANIUM ONTOGENY IN CLARIAS 231 ’80; Muller, ’87). Fixation of the cleithral tion between suspensorium and the ethmoid bone to the neurocranium, through an articu- region, through the palatine, is absent (Alex- lation, can be advantageous for this kind of ander, ’65; Gosline, ’75; Fink and Fink, ’81, mechanism. The formation of the posttem- ’96; Arratia, ’92). However, in most other poro-supracleithral bone can consequently teleosts, the entopterygoid appears prior to assist in the reinforcement of the hyoid the metapterygoid (Weisel, ’67; Verraes, ’73; mouth opening mechanism. Vandewalle et al., ’92; Cubbage and Mabee, The gradual strengthening of the man- ’96), although it is considered not to be of dibular articulation follows the onset of the sesamoid origin (Arratia, ’92). cannibalistic behavior (Fig. 25). Rather si- The following bones that are formed in- multaneously, the angular, articular, and ret- volve canal bones of the skull roof. A little roarticular bones are formed. The articular earlier, canal bones of the mandibular sen- facet of the initial bony mandibula is thus sory canal were already formed, possibly reinforced in order to withstand the increas- coupled to the early ossification of the other ing pressure during biting off catfish heads. mandibular bones. Apparently, the skull roof The transport of catfish bodies, however, is bones arise in close contact with the underly- also improved, as the lower pharyngeal tooth ing cartilaginous skull, although they are of plate is present. As the muscles acting onto dermal origin (cfr. ‘‘parachondral bone’’) (Ver- them are present, both pharyngeal jaws can raes, ’74; Verraes and Ismail, ’80). Initially, now become functional. This plays a crucial the chondrocranium becomes bordered, al- role in food fragmentation and transport most along its whole length: the frontal bone from the orobranchial chamber into the in the orbito-temporal region, followed by esophagus (Vandewalle et al., ’94). This the dermosphenotic, and eventually the der- transport also requires a subsequent suction mopterotic, which closes up the space be- activity, in which the hyoid bar plays an tween the dermosphenotic and the posttem- important role (Liem, ’90; Aerts, ’91). The poro-supracleithral bone. Consequently, the posterior ossification of the hyoid bar conse- main sensory canal is completely enclosed quently contributes to its additional rein- and protected. Such a close synchrony be- forcement, especially for the attachment of tween the ossification of these bones is found ligaments (ligamentum angulo-ceratohyale in many other teleosts (Kobayakawa, ’92; and ligamentum hyomandibulo-ceratohy- Vandewalle et al., ’92, ’95; Cubbage and ale). Mabbe, ’96; Vandewalle et al., ’97). In On- The ossification of the entopterygoid bone corhynchus mykiss, the frontal appears well in Clarias gariepinus, and perhaps other in front of the dermosphenotic and dermop- siluriform fish, may be related to increasing terotic bones (Verraes, ’73). The preopercu- tensions that are exerted onto the ligament, lar part of the preoperculo-mandibular ca- interconnecting the suspensorium with the nal is also protected at that moment, as the ethmoid region. As is proposed by Arratia preopercular bone encloses it. (’92), this entopterygoid is of sesamoid ori- Shortly after, two unpaired, bony com- gin, being the ossification of the middle part plexes are formed, covering the left and right of the formerly mentioned ligament. Miner- sides of the chondrocranium in the ethmoid alization is presumably induced by mechani- region and the occipital region, respectively. cal stress, whereas bone formation is in- In that way, the paired bones formed before duced at sites that experience an alternating are now closed off anteriorly and posteriorly mechanical load, e.g., bending, pressure, ten- by the mesethmoid and the parieto-supraoc- sion, torsion (Herring, ’93). The increasing cipital bones, respectively. Again, the der- use of mouth opening and mouth closure, as mal parts of these bones arise in close con- well as orobranchial expansions for feeding tact with the cartilage, which has become and respiratory purposes, will increasingly perichondrally ossified. At this moment, all load the suspensorium. Powerful biting will skull roof bones are present, and from here exert forces onto the suspensorium in an on, they can start enlarging medially and anteroposterior direction. As the articular laterally to close off the still unprotected facet of the suspensorium with the neurocra- part of the brain. Additionally, the increased nium is not yet secured, this ligament may contraction load on the insertion sites of contribute in restraining the suspensorium several muscles onto these bones, must play from being pulled backward, especially since an important role (e.g., levator arcus pala- in Siluriformes, in general, the direct connec- tini, dilatator operculi, levator operculi). 232 D. ADRIAENS AND W. VERRAES The development of the prevomeral tooth drag. This implies that forces, needed for plate coincides with the formation of the movements of the barbel, will have to in- opercular four bar-system, which is formed crease correspondingly, which involves in- at 11.1 mm SL. This bar-system plays a creasing load onto the skeletal elements of crucial role in mouth opening in many adult the palatine-maxillary mechanism. The max- teleosts (Liem, ’70; Aerts and Verraes, ’84; illary bone, enclosing the base of the barbel, Verraes, ’77; Westneat, ’90). A possible func- bears two well-developed articular facets, tional relation between an improved mouth for articulation with the anterior tip of the opening for food uptake and the ossification palatine (Adriaens and Verraes, ’97a). The of additional tooth plates is supported by the palatine itself becomes rod-like, articulating fact that at about this length (11.5 mm SL) medially with the ethmoid region (at the the digestive system becomes completely level of the orbito-nasal lamina). Posterior to functional (Fig. 25) (Verreth and Van Ton- this articulation, the extensor tentaculi geren, ’89; Haylor, ’95). This allows improve- muscle connects the palatine to the neurocra- ment of the carnivorous behavior of Clarias nium. It can thus be expected that autopala- gariepinus, as is demonstrated by the al- tine ossification provides: (1) a reinforced ready ongoing cannibalism (at 8 mm TL) insertion site for the extensor tentaculi (Hecht and Appelbaum, ’87). The increase in muscle, and (2) support for the rigidity of the prey size involves, apart from an improved palatine, in order to withstand bending mouth opening, an elevated load onto sev- forces, which would inactivate movement of eral elements like the lower jaw, and the the barbel in a rotating type of palatine- suspensorial and branchial elements. In- maxillary mechanism. creased tension on the tendon of the adduc- The last sensory canal to become enclosed tor mandibulae onto the lower jaw can be and protected by bones is the infraorbital related to the induction of its ossification at canal, as the antorbital and lacrimal bones that stage, i.e., the coronomeckelium. The are present at 11.6 mm SL. Late develop- suspensorium and the branchial basket ele- ment of these bones is probably related to ments enable orobranchial expansion, as well the late development of the infraorbital ca- as aid in prey transportation to the pharyn- nal itself, in relation to the other canals, as geal jaws. Reinforcement of the pterygoid occurs in other ostariophysan fishes (Le- process, at the level where the ligament kander, ’49). However, very early ossifica- running to the ethmoid region is attached, tion of the antorbital was observed in Galei- also occurs through ossification of the chthys felis (Siluriformes, Ariidae) (Bamford, metapterygoid. Manipulation of larger prey ’48). Possibly, the infraorbital canal is more requires that larger forces be exerted by protected between the eye and the man- those elements that enable orobranchial dibula than are the other canals. Ossifica- transport. Increased muscular activity onto tion of infraorbital canal bones proceeds an- the branchial floor may be related to ossifica- terocaudally. In other ostariophysans, the tion of the basibranchials and hypobranchi- antorbital bone develops earlier than the als at the level where muscles insert onto infraorbitals, which seems to be the case in them. Clarias gariepinus as well, although not so The active hunting behavior of Clarias distinct (Lekander, ’49). In relation to the gariepinus, although it is also able to per- supraorbital canal, the nasal bone is the last form filter feeding (Groenewald, ’64; Teu- to develop (Adriaens et al., ’97). The remain- gels, ’86), involves the use of well-developed ing exposed part of the preoperculomandibu- barbels for prey location, as well as for sam- lar sensory canal also becomes enclosed, as pling of potential food (Alexander, ’65, ’66; the suprapreopercular bone arises between Gosline, ’73). Although the muscular control the preopercular and the pterotic bones. of most of these barbels is restricted (Ghiot, As the chondrocranium enlarges, the unos- ’78; Adriaens and Verraes, ’97d), the maxil- sified space of the orbito-temporal region of lary barbel can be moved extensively, due to the skull floor expands. Fortification through the palatine-maxillary mechanism (Alex- ossification may consequently become neces- ander, ’65; Gosline, ’75; Adriaens and Ver- sary, as is demonstrated by the development raes, ’97a). In C. gariepinus, the maxillary of the pterosphenoids, the lateral ethmoids, barbel is rather large (up to 174% head and later, the orbitosphenoids. Additional length [Teugels, ’86]). The protraction and bridge formation between the pterosphenoid retraction of it will experience a substantial and parasphenoid contributes to reinforce- OSTEOCRANIUM ONTOGENY IN CLARIAS 233 ment of the sphenoid fenestra, which conse- believed to withstand re- and protraction quently becomes subdivided into two. Such a forces to a greater extent (Barel, pers. comm.; reinforcement is present in most catfish, as Adriaens and Verraes, ’77b). In Clarias gari- mentioned above. Important to mention is epinus, however, the articular ridge is rein- that in the siluriform chondrocranium, the forced through the formation of interdigita- commissura lateralis, which forms the lat- tions between the hyomandibular bone and eral wall of the trigemino-facial chamber the neurocranial bones, thus allowing lat- and subdivides the sphenoid fenestra, is ab- eral swinging of the suspensorium during sent (de Beer, ’37; Daget, ’64; Alexander, ’65). respiration and feeding (Adriaens and Ver- The absence of such a support between the raes, ’77b) but simultaneously prohibiting skull roof and skull floor is compensated by anteroposterior displacement during biting. this secondary, but bony, bridge. An even more extreme interdigitation can be The late ossifications of the third and found in some anguilliform clariids, which fourth infrapharyngobranchials can be also possess an enormous adductor mandibu- coupled to the increasing load exerted onto lae complex (Cabuy, pers. comm.). these elements. They play a crucial role in Variation in ossification sequence: a result the support of the upper pharyngeal jaws. of variation in functional demand? Additionally, muscles needed for manipula- tion of these jaws insert onto them. Ossifica- In their work, Mabee and Trendler (’96) extensively described the ossification se- tion of the dorsal hypohyal does not seem to quence of Betta splendens (Belontiidae), have any direct mechanical induction, as no which they compared with data from litera- muscles or ligaments insert onto it. Late ture of Oryzias latipes and Barbus barbus. It ossification may be related to the absence of appeared that the ossification sequence in a basihyal in siluriform fishes (Arratia and teleosts comprises sequences that are con- Schultze, ’90). served during evolution, whereas other se- Formation of the last canal bones, the quences have altered. Consequently, differ- splenials, is presumably related to the in- ent conserved sequence pairs, meaning a creasing length of an unprotected part of the fixed sequence of two bones within all the preoperculomandibar canal, with increasing teleosts studied, were present. As they men- body length. At 46.8 mm SL, one of these tioned that ossification sequence statistics bones could be found, whereas the number do not reflect a strict taxonomic coherence, reached three in larger specimens. In that other factors must be responsible. One of the way, the sensory canal becomes enclosed and possible determinants of ossification se- protected in a manner that still allows flex- quence can be the difference in functional ibility at that point, close to the mandibular demands of the different taxa. Differences in joint. early life histories of fish, reflected, for ex- Once the essential ossifications are pre- ample, by the difference in feeding type and sent, the increasing mechanical load onto food type, can play an inductive role in the the body parts, as body length and conse- differentiation of a certain set of bones quently also muscle size, contraction (Kohno et al., ’96a,b). For suction feeding strength, prey size, etc., increase, can be fish, other bones will of importance during supported by thickening of the bones. In initial feeding than for those performing suc- juveniles, the bones become rather thick tion-grasping feeding. Mechanical load will through spongy bone formation. The inter- act on different bones if feeding involves oral digitation becomes extremely complex, rein- manipulation, when compared with feeding forcing the connection between separate by pharyngeal jaw manipulation. Undoubt- bones. The skull roof becomes gradually edly, the ossification sequence has a genetic closed off completely, as a decrease of fonta- basis. However, this basis is subjected to nella size has been observed in larger speci- interspecific, as well as intraspecific varia- mens. A splendid example of additional rein- tion (Mabee and Trendler, ’96). Intraspecific forcement against mechanical load is the variation can in turn be seen as a response locking device of the suspensorium. Most to intraspecific variation in functional de- ostariophysans, i.e., Characiformes, Siluri- mands of developing larvae. formes and Gymnotiformes, have an articu- To conclude, some suggestions can be made lar ridge, instead of two articular condyles, concerning the relation of ossification se- for articulation with the neurocranium quence and the presence of certain func- (Arratia, ’92). Two articular condyles are tional demands. It seems clear that early 234 D. ADRIAENS AND W. VERRAES ossifications can be related to primordial Adriaens, D., and W. Verraes (1997d) Ontogeny of the respiration, as well as the formation of a hyoid and musculature in Clarias gariepinus, an Afri- can catfish (Burchell, 1822) Siluroidel: Clariidae). Zool. primordial feeding apparatus during the J. Linn. Soc 120:105–128. transition phase from endogenous to exog- Adriaens, D., W. Verraes, and L. Taverne (1997) The enous feeding. As larvae grow, the overall cranial lateral-line system in Clarias gariepinus mechanical load, related to skull support, (Burchell, 1822) Siluroidei: Clariidae): morphology and development of canal related bones. European J. Mor- muscle insertion, prey size, etc., will also phol. 35:1–28. increase. Such demands are coped with in Aerts, P. (1991) Hyoid morphology and movements rela- the growing Clarias gariepinus larvae as tive to abducting forces during feeding in Astatotila- fortifications arise, especially at the level of pia elegans (Teleostei: Cichlidae). J. Morphol. 208:323– articulations related to the feeding appara- 345. Aerts, P., and W. Verraes (1984) Theoretical analysis of a tus. Protection of the brain from the outside planar bar system in the teleostean skull: the use of initiates at the skull floor, prior to a dorsal mathematics in biomechanics. Annls. Soc. r. Zool. Belg. protection, which may be related to initial 114:273–290. food transport. 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