Cuad. herpetol., 18 (1): 3�15, 2004 3THREE-DIMENSIONAL RECONSTRUCTION OF THEHYOBRANCHIAL APPARATUS OF HYLA NANA TADPOLES(ANURA: HYLIDAE)

M ARÍA FLORENCIA VERA CANDIOTI 1 & ALEXANDER HAAS 21 Instituto de Herpetología, Fundación Miguel Lillo. Miguel Lillo 251. (4000) S. M. de Tucumán, Argentina.f l o r i v c @ h o t m a i l . c o m2 Zoologisches Institut und Zoologisches Museum Martin-Luther-King-Platz 3. 20146 Hamburg, Germany.a l e x a n d e r . h a a s @ u n i - h a m b u r g . d e

INTRODUCCIÓNTadpole skeleton and musculaturehave been described in numerous hylidspecies (i.a., De Sá, 1988; Fabrezi andLavilla, 1992; Fabrezi and Vera, 1997;Haas, 1996a; 1996b; 2001; 2003; Haasand Richards, 1998; Lavilla and Fabrezi,1987; Sokol, 1981). The technique mostfrequently employed for the analysis ofcartilage and bone structures in anuransis clearing and differential staining(Wassersug, 1976; Dingerkus and Uhler,1977; Taylor and Van Dicke, 1985).These protocols are both highly avail-able and useful, but dissection becomesdifficult in the case of complex orsmall-sized specimens. Sectioning andhistological analysis are also frequentlyused, but they imply loss of the 3D per-ception and integrity of specimens.

3Cuad. herpetol., 18 (1): 3�15, 2004

R E S U M E N. � En el presente trabajo se estudia el esqueleto hiobranquial y musculatura dela canastilla branquial de larvas de Hyla nana a través de reconstrucción tridimensional de seccio-nes histológicas. El esqueleto cartilaginoso se caracteriza por el marcado desarrollo de los cerato-hiales y la reducción de la canastilla branquial. La topología muscular es congruente con lo repor-tado para la mayoría de los hílidos, con características particulares como un m. subarcualis rectusI con sólo dos cabezas (dorsal y ventral), y un m. subarcualis rectus II-IV discontinuo e insertadoanterolateralmente en el ceratobranquial I. Estos caracteres no son frecuentes entre los hílidos,pero presumiblemente los comparten otras especies del grupo de H. microcephala.Palabras clave: esqueleto hiobranquial, musculatura branquial, renacuajos, Hyla nana, recons-trucción 3D.A B S T R A C T. � This study describes the hyobranchial skeleton and branchial musculature ofHyla nana tadpoles, employing three-dimensional reconstruction from histological sections. Thecartilaginous skeleton is characterized by a marked development of the ceratohyals and a reductionof the branchial basket. The muscular topology is congruent with the pattern found in most hylinehylids, with particular features such as the m. subarcualis rectus I with only two heads (dorsaland ventral), and a discontinuous m. subarcualis rectus II-IV, inserted anterolaterally in theceratobranchial I. Such features, infrequent among hylids, are presumably shared by other specieswithin the H. microcephala group.Key words: hyobranchial skeleton, branchial musculature, tadpoles, Hyla nana, 3Dreconstruction.

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M. F. VERA CANDIOTI & A. HAAS: Hyobranchial Apparatus of Hyla nana4 Haas and Fischer (1997) developed amethod of morphological reconstructionfrom serial histological sections, by us-ing commercially available product-de-sign software packages. Specimens arecut following standard histology proto-cols and then manually reconstructedwith software that models the samplesthree-dimensionally. This interactivetechnique is a reliable option for precisemorphological studies, and with ade-quate training, the time consumed toprepare a reconstruction is acceptable.Furthermore, virtual reconstructionsare not mere visual representations ofobjects, since they can be measured andanalyzed accurately.3D reconstruction methods are beingincreasingly used, not only in herpetolo-gy, but also in other fields of vertebrateand invertebrate morphology (v.g., Haasand Richards, 1998; Gorb and Beutel,2000; Adriaens et al., 2001; Sánchez-Vil-lagra et al., 2002).Hyla nana tadpoles are common in-habitants of lentic ponds in Chaco andLitoral regions from Argentina. The ex-ternal morphology has been extensivelydescribed by Lavilla (1990). The internalmorphology, particularly cartilaginousskeleton and cranial musculature, hasbeen also previously studied (Lavilla andFabrezi, 1992; Vera Candioti et al.,2004). Data on branchial muscles stillremained, which constitutes one of thereasons for the selection of this species.Furthermore, the reduced size of thespecimens ad their marked differenceswith the general morphological patternfound in other taxa derive in a particu-lar difficulty in the study of the saidmuscular group, and consequently, the3D reconstruction technique becomesvery helpful.This paper presents a morphologicalstudy of the hyobranchial skeleton andbranchial musculature of Hyla nana tad-poles, employing three-dimensional com-puter-aided reconstructions from seriallysectioned specimens.

MATERIAL AND METHODS

Hyla nana larvae in stages 31-36 (ac-cording Gosner, 1960) were examined.The study focused on the hyobranchialskeleton and branchial and spinal mus-cle groups (according to the classificationshown in Cannatella, 1999).Five larvae were collected in SantaFe City (Ciudad Universitaria, El Pozo,Santa Fe, Argentina), during January2001. The specimens were fixed in neu-tral 4% formalin. Part of them was pre-pared according to standard histologicalprotocols (Böck, 1989). For serial sec-tioning, specimens were decalcified (Di-etrich and Fontaine, 1975), dehydrated,embedded in paraffin, and sectioned at 7µm. Sectioning was performed on a Mi-crom HM360 rotary microtomeequipped with the Microm water trans-fer system. Sections were then stainedwith Azan trichrome (Böck, 1989) anddigitalized with a Nikon Coolpix 990camera on a Zeiss microscope. The pix-el files obtained were loaded into thesoftware package Alias/Wavefront Stu-dioTM 9.5 on a Silicon Graphics Octaneworkstation as background images (im-age planes). Contours of selected struc-tures were drawn manually on thebackground images. The resulting set ofcontours was used as a scaffold aroundwhich a surface was created manuallyto fit the contours tightly (Haas andFischer, 1997). The musculature wasreconstructed on one side only and nodifference to the other side musculaturewas found in histological sections.The remaining specimens were pre-pared according to the clearing andstaining protocol of Taylor and VanDicke (1985). To examine the muscula-ture, some individuals were removedbefore the digestion step (Lavilla, pers.commun.) and then stained with a lugolsolution (Böck and Shear, 1972). Dissec-tions were made employing a stereomi-croscope equipped with a camera lucida(Zeiss SV 11). The larvae so treatedserved as control against which the re-

Cuad. herpetol., 18 (1): 3�15, 2004 5

Figure 1. Hyla nana Stage 31. Hyobranchial skeleton. A. Ventral view. B. Anterolateral view.Legends: ca, condylus articularis; cb (I-IV), ceratobranchial; ch, ceratohyal; cII, copula II; phb,planum hypobranchiale; pr, pars reuniens.

M. F. VERA CANDIOTI & A. HAAS: Hyobranchial Apparatus of Hyla nana6

Cuad. herpetol., 18 (1): 3�15, 2004 7sults obtained with the 3D reconstruc-tion method were compared.The final virtual three-dimensionalmodel of the Hyla nana hyobranchialapparatus was animated with AliasWavefront Studio software and the re-sulting image series were composed toa movie with Apple Quicktime 6.1 Proon an Apple Macintosh G4 workstation.The terminology used for the follow-ing description corresponds to Haas(2003).RESULTSThe cartilaginous skeleton of Hyla

nana tadpoles is described in Lavillaand Fabrezi (1992) and Vera Candioti etal. (2004). Cartilaginous structures ofspecimens examined in the present workdo not show significant differences ascompared to former descriptions (Fig. 1).Although the cartilaginous branchialbasket is reduced in Hyla nana, all thebranchial and spinal muscles reportedfor hylids are present (Figs. 2 and 3).

Mm. levatores arcuum branchialium.These four muscles extend from the lat-eral and posterolateral margin of thebranchial basket to the posterior part ofthe palatoquadrate and otic capsule. InHyla nana all four muscles are distin-guishable.

M. levator arcuum branchialium I.Lateral margin of arcus subocularis-cer-atobranchial I. It is a wide muscle, withinsertion surfaces occupying a large areaon the ceratobranchial I and palatoquad-rate.M. levator arcuum branchialium II.

Larval processus oticus-commissura ter-minalis I and ceratobranchial II. It isseparated from the m. levator arcuumbranchialium I by a narrow gap in itsceratobranchial insertion, but the sepa-ration becomes less evident in the in-sertion into the palatoquadrate.

M. levator arcuum branchialium III.Lateral part of otic capsule-ceratobran-chial III.

M. levator arcuum branchialium IV.Posterolateral part of the otic capsule-ceratobranchial IV.Mm. constrictores branchiales. Theseare four muscles which extend betweenthe medial edge of their own ceratobran-chial and the commissurae terminales.In Hyla nana three of these four mus-cles are present.M. constrictor branchialis II. Processus

branchialis II-commissura terminalis I.M. constrictor branchialis III. Proces-

sus branchialis II-commissura terminalisII.

M. constrictor branchialis IV. Fibersare continuous with those of the anteri-or part of the m. subarcualis rectus II-IV. They insert on the commissura ter-minalis II and distal portion of cerato-branchial III.

Mm. subarcuales recti. This musclegroup extend from the ceratohyals to theceratobranchials, and between cerato-branchials. It can be divided into severalportions in anuran larvae. The musclesfound in Hyla nana are as follows:M. subarcualis rectus I. It is formedby a ventral and a dorsal slip. Bothoriginate in the lateral region of proces-

sus posterior hyalis. The ventral slip iswell developed and runs posteriorly tothe processus branchialis III, while inthe dorsal slip, fibers assume an obliquedirection and insert in the lateral prox-imal part of the ceratobranchial I.M. subarcualis rectus II-IV. It isformed by two parts, anterior and poste-rior. The anterior part originates in con-nective tissue next to the lateral side ofthe ceratobranchial I, approximately atmid-length. Its fibers are confluent withthose of the m. constrictor branchialis IVand m. subarcualis rectus II-IV, posteriorpart. The posterior part includes fibers ofthe anterior part and fibers which origi-nate in the processus branchialis III.This slip inserts in the ventromedialside of the ceratobranchial IV.M. subarcualis obliquus. Processus

urobranchialis-processus branchialis IIand III.

M. F. VERA CANDIOTI & A. HAAS: Hyobranchial Apparatus of Hyla nana8M. tympanopharyngeus. Lateral partsof the otic capsule-ceratobranchial IVand planum hypobranchiale.M. rectus cervicis. Peritoneum-proces-

sus branchialis III.M. geniohyoideus. Cartilago labialis

inferior-planum hypobranchiale.The attached CD shows a two-minuteanimation of the 3D reconstruction ofthe hyobranchial apparatus of Hyla nanatadpoles (see the file Readme.doc, alsoin the CD).DISCUSSIONAs mentioned by Lavilla and Fabrezi(1992) and Vera Candioti et al. (2004),the hyobranchial skeleton of Hyla nanatadpoles is considerably different fromthe one described for most hylids. Thelarge relative size of ceratohyals con-trasts with the evident reduction in sizeof the branchial basket. Features thatare commonly present in other speciesshow a different configuration or areabsent in H. nana. The ceratobranchi-als, for example, are almost straightbars, scarcely chondrified and devoid of

spicula and lateral projections. Lateralprojections from the ceratobranchialsare a unique character of Anura, andthey give support to the interbranchialsepta and filter epithelia (Haas, 2003).Spicula and lateral projections are alsoreduced or absent in carnivorous tad-poles of Hymenochirus boettgeri, Lepi-dobatrachus laevis, L. llanensis, Cer-atophrys ornata and C. cranwelli, mac-rophagous larvae such as Hyla micro-cephala and Anotheca spinosa, gastromi-zophorous larvae of Atelopus tricolor,suctorial larvae of Boophis sp. and en-dotrophic larvae of Cycloramphus stej-negeri, Eupsophus calcaratus, Flectono-tus goeldii and Rhinoderma darwini(Haas, 1996b; 2003; Haas and Richards,1998; Lavilla, 1987; 1991; Lavilla andFabrezi, 1992; Lavilla and de Sá, 2001;Sokol, 1962; Ruibal and Thomas, 1988;Wassersug and Hoff, 1979; pers. obs.).

As is the case with Hyla microcephala,and contrary to what has been observedin most hylids, the copula I and processusurobranchialis are also absent. Finally,commissurae proximales between cerato-branchials are absent, a character sharedby some hylid species (H. microcephala,H. cinerea, H. annectans, H. ebraccata,Litoria genimaculata, L. lesueuri, phyl-lomedusines; Haas, 2003; Haas and Rich-ards, 1998; pers. obs.).The configuration of the hyobranchialmusculature is not concomitant withcartilaginous skeleton reduction. All themuscles reported for representatives ofthe Neobatrachia are present, and thedifficulty in the observation and identifi-cation of each muscle is due to thesmall size of the individuals, ratherthan to poor development of the mus-culature itself. Compared to other spe-cies within the Hylidae, some featuresneed to be mentioned.Personal examination of Hyla micro-cephala tadpoles revealed a pattern ofhyobranchial musculature identical tothe one described here for H. nana.Tadpoles of species of the H. microceph-ala group share several characters inexternal morphology, cartilaginous skel-eton, musculature, buccal apparatus andbuccopharyngeal cavity (i.a., Duellman,1970; 2001; Haas, 1996a; Vera Candiotiet al., 2004; Wassersug, 1980). Previousdata on hyobranchial musculature ofthe other species integrating the groupare not available, but the similaritiesmight include this particular group ofmuscles. More extensive taxa sampleswill show whether or not these sharedcharacter states constitute apomorphiccharacter states suitable to define thespecies group.The mm. subarcuales recti show sev-eral peculiarities as compared to otherspecies of the family. The m. subarcua-lis rectus I has two bundles, ventral anddorsal. This is also the case of Aplasto-discus perviridis, Phrynohyas resinific-trix, Scinax ruber and Phyllomedusaspp. tadpoles (Haas, 2003). The presence

Cuad. herpetol., 18 (1): 3�15, 2004 9

Figure 2 (top). Hyla nana Stage 31. Branchial and spinal musculature. Some muscles of the hy-oid group are also shown. Ventral view. Legends: cb (II-IV), constrictor branchialis; gh, geniohy-oideus; ha, hyoangularis; ih, interhyoideus; lab (I-IV), levator arcuum branchialium; oh, orbitohy-oideus; rc, rectus cervicis; so, subarcualis obliquus; srI (d-v), subarcualis rectus I (dorsal head -ventral head); srII-IV (a-p), subarcualis rectus II-IV (anterior part - posterior part); tp, tympa-nopharyngeus.Figure 3 (bottom). Hyla nana Stage 31. Branchial and spinal musculature. Some muscles of thehyoid group are also shown. Ventrolateral view. Legends: cb (II-IV), constrictor branchialis; gh, ge-niohyoideus; ha, hyoangularis; ih, interhyoideus; lab (I-IV), levator arcuum branchialium; oh, or-bitohyoideus; rc, rectus cervicis; so, subarcualis obliquus; srI (d-v), subarcualis rectus I (dorsalhead - ventral head); srII-IV (a-p), subarcualis rectus II-IV (anterior part - posterior part); tp, tym-panopharyngeus.

M. F. VERA CANDIOTI & A. HAAS: Hyobranchial Apparatus of Hyla nana10

Cuad. herpetol., 18 (1): 3�15, 2004 11of two heads in the m. subarcualis rec-tus I, instead of three, is not a frequentfeature among hylids. In fact, among 25studied species, only eight have thisconfiguration, while the 70% corre-sponds to the alternative state. Thisproportion is also maintained consider-ing Anura as a whole (24 over a totalof 81 species studied by Haas, 2003).As to the m. subarcualis rectus II-IV,it also shows a rather atypical configu-ration as compared to other hylids. Sim-ilar to pseudines (Haas, 2003; Vera Can-dioti, 2004), the muscle in Hyla nana isdiscontinuous. Its fibers are interruptedat the level of processus branchialis III.In H. nana, however, this division isnot complete. Some fibers of the anteri-or part merge with those of the m. con-strictor branchialis IV, and other fibersjoin to the posterior part. All of theother hylid species examined have a sin-gle muscle that extends between theceratobranchial IV and the ceratobran-chials I or II (Haas, 2003).The m. subarcualis obliquus origi-nates in the ceratobranchials II and III,acquiring a clearly bifurcated shape.This is frequent among hylines (Haas,2003; pers. obs.). In pelodryadine hylidspecies such as Litoria genimaculata, L.nannotis, L. xanthomera, L. rheocolaand Nyctimystes dayi the m. subarcualisobliquus originates from ceratobranchi-als II and III and fibrous tissue betweenthem, acquiring a fan shape (Haas andRichards, 1998; Haas, 2003).The mm. levatores arcuum branchia-lium I and II are almost undistinguish-able, or even fused in several taxa(Haas, 2003) whereas in Hyla nana,even when these muscles are very closeto each other, it is still possible to tellthem apart. In like manner, the m.tympanopharyngeus and m. levator arc-uum branchialium IV are undistinguish-able in several taxa, whereas in H.nana, there exists a clear separation.H. nana features coincide with those ofhylid species, such as H. ebraccata, Sci-nax ruber, S. nasicus and Phyllomedusi-

nae (Haas; 2003; pers. obs.).Comparisons of the mentioned skele-tal and muscular characters amongHyla nana and other hylid tadpoles andnon-hylid tadpoles with similar feedinghabits are summarized in Table 1.Some skeletal characters in the hyo-branchial apparatus of Hyla nana, suchas large ceratohyals and hypobranchials,bar-like ceratobranchials with reduced orabsent spicula and lateral projections,seem to be linked to ecological habits(see also Alcalde and Rosset, 2003; VeraCandioti et al., 2004). They are associat-ed with a type of feeding which doesnot depend on the filtration capability ofthe hyobranchial apparatus (macrophagy,endotrophy), or with the generation ofgreat suction forces inside the buccalcavity (macrophagy, suctorial habits).They then appear in both taxonomicallyrelated and taxonomically unrelated spe-cies which share a similar functioning ofthose features (other macrophagoushylids, Ceratophryinae, endotrophic�Leptodactylidae�, Pipidae, Rhacoph-oridae, Rhinodermatidae). Other charac-ters, such as the commissurae proxi-males and copula I, even when theirabsence is shared by all the non-hylidmacrofages, cannot be accounted forsolely on ecological grounds due to theirvariable distribution within Hylidae andtheir co-occurrence with different feed-ing types.On the other hand, characters inbranchial muscles do not seem to berelated to feeding habits but to phyloge-ny, since muscular configuration in Hylanana and H. microcephala resemblesthat of Pseudinae rather than that ofother macrophagous tadpoles (pseudinesalso share other muscular characterswith H. microcephala group tadpoles;Vera Candioti, 2004). At least in a qual-itative study �i.e., without consideringmuscles weights, type of muscular fi-bers, etc.� there appear no noticeabledifferences among the species.Gradwell (1972) studied the function-al morphology of the hyobranchial appa-

M. F. VERA CANDIOTI & A. HAAS: Hyobranchial Apparatus of Hyla

na

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Table 1. Comparison among Hyla nana tadpoles and other hylids and non-hylids (only those non-hylids with similar feeding habits wereincluded). Left column show macrophages (Ma) and macrophages carnivores (MaC). Sr I = subarcualis rectus I, Sr II-IV = subarcualis rectus II-IV, So = subarcualis obliquus, Lab I and II = levator arcuum branchialium I and II, Lab IV and Tp = levator arcuum branchialium andtympanopharyngeous.

Cuad. herpetol., 18 (1): 3�15, 2004 13ratus of Rana catesbeiana tadpoles, dur-ing normal gill irrigation. He describedthe participation of some of the musclesin pharyngeal and branchial pumpmovements. The action of some otherbranchial muscles was established bySokol (1962). Furthermore, the morpho-logical variation of the hyobranchial ap-paratus has been correlated with slightmodifications in mechanisms of normalgill irrigation (i.a., Gradwell, 1971; 1972;1973; 1974). Formerly, hyobranchialmovements and muscle actions duringirrigation and feeding were consideredto be essentially the same (De Jongh,1968). However, electromyographic stud-ies have shown differential participationof certain muscles in such processes(Larson and Reilly, 2003). Thus, a moreprofound study of branchial musculaturewould be necessary to look for correla-tion between branchial muscles and eco-logical habits (not only feeding but alsogill irrigation). Or, as sustained by sev-eral authors, the anatomy mainly re-flects inherited characters, and morpho-logical designs are flexible enough tonot require modifications according spe-cific ecological habits (i.a., Gans et al.,1985; Gans and De Vee, 1987; Abdalaand Moro, 1996).ACKNOWLEDGEMENTSThis study was done during M. F.Vera Candioti�s research fellowship atthe Institut für Spezielle Zoologie undEvolutionsbiologie mit Phyletichem Mu-seum, Friedrich-Schiller Universität.Jena, Germany. We wish to thank theDeutscher Akademischer Austauschdi-enst for funding the fellowship. We aregrateful to M. S. Fischer, L. Olsson, K.Felbel, E. Gretscher and M. Roser fortechnical assistance, stimulating discus-sions and unconditional support. M. F.Vera Candioti would also like to thankC. Argot, D. Arnold, R. Beutel, S. Freit-ag, R. Hackard, M. Hasselmann, J.Hochbach, T. Kleinteich, K. Lilje, C.

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