803

THE HYOID O F NEOCERSTODUS AND A CONSIDERATION OF ITS HOMOLOGY IN URODELE AMPHIBIA

BY

HAROLD FOX

Department of Zoology and Coniparative Anatomy, University College London [Accepted 9th October, 19621

(With 2 plates)

The early development of the hyoid of Neoceratodus is described, and its structure and topography are compared with relevant components in various urodele larvae. These include ; Hymbius nebuloaua, Hynobius (Salamandrelkz) keyaerlingii, Hymbiua retardatus, Cryptobranchus japonicus, Cryptobranchus alleghaniensia, Salamandra ?naGUl08a, Ambystoma mexicanum. Necturus maculatua, Desmognathus fuscus fuscua, Deamognathw quadrimacu- latus, and Plethodon cinereua.

It is concluded that in Neoceratodua the stylohyale develops from the quadrate cartilage, and the otoquadrate cartilage derives from a pharyngohyale, which is recognized in urodeles as a post-palatine cartilage. The columella (and stilus) of the latter co-exists with a pharyngohyale, and thus may either derive from the hyoid blastema aui generia, or alter- natively from an epihyale-laterohyale component of an ancestral stock common to Dipnoi and Urodela.

CONTENTS

Introduction . . .. .. .. .. Material and methods . . .. .. .. Results . . .. .. .. .. .. The development of the hyoid of Neoceratodwr The otoquadrate cartilage and columella of urodeles Discussion . . .. .. .. .. .. Summary .. .. .. .. .. .. Acknowledgments .. .. .. .. References . . . . . . .. .. .. Explanation of plates . . .. .. ..

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INTRODUCTION

Urodeles unlike fishes possess a columella, considered to derive from the top of the hyoid (Kingsbury & Reed, 1901 ; Marcus, 1910 ; de Beer, 1937), but knowledge of its homologue in Neoceratodus is still recondite. In order to contribute to a solution of this problem the hyoid and some associated structures, in young specimens of Neoceratodus were examined, and comparisons were made with similar structures in a number of urodele larvae,

The evidence presented in this work from comparative morphology does not encourage the view that the columella of urodeles is the homologue of the otoquadrate (or pharyngohyale) of Neoceratodus.

804 HAROLD FOX

MATERIAL AND METHODS

Specimens investigated (the total lengths are included in brackets) include : Neoceratodus (11.5 mm, 13.5 mm, 14 mm, 15 mm, 16 mm, 27 mm., 34.5 mm- see Fox, 1963) ; Hynobius nebulosus (14 mm; 17 mm, 22 mm, 32 mm) ; Hynobius returdatus (27 mm, 37 mm,) ; Hynobiw (Salamandrellu) keyserlingii (18 mm, 19 mm, 21 mm) ; Cryptobranchusjaponicw (26 mm, 30 mm, 32 mm) ; Cryptobranchus alleghuniensis (37 mm) ; Salamandra muculosa (21 mm, 23 mm, 24 mm, 25 mm, 26 mm) ; Ambystoma mexicanum (13 mm, 17 mm, 21 mm, 25 mm) ; Necturus maculutus (22 mm, 24 mm, 29 mm) ; Desmognathus fuscus fwcus (28 mm) ; Desmognuthus quadrimaculutw (18 mm) ; Plethodon cinereus (17 mm, 19 mm).

All specimens were sectioned transversely at 10 microns, and variously stained with a number of common histological dyes (Fox, 1962).

RESULTS

The development of the hyoid of Neoceratodus The presumptive hyoid is already recognizable in an 11.5 mm larva, and

consists of a homogenous mass of darkly staining closely packed procartilaginous cells, with large nuclei and little yolk. It is situated behind the hyoid cleft and meets its opposite number ventrally in the mid line. The blastema extends dorso-laterally, behind the presumptive quadrate and Meckel’s cartilages, separate from the as yet unchondrified basal plate (Pl. 1 , fig. A). In a 13.5 mm specimen a small separate chondrified portion of the top of the hyoid blastema is in incipient cartilaginous connection with the now chondrified basal plate ; below the front of the chondrified auditory capsule (Pl. I, fig. B), and procartilaginous hyoid blastoma cells connect the top of the ceratohyale to this incipient chondrification-henceforth called the otoquadrate cartilage (Edgeworth, 1923, 1935).

Chondrified and demarcated ceratohyale, hypohyale and a median ventral basihyale are now recognizable. At 14 mm the otoquadrate is similarly arranged but larger than in the previous stage.

I n a 15 mm specimen cartilaginous continuity exists between the otoquad- rate and the outer edge of the basal plate, and from the upper surface of the ceratohyale dorso-laterally directed hyoid blastema cells are seen to be con- tinuous with the outer edge of the otoquadrate. Because of the relationship of this blastema to the otoquadrate it is henceforth called the epihyale (Pl. 1 figs. C, D), for in Neoceratodw 16 mm long there is some incipient chondrification.

The otoquadrate has been called a hyomandibula (Furbringer, 1904), or a pharyngohyale (Schmalhausen, 1923), and the more lateral epihyale has likewise been termed a hyomandibula, a symplectic or an interhyale (de Beer, 1937 ; see Bertmar, 1959).

In the 16 mm specimen a small cartilaginous protuberance is recognised on both sides, originating from the hind surfaoe of the quadrate cartilage and directed backwards, mesially to the ramus mentalis externus facialis - the nerve passes over it - in cellular continuity with the top of the ceratohyale (Pl. 1, figs. E-G). A separate epihyale is situated above the region of junction

THE HYOID OF NEOCERATODUS 805

of the backwardly directed quadrate process and the ceratohyale cartilages, and leading from the epihyale and extending postero-laterally to the levator hyoideus muscle, a small undemarcated laterohyale process is recognized.

When Neoceratodus is 27 mm long the hyoid components include a median ventral basihyale (probably derived from paired elements of either side), a hypohyale and a ceratohyale ; the latter in cartilaginous continuity with the previously mentioned backgrowth of the quadrate cartilage. The latter hind process is now larger and ultimately in a 34.5 mm specimen is recognized as a separate stylohyale, occupying a similar position as the same element illustrated by Bertmar (fig. 75, 1962) in his 34.5 mm specimen.

An epihyale leads dorso-posteriorly and its laterohyale portion, though still not yet demarcated, is similar though larger, than that in the 16 mm specimen. It extends below the crista parotica, mesially to the squamosal and laterally to the levator hyoideus (Pl. 2, figs. H, I). The uppermost otoquadrate cartilage extends outwards from the basal plate, and in front joins the inner surface of the quadrate cartilage. It ends behind as a small protuberance, but in the 34.5 mm specimen it fuses in front with the hind surface of the processus basalis, and behind with the ventral surface of the auditory capsule (Pl. 2, fig. H).

I n the 11.5 nim stage the vena capitis lateralis, or head vein, is situated dorso-laterally and the internal carotid artery mesially to the top of the hyoid blastema, the levator hyoideus muscle laterally and post-laterally, and the ramus hyomandibularis facialis leads outwards, over and hence lateral to it.

In later stages the facial nerve, vena capitis lateralis and the stapedial artery are situated above the otoquadrate cartilage, the two latter blood vessels being situated below and above the nerve respectively ; the internal carotid artery leads forwards below its inner margin (Pl. 2, fig. H).

The hyoid of a 34.5 mm larvae of Neoceratodus comprises a pharyngohyale (otoquadrate), epihyale and its laterohyale extension, stylohyale (probably derived from the quadrate cartilage), ceratohyale, hypohyale and basihyale. The arrangement of the recognizable components is similar to that illustrated in transverse section by Bertmar (1962), though in this present work no demarcated laterohyale is yet recognized, but development is known to be variable (de Beer, 1937).

The otoquadrate cartilage is considered to represent a compound infra- pharyngohyale-suprapharyngohyale, homologous with the lateral commissure of teleostomes (Bertmar, 1959).

The otoquadrate cartilage and columella of urodeles A well developed otoquadrate cartilage, similar in form and in position to

that of Neoceratodus, is recognizable in all urodeles examined, with the exception of young larvae of Hynobius nebulosus (14 mm), and Ambystoma mexicanum (13 mm), and all stages of Necturus smaller than the 29 mm specimen (Pl. 2, figs. J, K, M, N).

In a 32 mm Hynobius nebulosus or in Salamundra muculosa the otoquadrate originates mesially from the junction of the inner margin of the auditory capsule

806 HAROLD FOX

and the basal plate, to lead postero-laterally below the ramus hyomandibularis facialis, vena capitis lateralis and the stapedial artery ; the internal carotid artery is situated mesially or mesio-ventrally to it (Pl. 2, fig. K) (FOX, 1959). A reduced otoquadrate is recognized in Cryptobranchus japonicus, Desmognathus quadrimaculatus and Plethodon cinereus in the same position. It has a similar topographical relationship to nerves, blood vessels and other associated structures as in the other specimens, but does not extend backwards below the auditory capsule, for it comprises the mesial and posterior boundary of the facial foramen and the ventro-mesial surface of the auditory capsule ; between the latter and the basal plate at about the level of the foramen abducentis.

The otoquadrate cartilage is in fact incorporated into the mesial floor of the auditory capsule. In Neoceratodus, in contrast, it extends below a complete cartilaginous capsule floor (Pl. 1, fig. D, P1. 2, fig. H ; see fig. 453, Goodrich, 1930).

The otoquadrate is ossified in Desmognathus fuscus fuscus. Furthermore, as seen typically in Hynobius, Salamandra and Necturus, after the ramus palatinus facialis has emerged through the palatine foramen, to lead forwards below the basal process, the front inner end of the otoquadrate fuses with the median boundary of the floor of the auditory capsule against the outer edge of the basal plate, to separate the ramus hyomandibularis facialis and the ramus palatinus facialis.

If indeed the urodele otoquadrate is the homologue of its namesake in Neoceratodus, then this post-palatine cartilage, incorporated in part into the floor of the auditory capsule mesial to the facialis foramen, is in reality the top of the hyoid, or the pharyngohyale of Neoceratodus.

A chondrified columella auris is juxta-posed against the foramen ovale in specimens of Hynobius nebulosus 22 mm and 32 mm long (Pl. 2, fig. L), Hynobius retardatus. H . (Salamandrella) keyserlingii and Salamandra, 30 mm and 32 mm specimens of Cryptobranchus japonicus, Cryptobranchus alleghaniensis (PI. 2, fig. 0), 24 mm and 29 mm Necturus muculatus, 25 mm Ambystomu mexicanum (though the columella cartilage is here incipient), Desmognuthus fuscus fuscus, and Plethodon cinereus (Fox, 1954, 1959, 1962). In all other specimens it is either procartilaginous, or not yet apparent in serial section.

A cellular stilus leads forwards from the lateral front end of the columella in a 22 mm Hynobius nebulosua, Hynobius retardatus, Cryptobranchus japonicus, a specimen of Necturus muculatus 29 mm long, and in Desmognuthus fuacus fuscus but it is not yet recognizable in Salamandra maculosa, Hynobius (Salamandrella) keyserlingii and Ambys tma mexicanum. The stilus is chondrified in a 32 mm Hynobius nebulosus and in Cryptobranchus alleg- haniensis (PI. 2, figs. L, 0), and in a 19 mm Plethodon cinereus. Chondrified or non-chondrified cells of the stilus seem to terminate against the base of the squamosal, just behind the quadrate cartilage, except in Plethodon, where the front end is in almost complete cartilaginous continuity with the quadrate cartilage. In the 32 mm larva of Cryptobranchus japonicus and in Cryptobranchus alleghaniensis merely 50 microns of procartilaginous tissue separates stilus and quadrate, and in the 32 mm Hynobius nebulosus cartilaginous union is complete (Fox, 1959).

THE HYOID OF NEOCERATODUS 807

The vena capitis lateralis lies ventrally to the columella in a 14 mm Hynobius nebulosus, but in older specimens it is usually situated either ventro-laterally, laterally, but in most cases dorso-laterally or dorsally to it. A main venous division lies above and another below it in Cryptobranchus. The internal carotid artery is usually situated ventrally or ventro-mesially, the ramus hyomandibularis facialis anteriorly, the digastricus laterally and the squamosal antero-laterally to the columella.

Similarly the vena capitis lateralis lies dorsally and thence mesially to the columella stilus, the internal carotid ventro-mesially to it below the auditory capsule and the digastricus laterally. A stapedial artery, from the lateral dorsal aorta, continues forwards mesially to the stilus, lateral to the head vein and over the facialis nerve, to lead mesially to the otic process, through the cranio-quadrate passage to the orbit. The facialis nerve emerges ventrally to the cellular or chondrified stilus, in all forms examined, except in Necturus where the ramus jugularis facialis crosses over it, though components of the ramus mandibularis facialis are situated below (Fox, 1954).

DISCUSSION

The ontogeny of the hyoid of Neoceratodus has been fully and excellently described by Bertmar (1959), from egg stages to larvae 34.5 mm long. I n general, with merely one exception, the descriptions of Bertmar and in this work are in substantial agreement. I n a 24 mm specimen Bertmar describes the laterohyale and epihyale to chondrify in connection to form a club-shaped element (hyomandibula), the epihyale forming the shaft. At 34.5 mm occasionally there are separate chondrifications. Furthermore the stylohyle derived in major part from the hyoid blastema, and in part from a subepidermal blastema. From the results in this present work however, it is concluded that a stylohyle originated in the 16mm specimens (three were examined) as a backgrowth of the quadrate cartilage, which eventually in a 34.5 mm specimen had separated from the latter to become incorporated into the hyoid, between the ceratohyale below and the epihyale above. It is significant that in a larval stage of Neocerutodus somewhat older than the Semon stage 47 (15.7 mm long, see Edgeworth, 1923), Sewertzoff (fig. 2B, 1902) illustrates a connection between the quadrate cartilage and the ceratohyale, below the hyomandibula, and writes ; '' Das Quadratum hat einen Fortsatz, welcher zur Angliederung des hyoids dient. ''

Nevertheless contribution to the stylohyale by cells of the hyoid blastema cannot be ruled out, for the region where the stlyohyale is recognized is within the hyoid blastema field. The two described types of development could be reconciled by assuming a close association of presumptive quadrate and hyoid blastemata ; chondrification within the latter proceeding from the hind surface of the quadrate backwards, to attain its recognized location as a separate stylohyoid cartilage.

In urodeles a columella and stilus co-exist in space and time with an otoquadrate cartilage (Pl. 2, figs, K, L), a result which seems to preclude a view that the urodele columella apparatus is the homologue of the pharyngo- hyale of Neoceratodus.

808 HAROLD FOX

However a columella and an epihyale-laterohyale are hyoidal elements which never co-exist in urodeles. The latter joins the quadrate cartilage by a stylohyale in Neoceratodus, and the former cartilage joins the quadrate by a cartilaginous or cellular stilus in urodeles, Both columella and epi-laterohyale are situated mesially to the squamosal.

In Neoceratodus a retractor mandibulae muscle depresses the lower jaw, but in urodeles a large digastricus (depressor mandibulae) muscle develops from the levator hyoideus to fulfill the same purpose (Schmalhausen, 1955). In the phylogeny of tetrapods the procartilaginous epi-laterohyale blastema may thus have extended backwards mesially to the digastricus, instead of laterally to the levator hyoideus as in Neoceratodus, thus to chondrify in the typical columella situation, although still retaining its connection with the quadrate cartilage by the stilus. In order for the columella to become a sound conducting apparatus, against the foramen ovale of the auditory capsule in the presence of a large digastricus, it would of necessity need to be situated mesial to the latter. The particular relationships of the associated blood vessels and nerves would be a consequence of this development.

SUMMARY

1. The stylohyale of Neoceratodus develops from the quadrate cartilage. 2. The otoquadrate cartilage of Neoceratodus derives from a pharyngohyale,

which is recognized in urodeles as a post-palatine cartilage. 3. The columella (and stilus) of urodeles co-exists with a pharyngohyale

and thus may derive either from a hyoid blastema sui generis, or alternatively from the epi-laterohyale component of an ancestor common to Dipnoi and Urodela. The altered topographical situation of the columella, to become a sound conducting organ, may be in part influenced by the phylogenetic devel- opment pari passu of a large depressor mandibulae, derived from the levator hyoideus muscle.

ACKNOWLEDGMENTS

It was most kind of Professor H. Gruneberg of the Department of Genetics, University College, to provide facilities to produce the photomicrographs used in this work. These were prepared by Mr C. Atherton whose assistance is warmly appreciated.

REFERENCES

BERTMAR, G. (1959). On the ontogeny of the chondral skull in Characidae with a discussion on the chondrocranial base and the visceral chondrocranium in fishes. Acta zool., Stockh.

BERTMAR, G. (1962). Homology of ear ossicles. Nature, Lond. 198 : 393-4. DE BEER, G. R. (1937). The development of the vertebrate akull. Oxford University Press. EDQEWORTR, F. H. (1923). On the development of the hypobranchial, branchial and laryngeal

muscles of Ceratodua. With a note on the development of the quadrate and epihyale. Quart. J . micr. Sci. 67 : 326-368.

40 : 203-364.

EDGEWORTH, F. H. (1935). Fox, H. (1954). Development of the skull and associated structures in Amphihia with special

The cranial muaclea of vertebratea. Cambridge University Press.

reference to the urodeles. Trans. zool. SOC. Lond. 28 : 241-304.

THE HYOID OF NEOCERATODUS 809

Fox, H. (1959). A study of the development of the head and pharynx of the urodele Hyrwbiva and its bearing on the evolution of the vertebrate head. Phil. Tram. B. 242 :

A study of the evolution of the amphibian and dipnoan pronephros by an analysis of itJs relationship with the anterior spinal nerves. Proc. zool. SOC. Lond.

151-204. Fox, H. (1962).

188 : 225-256. Fox, H. (1963). Prootic anatomy of the Neoceratodus larvae. Acta anat. 52 : 126-129 FURBRINGER, K. ( 1904). Beitrage zur Morphologie des Skelettes der Dipnoer, nebst Bemerkungen

iiber Pleuracanthiden, Holocephalen, und Squaliden. Semon’a Forachungar. I. Lief. IV : 425-510.

GOODRICH, E. S. (1930). Studies on the structure and development of oertebrates. London : Macmillan.

KINGSBURY, B. F. & H. D. REED. (1909). The columella auris in amphibia. J . MOT& 20 :

MARCUS, H. (1910). Beitrage zur Kenntnis der Gymnophionen. 111. Zur Entwicklungsgeschichte

SCHMALHAUSEN, J. J. (1923). Der suspensorialapparat der Fische und das Problem Gehorkno-

SCHMALHACSEN, J. J. (1955). Development of the visceral musculature of tailed Amphibia.

SEWERTZOFF, A. N. (1902). Anat. Anz.

549-628.

des Kopfes. MOT&. Jb. 40 : 105-183.

chelchen. Anat. Anz. 56 : 534-543.

Zool. Zh. 84 : No. I : 162-174. Zur Entwickelungsgeschichte des Ceratodus forsteri.

21 : 598-608.

EXPLANATION OF PLATES

All photomicrographs are of transverse sections 10 p thick.

PLATE 1

Sections are of Neoceratodus, mostly featuring views from the left side except for figure G, which is of the right. Fig. A-Photomicrograph of an 11.5 mm specimen (nose-to-cloaca1 length 7.88 mm) ; 1.23 mm from the tip of the snout, at the level of the presumptive hyoid arch. Fig. B-Photomicrograph of a 13.5 mm specimen (nose-to-cloaca1 length 8.45 mm) ; 1.46 mm from the tip of the snout, at the level of the origin of the otoquadrate cartilage from the top of the hyoid blastema. Fig. C-Photomicrograph of a 15 mm specimen (nose-to-cloaca1 length 9.09 mm) ; 1.64 mm from the tip of the snout, a t the level of the origin of the epihyale blastema. Fig. D-Photomicrograph of same specimen as in figure C, 1.73 mm from the tip of the snout, a t the level of the incipient cartilaginous otoquadrate cartilage. Fig. E-Photomicrograph of a 16 mm specimen (nose-to-cloaca1 length 10.28 mm) ; 2.08 mm from the tip of the snout, a t the level of the origin of the cartilaginous backgrowth of the quadrate cartilage. Fig. F-Photomicrograph of same specimen as in figure E , 2.11 mm from the tip of the snout, at the level of the hind process of the quadrate cartilage. Fig. G.-Photomicrograph of another 16 mm specimen (nose-to-cloaca1 length 8.90 mm); 1.75 mm from the tip of the snout, a t the level of the origin of the cartilaginous backgrowth of the quadrate cartilage.

PLATE 2

Fig. H-Photomicrograph of a 34.5 mm specimen of Neoceratodus (nose-to-cloaca1 length 17.55 mm) ; 3.78 mm from the tip of the snout, a t the level of the otoquadrate cartilage, dorsal hyoid elements and the origin of the facial nerve. Fig. I-Photomicrograph of same specimen as in figure H, 4.05 mm from the tip of the snout, showing the lateral process (laterohyale) of the epihyale situated laterally to the levator hyoideus muscle.

810 THE HYOID OF NEOCERATODUS

Fig. J-Photomicrograph of a 17 mm specimen of Hynobiw nebuEocnu, (nose-to-cloaca1 length 7.6 mm) ; 1.55 mm from the tip of the snout, at the level of the incipient otoquadrate cartillage. Fig. K-Photomicrograph of a 32 mm specimen of Hynobizca nebuloaw (nose-to-cloaca1 length 14.4 mm) ; 2.61 mm from the tip of the snout at the level of the otoquadrate cartilage and emer- gent facial nerve. Fig, L-Photomicrograph of same specimen as in figure K, 2.87 mm from the tip of the snout, at the level of the origin of the stilus from the columella. Fig. M-Photomicrograph of a 27 mm specimen of Hynobiw retardatua (nose-to-cloaca1 length 12.8 mm) ; 2.78 mm from the tip of the snout, at the level of the otoquadrate cartilage and the emergent facial nerve. Fig. N-Photomicrograph of a 26 mm specimen of Salamandra maculoua (nose-to-cloaca1 length 13.7 rnm) ; 3.1 mm from the tip of the snout, at the level of the otoquadrate cartilage and the emergent facial nerve. Fig. 0-Photomicrograph of a 37 mm specimen of Cryptobranchua alleghanieneb (nose-to-cloaca1 length 17.32 mm) ; 4.01 mm from the tip of the snout, a t the level of the origin of the cartilaginous stilus from the columella.

KEY TO LETTERING

ax. , auditory capsule ; b.p., basal plate ; c., ceratohyde ; co., columella ; c.P., crista parotica ; c.s., columella stilus; d., digastricus; e., epihyale; f.ab., foramen abducentis; f.g., facialis ganglion; fm., facial nerve ; f.o., foramen ovale ; g.f.I., gill filament I ; g.s.I., gill slit I ; h.b., hyoid blastema ; i.c.a., internal carotid artery ; int., interhyoideus muscle ; l., lateral process of epihyale (laterohyale) ; I.h., levator hyoideus muscle ; m., masseter muscle ; m.a., mandibular artery ; m.b., mid-brain ; not., notochord ; o,, otoquadrate ; ph., pharynx ; p.n., palatine nerve ; q., quadrate cartilage ; q,p., quadrate process ; r,c., rectus cervicis muscle ; s., stylohyale ; sq., squamosal ; s.a., stapedial artery ; t.f.g., trigemino-facialis ganglion ; v.c.1.. vena capitis lateralis.