Olfactory and Vomeronasal Systems of Caecilians (Amphibia: Gymnophiona)

Home , Boulengerula boulengeri, Epicrionops petersi, Gymnopis, Gymnopis multiplicata, Olfactory mucosa, Oscaecilia

JOURNAL OF MORPHOLOGY 205255-268 (1990)

Olfactory and Vomeronasal Systems of Caecilians (Amp hibia:Gymnophiona)

ANDREA SCHMIDT AND MARVALEE H. WAKE Department ofBiology, University of Bremen, 2800 Bremen 33. Federal Republic of Germanj-fAS.);Department of Integrative Biology, and Museum of Vertebrate Zoology, University of California,Berkeley, California 94720 (M.H. W.)

ABSTRACT The morphology of both the main nasal cavity and the vomeronasal organ differs among species representing six families of caecilians. The main nasal cavity is either divided or undivided. The vomeronasal organ differs in position (mediolateral, lateral), size (large vomeronasal organ in the aquatic species), and shape (mediolateral extension, vomeronasal organ with a lateral rostral projection). The great amount of respiratory epithelium of the main nasal cavity, the large vomeronasal organ, and its extensive innervation in typhlonectids may reflect both phylogeny and habitat adaptation, for these taxa are secondarily aquatic or semiaquatic and have several concomitant morphologicaland physiological modifications. The vomeronasal organ is associated with the caecilian tentacle as the tentacular ducts open into it. This association is further evidence for the involvement of the caecilian tentacle in vomeronasal chemoperception and may represent the mechanism by which these animals smell though the main nasal cavity is closed during burrowing or swimming. Labelings of primary olfactory and vomeronasal projections by means of horseradish peroxidase reaction reveal that the pattern of vomeronasal projections is similar in Ichthyophis kohtaoensis,Dermophis mexicanus, and Typhlo- nectes natans, even though T. natans possess stronger vomeronasal projections relative to olfactory projections than I. kohtaoensis and D. mexicanus. However, there are differences with respect to the patterns of olfactory projections. The olfactory projection of I. kohtaoensis is characterized by many displaced glomeruli. T. natam has the smallest olfactory projection. The nervus terminalis is associated with the olfactory system as shown by selective labelings of olfactory projections. Six characters potentially useful for phylogenetic analysis emerge from this study of comparative morphology. The characters were subjected to analysis using PAUP to see 1) if any resolution occurred and 2) if any groups were distinguished, whether they corresponded to phylogenetic arrangements based on other morphological characters. The characters are too few to produce nested dichotomous sets for all cases, but they do support the two typhlonectid genera examined and Dermophis and Gymnopis as sister taxa discrete from other groups, and they show that species within genera cluster together.

Comparative studies of the olfactory and been discussed either as an adaptation to terres- vomeronasal systems provide useful data with trial life (Bertmar, '81) or as an originally aquatic which to consider the question of a monophy- olfactory organ resembling the olfactory mucosa letic or polyphyletic origin of terrestrial verte- of fishes (Broman, '20). The vomeronasal organs brates. Neuroanatomical studies on phyloge- of members of the three extant amphibian or- netic aspects of the olfactory and vomeronasal ders differ with regard to size and location. systems of amphibians thus far have dealt largely In the gymnophione amphibians, the caecil- with the morphology of the nasal cavities (e.g., ians, the olfactory and vomeronasal systems are Parsons, '67; Jiirgens, '71) and olfactory and nearly completely separated. There is only a vomeronasal projections in salamanders small connection between the primary olfactory (Schmidt et al., '88). The vomeronasal organ has cavity and the vomeronasal organ. Chemorecep-

o 1990 WILEY-LISS, INC. 256 A. SCHMIDT AND M.H. WAKE! tion in the vomeronasal organ is considered to be and their sensory and respiratory epithelia were facilitated by the caecilian tentacle (summarized digitized from drawings of five sections that in- in Badenhorst, ’78; Billo and Wake, ’87). cluded the greatest extent of each component to The present study is directed to the question provide comparative data. These measurements, of whether the anatomy of the olfactory and however, do not reflect the volumes of the struc- vomeronasal systems, e.g., the anatomy of the tures because all extensions could not be in- nasal cavities and primary olfactory and vomer- cluded. onasal projections, might provide information The primary projections of the olfactory and on phylogenetic relationships and/or correlate vomeronasal mucosae were demonstrated using with different habitats of caecilians. Our results the horseradish peroxidase (HRP) method. The offer the basis for a comparison of the olfactory animals were anesthetized in a 1:150 (w/v) solu- and vomeronasal systems in the three amphib- tion of tricaine methanesulfonate (MS 222). A ian orders. We also further investigate the rela- solution of 30% (w/v) HRP (HRP Boehringer, tionship of the caecilian tentacle to chemorecep- grade 1) in 0.8% (w/v) NaCI, 5% (v/v) Nonidet tion by the vomeronasal system. (P 40), and 2% (v/v) dimethylsulfoxide was injected by inserting a syringe medially and later- MATERIALS AND METHODS ally through the naris into the main nasal cavity. Species examined A selective staining of olfactory projections was The morphology of the nasal cavities was stud- achieved by injecting HRP exclusively into the ied in representatives of all six families included medial olfactory sac. In order to stain vomerona- in the Order Gymnophiona (see Duellman and sal projections selectively, we removed the skull Trueb, ’86).Species studied include Family Rhi- and tissues covering the vomeronasal organ and natrematidae (northern South America),Epicri- injected HRP precisely into the vomeronasal onops petersi; Ichthyophiidae (southeast Asia), organ. We reacted the nasal cavities as whole Ichthyophisglutinosus and I kohtaoensis; Urae- mounts in order to ascertain that HRP was in- otyphlidae (India), Uraeotyphlus narayani; Cae- jected only into the vomeronasal organ. We also ciliaidae, (Central and South America, Africa, injected HRP into the tentacle sac to investigate Seychelles, India), subfamily Caeciliainae, Cae- the connection between the tentacle and the cilia occidentalis (Peru) and Oscaecilia ochro- vomeronasal organ. cephala (Panama), subfamily Dermophiinae, The animals were sacrificed after 48 to 72 Dermophis mexicanus (Central America), Gym- hours by overdose of anesthetic. They were nopis multiplicata (Central America),Hypogeo- transcardially perfused with a 0.8% (w/v) solu- phis rostratus (Seychelles), Geotrypetes sera- tion of NaCl followed by a solution of 2 % (v/v) phini (Ghana), Syluacaecilia grandisonae glutaraldehyde and 1%(w/v) paraformaldehyde (Ethiopia), Afrocaecilia taitana (Kenya), Bou- in 0.1 M phosphate buffer (pH 7.4) and 2.5% Lengerula boulengeri (Kenya), and Idiocranium sucrose for fixation. The brain was removed, russeli (Cameroon); Scolecomorphidae (East postfixed for 30 minutes, and rinsed for at least 2 and West Africa), Scolecomorphus kirkii and hours in 0.1 M cacodylate buffer (pH 7.2). The Scolecomorphus uluguruensis; Typhlonectidae, brains were reacted as whole mounts in 0.2% Typhlonectes natuns and Chthonerpeton indis- (wh) diaminobenzidine (Sigma) in 0.1 M caco- tinctum. dylate buffer (pH 5.45) and 0.015% hydrogen Primary olfactory and vomeronasal projec- peroxide for 60 minutes. The reactions were per- tions were examined in an ichthyophiid, I. formed according to the protocols of Fritzsch kohtaoensis (five animals), a caeciliaid,D. mexi- (’80, ’81). Whole mounts were embedded in His- cunus (five animals), and a typhlonectid, T. na- tosec or Epon and cut into 30 pm sections. turn (one animal). RESULTS METHODS Morphology of nasal cavities The morphology of nasal cavities was exam- The morphology of nasal cavities differs in ined in heads of adults, which were serially sec- size, shape, and epithelial distribution in the tioned (transversely, sagittally, or frontally) at caecilians examined. The differences concern 7-10 pm. Every third slide was stained with both the main nasal (primary olfactory) cavity, picroponceau, hematoxylin-eosin, or Mallory’s the “Hauptnase” (e.g.,Wiedersheim, 1879; Sara- azan. Sections were drawn with the aid of a sin and Sarasin, 1887-1890), as well as the vo- camera lucida. Measurements of circumferences meronasal organ, the “Nebennase.” (Through- of the main nasal cavity, the vomeronasal organ, out this work, we will refer to these paired CAECILIAN OLFACTORY SYSTEM 257 structures in the singular for simplicity of In S. kirkii, S. uluguruensis, I. glutinosus, I. expression.) Badenhorst ('78) reviewed the liter- kohtaoensis, A. taitana, D. mexicanus, Gym. ature on these structures. He resolved some of multiplicata, Geo. seraphini, and E. petersi the the inconsistencies of terminology and descrip- ventral main nasal cavity is divided into medial tion found in several papers that described the and lateral cavities (type B; Fig. 1, Table 1).In morphology of the heads, including olfactory these species the lateral cavity is lined by respira- components, of various species. We follow his tory epithelium caudally and sensory epithelium usage. rostrally. The medial cavity is coated with sen- Two types of main nasal cavities are distin- sory epithelium. In Epicrionops the division of guished (types A [undivided] and B [divided]; the main nasal cavity is not complete, i.e., the Table 1, Fig. 1) In T. natans, C. indistinctum, B. lateral and medial cavity join at its caudal part boulengeri, S. grandisonae, H. rostratus, C. occi- (Fig. 1).Most of the lateral cavity is covered by dentalis, U. narayani, I. russeli, and 0. ochro- respiratory epithelium. Afrocaecilia has a slightly cephala (the latter ascertained with difficulty divided nasal cavity. due to preservation artifact), the main nasal The connection between the main nasal cavity cavity is uniform and undivided (type A; Table and the vomeronasal organ is very small in caecil- 1, Fig. 1).Respiratory epithelium covers the lat- ians. The size and position of the vomeronasal eral part of the caudal cavity. The medial cavity organ differ in the species examined. In accord and the lateral rostral cavity are lined with sen- with earlier studies in amphibians (Parsons, '67; sory epithelium. The only exceptions are T. na- Badenhorst, '78, Seydel, 1895), the size and the tans and C. indistinctum. In these species sen- position of the vomeronasal organ are described sory epithelium is found only in the rostral fourth relative to the main nasal cavity. These relations of the cavity (Fig. 1). Most of the main nasal do not reflect the position relative to the axis of cavity is covered by respiratory epithelium. In the head, because the species examined differ contrast to all other species examined, the longi- with regard to head shape and to the rostrocau- tudinal axis of the main nasal cavity in B. bou- dal extension of nasal cavities. S. kirkii, S. ulugu- lengeri is not parallel to the longitudinal axis of ruensis, I. glutinosus, I. kohtaoensis, and A. the head. The axes form an angle of about 60'. taitana possess a vomeronasal organ that is situ-

TABLE 1. Morphology of the main nasal cavity and the uomeronasal organ ingymnophiones' Vomeronasal Main nasal Family, genus, species organ cavity Rhinatrematidae Epn'crionops petersi Lateral, extends caudad B Ichthyophidae Ichthyophis kohtaoensis Mediolateral I B Ichthyophis glutinosus Mediolateral I B Scolecomorphidae Scolecomorphus uluguruensis Mediolateral I B Scolecomorphus kirkii Mediolateral I B Caeciliaidae Subfamily Dermophiinae Afrocaecilia taitana Mediolateral I Boulengemla boulengeri MediolateralI1 Syluacaecilia grandisonae MediolateralI1 Gymnopis multiplicata Lateral Dermophis mexicanus Lateral Geotrypetes seraphini Mediolateralwith lateral rostral projection Hypogeophis rostratus Mediolateralwith lateral rostral projection Idiocranium russeli Mediolateral,probably with lateral rostral projection Subfamily Caeciliainae Caecilia occidentalis Mediolateralwith lateral rostral projection Oscaecilia ochrocephala Mediolateralwith lateral rostral projection Uraeotyphlidae Uraeotyphlus narayani Mediolateralwith lateral rostral projection A Typhlonectidae Typhlonectes natans MediolateralI1 A Chthonerpeton indistinctum Mediolateral I1 A

'A, uniform cavity; B, divided cavity; mediolateral I, vomeronasal organ lies between the medial and lateral cavity of the main ndcavity; mediolateral 11, vomeronasal organ lies beneath the uniform main nasal cavity. 258 A. SCHMIDT AND M.H. WAKE

b I-m Figure 1 CAECILIAN OLFACTORY SYSTEM 259 ated between the medial and the lateral cavities Olfactory and vomeronasal projections of the main nasal cavity (Fig. 1) and extends Horseradishperoxidase experiments show that mediolaterally ventral to the lateral cavity of the in I. kohtaoensis and mexicanus, paired dor- main nasal cavity in I. glutinosus, I. kohtaoen- D. sis, and A. taitana. In S. ulugurensis the vomer- sal and ventral nervi olfactorii originate in the onasal organ extends rostrocaudallybetween the olfactory mucosa and enter the bulbus olfacto- lateral and medial cavities of the main nasal rius dorsally and ventrally, respectively (Figs. 5, cavity and shows only a slight mediolateral exten- 6). T. natans does not have a clear separation of sion. In T. natans, C. indistinctum, B. bouleng- the dorsal and ventral nervi olfactorii where they eri, and S. grandisonae the vomeronasal organ enter the bulbus olfactorius. The bulbus olfacto- extends mediolaterally ventral to the undivided rius constitutes the rostral telencephalon and is main nasal cavity (Fig. 1). D. mexicanus and concentrically laminated (Fig. 6b). Fibers of the Gym. muttiplicata possess the smallest vomero- dorsal nervus olfactorius originate exclusively in nasal organs; they lie ventral to the lateral cavity the olfactory mucosa. The ventral nemolfacto- of the main nasal cavity and extend laterally rius consists of fibers which innervate both the (Fig. 1). In Epicrionops the entire vomeronasal olfactory mucosa and the vomeronasal mucosa. organ extends caudad, lateral to the main nasal Ventral fibers of the ventral nems olfactorius cavity. The vomeronasal organ joins the main come from the olfactory mucosa while dorsal nasal cavity at the lateral wall of the lateral fibers of the ventral news olfactorius originate cavity (Fig. 1).A caudal extension of the vomero- in the vomeronasal mucosa. Vomeronasal fibers nasal organ, as occurs in Epicrionops, has not cross the bulbus olfactorius caudally to enter the been observed in any other taxa. Geo. seraphini, bulbus olfactorius accessorius (Figs. 4-6). The H. rostratus, C. occidentalis, 0. ochrocephala, bulbus olfactorius accessorius lies caudal to the U. narayani, and I. russeli also possess a vomer- bulbus olfactorius and forms only a part of the onasal organ that extends mediolaterallyventral lateral telencephalic wall (Fig. 6). to the main nasal cavity and bend laterally to form a rostral extension (Figs. I, 2; Table 1).7'. Olfactoryprojections natans and C. indistinctum are characterized by a very large vomeronasal organ, which is corre- In D. mexicanus and I. kohtaoensis olfactory lated with a very extensive innervation. Whole- projections cover the bulbus olfactorius entirely mount preparations of the nasal cavities indicate (Fig. 4). The extensions of the olfactory projec- a patent connection between the tentacular ducts tions are similar in the two species. The area of and the vomeronasal organ (Fig. 3), as also shown termination of olfactory fibers is smaller in by other workers (see summaries in Badenhorst Typhlonectes than in Dermophis and Ichthyo- "781 and Billo and Wake "871). phis. In Typhlonectes olfactory projections cover only the rostral bulbus olfactorius (Fig. 4). In Zchthyophis we find many glomeruli outside the main olfactory and vomeronasal projection areas Fig. 1. Line drawings of the morphology of a: main nasal (Figs. 4,5a). These displaced glomeruli surround cavities (sensory epithelium is shaded; respiratory epithelium is unshaded) in T.n, Typhlonectes natans; U.n, Ureaotyph- the olfactory and vomeronasal projection area at lus narayani; S.k, Scolecomorphw kirkii; and b vomerona- their caudal extent and consist exclusively of sal organs (black) in E.p, Epicrionops petersi; G.m, Gymno- olfactory fibers, as demonstrated by selective pis multiplicata; G.s, Geotrypetes seraphini; H.r, stainings of olfactory projections (Fig. 5b). The Hypogeophk rostratus; T.n, Typhlonectes natans;A.t, Afro- caecilia taitana; S.k,Scolecomorphw kirkii all at the level of T. natans examined has only one displaced glom- greatest extensions. Lateral is to the left and medial to the erulus in the dorsal bulbus olfactorius, and D. right. Two types of main nasal cavities can bedistinguished: a mexicanus does not possess any displaced glom- uniform cavity (T.n and U.n) and a divided cavity (S.k). The main nasal cavity of T.n is characterizedby a great amount of eruli. respiratory epithelium. The vomeronasal organ is characterized with respect to extension and position relative to the Vomeronasal projections main nasal cavity: a lateral vomeronasal organ extending caudally in E.p, a small lateral vomeronasal organ with a In all species vomeronasal fibers cross the ol- slight mediolateral extension in G.m, a vomeronasal organ factory projection area dorsolaterallyand termi- that extends mediolaterally and bends laterally to form a lateral rostral projection in G.s and H.r, a mediolateral vom- nate caudal to the bulbus olfactorius within the eronasal organ in T.n and A.t extending mediolaterally be- bulbus olfactorius accessorius,which is well sep- neath the undivided main nasal cavity in T.n and extending arated from the bulbus olfactoriusin Z. kohtaoen- mediolaterally ventral to the lateral cavity of the main nasal sis and D. mexicanus and less in T. natans. All cavity in A.t, and a vomeronasal organ extending mainly rostrocaudally between the lateral and the medial main nasal species examined have only one vomeronasal cavity in S.k. 1, lateral; m, medial. Bars = 1mm. projection field (Fig. 4). Injections of HRP into 260 A. SCHMIDT AND M.H. WAKE

Fig. 2. Horizontal sections of nasal cavities in a: Ichthyo- nasal cavity; lateral, small vomeronasal organ); and d Geotry- phis kohtaoemis (divided main nasal cavity, vomeronasal petes seruphini (divided main nasal cavity, mediolateral organ between the medial and lateral main nasal cavity); b vomeronasal organ with a lateral rostral projection). 1, lateral Typhlonectes natam (uniform main nasal cavity, large vo- cavity; m, medial cavity; MC, main nasal cavity; VO, vomero- meronasal organ); c: Dermophis mexicanus (divided main nasal organ. Bars = 1mm.

the tentacular sac produce selective stainings of fibers that branch off the displaced glomeruli vomeronasal projections. (Figs. 4, 5b). These fibers run caudad to the anterior commissure and extend to the hypothal- Neruus terminalis amus. T. natans has fibers which leave the olfac- Selective stainings of fibers that originate in tory projection area medially and run caudad to the olfactory epithelium in I. kohtaoensis reveal the septum. The course of all these fibers and CAECLIAN OLFACTORY SYSTEM 261

Fig. 3. Whole-mount preparation of the nasal cavities in higher magnification of the connection at the left side in a. Typhlonectes natans to show the connections between the MC, main cavity; T, tentacle; TD, tentacle duct; VO, vomero- tentacle duds and the vomeronasal organ (arrows). The ten- nasal organ. Bars = 1 mm. tacle and the tentacle duct are cut at the left side. b shows a their terminations within the septum and the E. petersi; and one mediolateral with a lateral hypothalamus suggest that they are fibers of the rostral extension in U.narayani, H. rostratus, C. nervus terminalis, as described for many verte- occidentalis, 0. ochrocephala, and G. seraphini. brates (Herrick, '09; Johnston, '13; Crapon de E. petersi is the only species examined that Caprona and Fritzsch, '83; Demski and North- has a vomeronasal organ which lies completely cutt, '83; Miinz et al., '82; von Bartheld and lateral to the main nasal cavity and which enters Meyer, '86; Schmidt et al., '88). Primary olfac- the main nasal cavity laterally. This position tory and vomeronasal projections terminate ex- resembles that found in urodeles, although in clusively within the olfactory bulb and the acces- urodeles the vomeronasal organ forms a lateral sory olfactory bulb, respectively. Fibers of the outpocketing of the main nasal cavity with a nervus terminalis are not labeled by selective continuous connection between the main cavity stainings of vomeronasal projections. and the vomeronasal organ. In Epicrionops the main nasal cavity and the vomeronasal organ are DISCUSSION almost completely separated. In most anurans Morphology of nasal cavities the vomeronasal organ lies ventromedially, Gymnophione species exhibit differences in though Pipa has a lateral vomeronasal organ the morphology of nasal cavities with regard to (Parsons, '67). Jiirgens ('71) hypothesized that in both the main nasal cavity and the vomeronasal anurans a shifting of the vomeronasal organ from organ. We distinguish two morphological types anterolateral to anteromedial has taken place of main nasal cavity: an undivided cavity and a during phylogeny. The phylogenetic implica- cavity that is divided into medial and lateral tions of these morphological patterns are dis- components. The vomeronasal organ differs with cussed below. regard to shape, position, and sue. We recognize The main nasal cavities of T. natans and C. a mediolateral position of the vomeronasal organ indistinctum have less sensory epithelium than in I. glutinosus, I. kohtaoensis, A. taitana, B. respiratory epithelium. In other species the main boulengeri, S. grandisonae, S. Kirhii, S. uluguru- nasal cavity mostly is covered by sensory epithe- ensis, T. natans, and C. indistinctum; a lateral lium. These results likely reflect the phyloge- position in G. multiplicata, D. mexicanus, and netic history, and particularly adaptation to the 262 A. SCHMIDT AND M.H. WAKE

Fig. 4. Line drawings of olfactory (shaded)andvome- ronasal projections in a: Ichthyophis kohtaoemsis, b Dermophis mexicanus, and c: Typhlonectes natans, revealed by injections of HRP into the olfactory and vomeronasal ma. A, lateral view; B, dorsal view; C, ventral view. Note that the olfactory projection of Ich- thyophis is characterizedby several displaced glomeruli, which surround even the vomeronasal projection area. The nemterminalis is in contact with these displaced glomeruli. Bulb. olf.acc., accessory olfactory bulb Bulb. OK., Olfactory bulb; D, diencephalon; DGL, displaced glomeruli; HY,hypophysis; ME, medulla oblongata; N.t, newterminalis; TEL, telencephalon; TO, tectum opti- cum; I, newolfactorius. Bars = 1 mm. CAECILIAN OLFACTORY SYSTEM 263

Fig. 5. Dorsal (a) and lateral (b)views of the rostral Selective staining of vomeronasal projections by application telencephalon in Ichthyophis kohtaoensis. In a, olfactory of HRP to the vomeronasal mucosa is shown in c; selective projections are stained at both olfactory bulbs while vomero- staining of olfactory projections (black arrow) in Dermophis nasal projections are stained only at the left side (arrow). In b, mexicanus by application of HRP to the olfactory mucosa is the newterminah joins fibers that run around the spared shown in d. Vomeronasal projections are unstained (white vomeronasal projection field at the right side. Lateral views arrow). DGL, displaced glomeruli; N.t, nem terminalis. (c,d)of the rostral telencephalon in Dermophis mexicanus. Bars = 500~m. aquatic to semiaquatic habitat, of these animals. eral lungs and to a requirement for extensive It is assumed that the typhlonectid ancestor was gaseous exchange epithelium in low oxygen ten- terrestrial and that extant species are second- sion aquatic habitats. Typhlonectids come to the arily aquatic. Several morphological features surface to breathe and stay submerged for long unique among caecilians are thought to be asso- periods of time. ciated with aquatic life. For example, typhlonec- There are different opinions of the evolution- tids are the only caecilians that have extensively ary history of the vomeronasal organ. Broman bilateral lung development, though the length of (’20) and Parsons (’67) hypothesize that the the second lung varies among taxa. Species of all vomeronasal epithelium correspondsto the olfac- other families characteristically have only the tory epithelium of fishes. Bertmar (’81) and Jiir- right lung well developed, though some have gens (’71) regard the vomeronasal organ as an small “tracheal” lungs as well. The great amount adaptation to terrestrial life. This was indicated of respiratory epithelium in typhlonectids may by Anton (’12) and Bruner (’14)’ who found a be related functionally to the existence of bilat- small vomeronasal organ in aquatic urodeles, 264 A. SCHMIDT AND M.H. WAKE

Fig. 6. Transverse sections of the accessory olfactory bulb (a) and the olfactory bulb (b) in Ichthyophis kohtaoensis. The glomeruli of both the olfactory bulb and the accessory olfactory bulb are stained by application of HRP to the olfactory and the vomeronasal mucma, respectively. GCL, granule cell layer; GL, glomeruli; MC, mitral cell layer. Bars = 100 Fm.

and by Medvedeva ('67), who described a reduc- epithelium. The olfactory projection of I. tion of the vomeronasal organ in animals that kohtaoensis is characterized by many displaced returned to water secondarily. However, this is glomeruli which surrounded the olfactory and not the case in T. natans and C. indistinctum. vomeronasal projection area at their caudal ex- Our studies show that the typhlonectids exam- tent. It remains unresolved whether these dis- ined have a very large vomeronasal organ. We placed glomeruli represent a functional special- believe that Medvedeva's conclusions regarding ization or whether they are the beginning of an a loss of functional demands for the vomeronasal extended projection. Displaced glomeruli, al- organ in animals which return to water second- though fewer in number, have been found in arily may not be appropriate. We question that urodeles as well (Schmidt et al., '88). In urodeles there is a loss of functional demand because the the displaced glomeruli appeared to be in con- vomeronasal system may well facilitate the per- tact with the nervus terminalis. Our experiments ception of water soluble odorants. show that this is also the case in caecilians. Staining of the nervus terminalis was achieved Olfactory and vomeronasal projections by selective stainings of olfactory projections. Though the vomeronasal projections are simi- HRP labelings of vomeronasal projections did lar in the species examined via HRP stainings, not label fibers of the nemterminalis. The there are differences in olfactory projections. T. nervus terminalis is thought to be involved in the natans has the smallest olfactory projection area, perception of pheromones (see Demski and corresponding to its small amount of olfactory Northcutt, '83, for summary). The terminations CAECILIAN OLFACTORY SYSTEM 265 of the newterminalis within the septum, the tactile. Billo ('86) found Merkel cells, which are preoptic region, and the hypothalamus, which thought to be tactile, in the tip of the tentacular have been shown in many vertebrates (Herrick, fold, though he did not state which of the species '09; McKibben, '11; Johnston, '13; Crapon de (I.kohtaoensis and T.compressicauda) he exam- Caprona and Fritzsch, '83; Demski and North- ined. Fox ('85) stated that Merkel cells are ab- cutt, '83; Miinz et al., '82; von Bartheld and sent in I. kohtaoensis and I. orthoplicatus. The Meyer, '86; Schmidt et al., '88) and evidence for studies of development and adult structure of gonadotropin-releasingfactor immunoreactivity the tentacle and the vomeronasal organ by Mar- in neurons of the newterminalis (summarized cus ('30) on Hypogeophis, by Badenhorst ('78) in Muske and Moore, '88) lend credence to the on Ichthyophis, and by Billo and Wake ('87) on hypothesis of pheromone perception. In this con- Dermophis and Gymnopis clearly show that text, it is surprising that fibers of the news there is a connection between the tentacle and terminalis stained in our experiments originated the vomeronasal organ. Billo and Wake ('87) exclusively from the olfactory projection. demonstrate that patent ducts from the tentacu- Olfactory projections in caecilians are dif- lar sac join the vomeronasal organ in fetuses that ferent from those of urodeles and anurans. In are near birth size (approximately 105 mm total urodeles primary olfactory projections form an length [TL] in Dermophis; 84 mm TL in Gymno- oval shaped lateral termination area (Schmidt et pis). al., '88). In anurans primary olfactory projec- Our experiments verify that there is a patent tions enter the olfactory bulb ventrally (North- connection between the tentacular ducts and the cutt and Kicliter, '80). vomeronasal organ in adults of all species exam- The vomeronasal projections that we have ined. Injections of HRP into the tentacular sac shown for three caecilian species bear resem- led to selective stainings of vomeronasal projec- blance to vomeronasal projections found in tions. This is further evidence that the tentacle is anurans, forming one vomeronasal projection functionally related to the vomeronasal organ. field (Weiss, personal communication) and to Odorants entering the tentacular lumen may be those found in urodelan species that are consid- led via the tentacular ducts to the vomeronasal ered to be derived. Even though many urodeles organ. Therefore animals would be able to smell show several vomeronasal projection areas, other even if the main nasal cavity is closed by constric- urodeles such as the strictly terrestrial Pleth- tion of the nostrils during burrowing or swim- odontini and Bolitoglossini (highly specialized ming. plethodontids: Wake, '66) have vomeronasal projection fields that are reduced in number and Phylogenetic relationships extent (Schmidt et al., '88). The reduction of We believe that when surveys of morphology vomeronasal projection fields in Plethodontini across taxa are conducted, the data should be and Bolitoglossini is due to a reduction of the examined to see if characters of systematicsignif- entire vomeronasal system of these animals (un- icance emerge. Our data on olfactory and vomer- published results) and may be correlated with onasal organ morphology contribute new charac- the terrestrial life of these groups. In caecilians ters potentially useful for assessment of the number of vomeronasal projections relative phylogenetic relationships. to the amount of olfactory projections differed. Six characters emerged from analysis of olfac- T.natans has the strongest vomeronasal projec- tory and vomeronasal morphology (the charac- tion of the species examined. This projection size ter states and the data matrix are presented in is correlated with the large vomeronasal organs Appendix A). Characters were polarized accord- we found in typhlonectid species. Even though ing to the condition in the outgroup to caecilians T. natans has only one vomeronasal projection (urodeles) and to the ontogeny of the character field, it was very enlarged compared to those of I. (if known). Characters include 1) shape of the kohtaoensis and D. mexicanus. main nasal cavity; 2) orientation of the main nasal cavity to the axis of the head; 3) extent of Role of the caecilian tentacle in uomeronasal respiratory epithelium lining; 4) vomeronasal or- perception gan position; 5) vomeronasal with or without a Opinions differ on the functional role of the caudal extension; and 6) size of vomeronasal caecilian tentacle. Some authors have consid- organ. ered it tactile (Sarasin and Sarasin, 1887-1890; The six characters were subjected to a PAUP Engelhardt, '24; Laubmann, '27; Marcus, '30; computer analysis (Swofford, '84)to see whether Fox, '85). The investigations of Badenhorst ('78), character states of the olfactoryhomeronasalsys- Billo ('86)' and Billo and Wake ('87) indicate tem showed correlations with each other and that the tentacle is chemosensory as well as with the current associations of genera in fami- 266 A. SCHMIDT AND M.H. WAKE lies based on other characters. We recognize that recently congeneric (Wake, ’87), do not have six characters for 18 species do not have real similar olfactory morphology at all. However, we resolution power, especially since some derived suggest that typhlonectid morphology-the large states are shared broadly (see Table 1 and Ap- size of the vomeronasal organ, its extensive inner- pendix A). Characters 1 and 4 (and to some vation, its limited olfactory projections, and the degree 5) seemed in conflict in terms of numbers reduced sensory epithelium but increased respi- of reversals or convergences. Running the pro- ratory epithelium of the main nasal cavities-is gram with characters 1 and 4 (see above) unor- in contrast to the condition in all other species dered and unpolarized did not produce further examined and likely is correlated with their sec- resolution. When the analysis is run with charac- ondarily aquatic mode of life, including effective ter 4 polarized in accord with the outgroup state, function of the vomeronasal organ. Gymnopis and Dermophis are sister groups with The analysis does show, however, that species Epicrionops their sister taxon, and the group is in the same genus, at least in the cases we tested nested high in the tree; the polychotomy of Idio- (Ichthyophis and Scolecomorphus), share the cranium, Hypogeophis, Uraeotyphlus, Oscae- same morphology, as do the genera Dermophis cilia, and Caecilia is lowest in the tree. When the and Gymnopis, terrestrial viviparous Central analysis is run with characters 1,4,and 6 unor- American dermophiines considered closely re- dered, or with all characters unordered, Epicri- lated based on other characters (Case and Wake, onops is the most primitive taxon and Gymno- ’77). Indications that closely related taxa share pis-Dermophis its sister group. This grouping is the same morphology suggest that changes in in better accord with the phylogenetic hypothe- the olfactory and vomeronasal organs are rela- sis based on other morphological criteria (cf. tively old. Further, since genera assigned to a Duellman and Trueb, ’86). Depending on how diversity of families based on other data share the characters were ordered, three to six equally certain characters of the olfactory system in a parsimonious trees were produced. mosaic fashion, we infer that change is relatively Character 2, the angled main nasal cavity, is constrained and that many features are in com- an autapomorphy unique to Boutengerula, hence mon. uninformative. Characters 3 and 6 are autapo- We recognize that while the position of the morphies for Typhlonectes and Chthonerpeton, vomeronasal organ suggests that caecilians and the two members of the family Typhlonectidae urodeles are sister taxa, the anatomy of the olfac- examined (see below). The state of character 5 is tory bulb and the olfactory projections in the shared by all taxa except Epicrionops, which is three taxa that we examined (Ichthyophis,Der- primitive on other grounds. Similarities among mophis, and Typhlonectes) is different from the the PAUP trees and that from a compatibility urodelan condition. analysis indicate that Dermophis and Gymnopis We are accumulating information that pro- are closely allied; one large, poorly resolved block vides new characters from morphological of taxa includes Ichthyophis, Scolecomorphus, “systems” not previously examined compara- and Afrocaecilia, with Boulengerula and Sylua- tively. Neuroanatomical data are not often used caecilia less closely allied; the other large, poorly in phylogenetic analysis. While data from any resolved block consistently includes Uraeotyph- one “system” may not be conclusive, an examina- lus, Geotrypetes, Idiocranium, Hypogeophis, tion of overall neuroanatomical patterns is likely Caecilia, and Oscaecilia, with the typhlonectids to be useful in understanding both phylogeny their sister group. and adaptive radiation. Our data on the morphol- The postulated relationships show some vari- ogy of the olfactory and vomeronasal system of ations of interest. For example, the Old World selected caecilian species facilitate testable hy- potheses of function and life history and of some ichthyophiids and scolecomorphids, and the phylogenetic correlates. “caeciliaid” Afrocaecilia cluster together, with Boulengerula, sometimes postulated to be related to Afrocaecilia, less closely associated. Ich- ACKNOWLEDGMENTS thyophiids are considered primitive in a number We thank Gloria Wurst, Kath Thomas, Willy of characters of osteology and reproductive biol- Bemis, Kristen Nygren, and Enrique Lessa for ogy, while Afrocaecilia and scolecomorphids are preparing specimens; Monika Ernst for photo- derived. Uraeotyphlids are not allied in any way graphic assistance; Ted Papenfuss, Robert Seib, to ichthyophiids by the morphology of the olfac- David Wake, and Thomas Wake for collecting tory system, though many other kinds of morpho- Dermophis mexicanus; Werner Himstedt for logical characters suggest a relationship. Fur- permission to study his Ichthyophis kohtaoen- ther, Sylvacaecilia and Geotrypetes, until sis sections; and Ralph Billo for valuable discus- CAECILIAN OLFACTORY SYSTEM 267 sions. We also thank David Good for computer Herrick, C.J. (1909) The newterminalis (nerve of Pinkus) in the frog. J. Comp. Neurol. 37(3):17&190. assistance with the phylogenetic analysis. Jay Johnston, J.B. (1913) Nervus terminalis in reptiles and mam- Savage and Douglas Rossman kindly gave per- mals. J. Comp. Neural. 23t15C-156. mission to section material from Gymnopis mul- Jiirgens, J.D. (1971) The morphology of the nasal region of tiplicata and Epicrionops petersi and E. bicolor, Amphibia and its bearing on the phylogeny of the group. respectively. A.S. expresses her sincere thanks to Ann. Univ. Stellenbmch Ser. A, No. 2,46:1-146. Laubmann, W. (1927) Uber die Morphogenese vom Gehirn Gerhard Roth for his steady support. We thank und Geruchsorgan der Gymnophionen (Beitragzur Kennt- the National Science Foundation for support of nis der GymnophionenNr. 10). Z. Anat. Entw. 84:597-637. this research (BSR 83-05571 and 87-06135 to Marcus, H. (1930) Uber die Bildung von Geruchsorgan, Ten- M.H.W.). AS.%research period in Berkeley was take1 und Choanen bei Hyphogeophis, nebst Vergleich mit Dipnoem und Polypterus (Beitrag zur Kenntnis der Gym- funded by the DAAD (Deutscher Akademischer nophionen Nr. XIII). Z. Ges. Anat. 1: Z. Anat. Entw. Gesch. Austauschdienst). The manuscript was com- 91:657491. pleted while M.H.W. was a Short-Term Visitor McKibben, P.S. (1911) The new terminalis in urodele amphibia. J. Comp. Neurol. 21261-309. at the Smithsonian Tropical Research Institute Medvedeva, LM. (1967) Die Homologie des Jacobsonschen in Panama and with the support of a John Si- Organs bei Anura und Urdela. In T. Orvig (ed.): Current mon Guggenheim Foundation Fellowship. Problems of Lower Vertebrate Phylogeny. Stockholm: Almquist & Wiksell, pp. 331-341. LITERATURE CITED Miinz, H., B. Claas, W.E. Stumpf, and L. Jennes (1982) Centrifugal innervation of the retina by luteiniziig hor- Anton, W. 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Characters and character code matrix Character 1: Main nasal cavity undivided (O), slightly divided (la),divided (lb) Character 2 Longitudinal axis of main nasal cavity parallel to longitudinal axis of the head (01, longitudinal axis of main nasal cavity at 60' angle to longitudinal axis of the head (1) Character 3: Respiratory epithelium concentrated in lateral part of caudal region of main nasal cavity (O), respiratory epithelium covering most of main nasal cavity (1) Character 4: Vomeronasal organ mediolateral with lateral projection (O), mediolateral (la), lateral (lb) Character 5 Vomeronasal organ with caudal extension (0),without caudal extension (1) Character 6 Vomeronasal organ moderate in size (O), vomeronasal organ large (1) Character Species 1 2 3 4 5 6 Epicrionops petersi la 0 0 lb 0 0 Ichthyophis glutinosus lb 0 0 la 1 0 Ichthyophis kohtaoensis lb 0 0 la 1 0 Uraeotyphlus narayani 0 0 0 0 1 0 Caecilia occidentalis 0 0 0 0 1 0 Oscaecilia ochrocephala 0 0 0 0 1 0 Afrocaecilia taitana Ib 0 0 la 1 0 Boulengerula boulengeri 0 1 0 la 1 0 Hypogeophis rostratw 0 0 0 0 1 0 Syloacaecilia grandisonae 0 0 0 la 1 0 Geotrypetes seraphini Ib 0 0 0 1 0 Idiocranium russeli 0 0 0 0 1 0 Gymnopis rnultiplicata lb 0 0 lb 1 0 Dermophis rnexicanus Ib 0 0 lb 1 0 Scolecomorphus kirkii lb 0 0 la 1 0 Scolecomorphus uluguruensis lb 0 0 la 1 0 Typhlonectes natans 0 0 1 la 1 1 Chthonerpeton indistinctum 0 0 1 la 1 1