zoology

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ZOOLOGY Zoology 109 (2006) 26–42 www.elsevier.de/zool

Extreme tadpoles: The morphology of the fossorial megophryid larva, Leptobrachella mjobergi Alexander Haasa,, Stefan Hertwigb, Indraneil Dasc a

Biozentrum Grindel und Zoologisches Museum, Martin-Luther-King-Platz 3, D-20146 Hamburg, Germany Institut fu¨r Spezielle Zoologie und Evolutionsbiologie, Erbertstr. 1, D-07743 Jena, Germany c Institute of Biodiversity and Environmental Conservation, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia b

Received 15 June 2005; received in revised form 9 September 2005; accepted 30 September 2005

Abstract The bizarre larvae of Leptobrachella mjobergi are fossorial and live in the gravel beds of small streams. These tadpoles are vermiform in body shape. Here we present details on their skeleton and musculature, particularly of the head. The entire cranium and its associated musculature are reconstructed in three dimensions from serial histological sections. The hyobranchial apparatus is highly reduced. The head of the L. mjobergi larva is more mobile than in other anuran species. This mobility can largely be ascribed to the exclusion of the notochord from the cranial base and an articulation of the foramen magnum floor with the atlas of the tadpole. The articulation is unique among anuran species, but design parallels can be drawn to salamanders and the articulation between atlas and axis in mammals. In L. mjobergi, the atlas forms an anterior dens that articulates with the basal plate in an accessory, third occipital articular face. The muscle arrangements deviate from the patterns found in other tadpoles: For instance, epaxial and ventral trunk muscles reach far forward onto the skull. The post-cranial skeleton of L. mjobergi is considerably longer than that of other anurans: it comprises a total of 35 vertebrae, including more than 20 post-sacral perichordal centra. Despite a number of features in cranial and axial morphology of L. mjobergi, which appear to be adaptations to its fossorial mode of life, the species clearly shares other features with its megophryid and pelobatid relatives. r 2005 Elsevier GmbH. All rights reserved. Keywords: Pelobatidae; Megophryidae; Anuran larvae; Cranial morphology; Cranial musculature; Fossorial tadpole

Introduction With over 5000 extant species, anurans are the most successful group of lissamphibians (Frost, 2004). The remarkable diversity of reproductive modes, larval forms and adaptive strategies exhibited by anurans are unquestionably major determinants of their evolutionCorresponding author.

E-mail address: [email protected] (A. Haas). 0944-2006/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.zool.2005.09.008

ary success (McDiarmid and Altig, 1999). Ecomorphological guilds have been described containing distantly related species indicating much of convergence in the evolution of adaptive types and resource use. In different regions of the world equivalent microhabitats are used by similar larval ecomorphs belonging to different frog taxa (Orton, 1953; Altig and Johnston, 1989; Altig and McDiarmid, 1999a). An elongate and slender larval ecomorph has evolved in the megophryid genus Leptobrachella (Inger, 1983,

ARTICLE IN PRESS A. Haas et al. / Zoology 109 (2006) 26–42

1985). Species of Leptobrachella are restricted to Borneo and the Natuna Island where the adults live along river banks (Dring, 1983; Inger, 1983; Inger and Stuebing, 1991). Currently, seven species are recognized (Frost, 2004). The body can be described as vermiform or eellike, and has an almost seamless transition from the narrow, cylindrical trunk into the strong tail. The tail fin is very low (Fig. 1). The combination of these unusual external features is indicative of a fossorial life style. The external morphology and details of the bucco-pharyngeal cavity of Leptobrachella mjobergi larvae have been described by Inger (1983, 1985). A similarly elongate body form is also known from the closely related Leptolalax (Inger, 1985). Apart from megophryids, slender tadpoles have been described for unrelated taxa, such as the ranid Staurois (Inger and Wassersug, 1990; Malkmus et al., 1999), and centrolenids (MijaresUrrutia, 1990; Jaramillo et al., 1997; Ibanez et al., 1999; Noonan and Bonett, 2003; Altig and McDiarmid, 1999a).

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We collected L. mjobergi tadpoles from a small stream in a secondary forest patch, within a largely cultivated landscape. Tadpoles were found among the superficial layer of gravel, particularly in riffles in the middle of the stream (see Fig. 1 with gravel from the collection site). Syntopic amphibian fauna included Ichthyophis sp., Staurois guttatus, Pelophryne signata, Ansonia minuta, Megophrys nasuta, Limnonectes malesianus, and Nyctixalus pictus. The morphology of the musculo-skeletal apparatus in anuran tadpoles has been reviewed recently (Cannatella, 1999; Rocˇek, 2003). The musculo-skeletal architecture of the cranium is profoundly different in the tadpole and frog stage (Gaupp, 1893). The one is transformed into the other in a dramatic metamorphosis (de Jongh, 1968). Although interspecific variation in tadpole morphology has been documented and its usefulness for phylogenetic reconstruction proven for a variety of species (Pu´gener et al., 2003; Haas, 2003), the limits to which the general tadpole body plan has been modified in evolution in various groups have not been explored at depth. The study of tadpoles with extreme life histories is essential to understand innovative adaptations and alterations in larval evolution. An account on cranial skeletal and muscular features of L. mjobergi tadpoles is not available. In the present study, we give a detailed description of the cranial musculoskeletal system of L. mjobergi and address the following questions: (1) Which features distinguish the tadpole of L. mjobergi from other anurans? (2) Can these features be related with a fossorial lifestyle and a particular feeding mode? (3) Finally, do the special features of L. mjobergi obscure its phylogenetic relatedness to other megophryids? Although cranial morphology is the main focus, we will give preliminary data on postcranial features.

Materials and methods

Fig. 1. Movements of living Leptobrachella mjobergi. Two individuals (a and b/c) were captured and transferred to an aquarium. The specimen in (a) shows strong dorsal extension of the trunk vertebral column. The specimen in (b) and (c) demonstrates burrowing abilities in (b) and ventral flexion of head in (c). The gravel in (b) and (c) was taken from the collection site at Annah Rais.

Specimens of L. mjobergi tadpoles were collected from a small stream within a secondary forest at the foothills of Gunung Penrissen (N 011090 2600 , E 1101170 2900 ) near Anna Rais village, Sarawak State, Malaysia (Western Borneo). Adults of L. mjobergi were found calling in numbers along the banks of the brook. Assignment of larvae to the species was based on the descriptions by Inger (1983, 1985), and the abundance of adult L. mjobergi in the habitat. Tadpoles were anesthetized and killed in chlorobutanol (Sigma T-5138), fixed and stored in neutral buffered formalin (4%). Four sources of information were used for this study: (1) plain preserved specimens (external characters); (2) serially sectioned specimens (soft tissue, musculature and skeletal characters); (3) manually

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A. Haas et al. / Zoology 109 (2006) 26–42

dissected specimens (soft tissue, musculature); and (4) cleared and stained whole-mount preparations (mostly skeletal characters). Whole-mount specimens were processed according to the protocol of Taylor and van Dyke (1985). For serial sectioning, two specimens were decalcified in 2% ascorbic acid (Dietrich and Fontaine, 1975), dehydrated, embedded in paraffin and sectioned at 7 and 8 mm thickness, respectively. Two other specimens were embedded in Historesins (Leica) and sectioned at 3 mm. Sectioning was performed with a Microm HM360 rotary microtome equipped with the Microm water transfer system for paraffin, and a glass knife for resin sectioning, respectively. Paraffin sections were stained with Heidenhain’s Azan (Bo¨ck, 1989); plastic resin sections in methylene blue and basic fuchsin solution (6 parts ethanol 100%, 4 parts methylene blue 0.13%, 3 parts basic fuchsin 0.13%, 8 parts sodium borate 1%). Specimens for manual dissection were prepared by applying only the first staining step (Alcian blue) of the clearing and staining protocol with subsequent rinsing and transfer to 70% ethanol. This procedure contrasts dark blue stained cartilages against white muscles. Drawings were made either from digital photographs (Zeiss SV11 stereomicroscope equipped with a digital video camera ColorView 12, software analySISTM; both Soft Imaging System GmbH, Germany) or with a camera lucida on a Zeiss SV11. Data on individuals examined are summarized in Table 1. The serially sectioned specimen BrachellaM1 was chosen for a three-dimensional reconstruction of the complete cranium and associated musculature. Only the muscles of the right side of the body were reconstructed. Every second histological section was photographed digitally with a Canon Powershot S50 camera mounted on a Leica MZ 9.5 stereomicroscope and connected to a Apple Macintosh G5. Similar to an account given in Haas and Fischer (1997), digital images were imported as background image planes into Alias-Wavefront Mayas 5.01 software. Contour lines of bones, cartilages and muscles were digitized manually from image planes. Contour lines were aligned at the proper distance to each other and moved and rotated manually in the transverse plane to generate best fit. A cleared specimen Table 1.

was used as additional reference for alignment. Subsequently, surfaces were built starting with shape primitives and forming and refining them with various tools in Mayas to closely fit the surfaces to the contour line stacks. Reliable reconstruction is impossible when a muscle has widely spaced fibers or fiber bundles and when the artifacts and distortions due to histological sectioning are much greater than the fibers’ diameters. This was particularly true for the extremely fine and spaced fibers of the m. interhyoideus posterior. This muscle was not reconstructed. In the m. levator arcuus branchialis (I+II) the muscle’s fibers were approximated in position, length, and number as closely as possible. Others, namely the m. mandibulolabialis and the m. transversus ventralis IV had less fiber spacing than the previous two muscles and were reconstructed as closed surface for simplicity. Final 3D surface reconstructions were rendered with Mayas’s software. The anatomical terminology largely follows the summary in Rocˇek (2003) for skeletal structures and Haas (1997, 2001, 2003) for hyobranchial and jaw musculature. Post-cranial muscles were identified according to Gaupp (1896). A supplementary movie file is available on the journal’s pages (www.elsevier.de/zool) for download. The movie shows an animation of the computerreconstructed skull of a Leptobrachella mjobergi larva (Quicktime 7 required).

Results External features The body is vermiform. Head and trunk are oblong, and together account for up to one-third of the total length (Fig. 1). The trunk is approximately cylindrical in cross section but the head is bluntly conical and slightly depressed. The trunk is capable of considerable hyperextension (Fig. 1a). The pigmentation is uniformly dark brown above. Small specimens o20 mm are only faintly brown with overall white-bluish to pinkish appearance. The venter is light blue in life and the gut coils are only

List of specimens examined for this study

Study identification

Head–body length

Total length

Preparation

BrachellaM BrachellaM BrachellaM BrachellaM BrachellaM BrachellaM

9.0 7.5

27.4 22.3 21.0 17.0 26.6 25.5

Serial section, paraffin, 3D Serial section, paraffin Serial section, plastic resin Serial section, plastic resin Micro-dissection Cleared and stained

1 2 3 4 5 6

¼ no measurements available.

9.0 8.8


faintly visible. The oral disk is distinctly protruding antero-ventrally and funnel-shaped with deep dorsal and ventral disk emarginations that incompletely subdivide the oral funnel into right and left halves. The oral disk is without pigmentation; keratodonts are absent. The marginal papillae stand in an uniserial line at the margin of the oral disk. Some submarginal papillae are present around the mouth orifice. The naris is closer to the snout than to the eye. The eyes are positioned dorsolaterally and are relatively small and sunken in; they hardly protrude beyond the contour of the body. The spiraculum is sinistral, long and tubular, clearly protruding from the body wall. The vent is dextral. The dorsal fin originates slightly posterior to the trunk–tail junction. It is low on the proximal half of the tail, expanding only posteriorly. The tip of the tail is blunt. The muscular part of the tail is strong and myotomes are clearly visible. The specimens examined ranged from 17 to 27.5 mm in total length and would fall into stage 25 according to Gosner (1960) (Table 1). Application of this staging table, however, is not meaningful in L. mjobergi, because hind limb development appears to be more delayed relative to growth and the development of internal structures than in most other tadpole species. The maximum head–body length is 13.8 mm (Inger, 1983).

Cranial skeleton Neurocranium and first visceral arch The neurocranium is oblong, narrow, and approximately parallel sided (Fig. 2). The lateral contours do not expand but rather narrow at the posterior part of the palatoquadrate. The widest point of the neurocranium is at the mid-level of the ovoid otic capsules. Arcus occipitalis and tectum synoticum form the foramen magnum and connect the otic capsules to the planum basale and to each other. The arcus occipitalis also delimits the foramen jugulare posteriorly. The otic capsule is well chondrified and has four foramina: the foramen endolypmphaticum inferius posteriorly, the foramen endolymphaticum superius posteromedially, the foramen acusticum medially close to the planum basale, and the foramen endolymphaticum dorsally in the medial capsular wall. The fenestra ovalis is formed but no operculum is present. The medial wall of the otic capsules is confluent with the planum basale. The posterior margin of the planum basale is deeply emarginated. The notochord does not enter the planum. The planum is approximately as wide as long. Anteriorly it gives rise to the trabeculae cranii. They form an elliptical arch and meet anteriorly at the planum trabeculare anticum (Figs. 2–4). The trabeculae cranii encircle the fenestra intertrabeculare, which is unchon-

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drified. However, the bony ring of the parasphenoid fits into the intertrabecular opening. The cornua trabeculae originate from the planum trabeculare anticum anteriorly (Figs. 2 and 3). They are fused for most of their lengths. Distally they diverge, curve laterad and form a pronounced lateral process for articulation with the cartilago labialis superior. None of the specimens has chondrified nasal structures. Anteriorly, the palatoquadrate is connected to the trabecula cranii by the commissura quadrato-cranialis anterior. Posteriorly, the upward curving processus ascendens quadrati connects to the pila antotica of the cranial sidewall (Figs. 2 and 5). A processus oticus is absent. The palatoquadrate is widest anteriorly and thin and slender at the processus ascendens. Anteriorly, the pars articularis quadrati—mostly its lateral margin— articulates with the cartilago meckeli. The palatoquadrate articulates with the ceratohyale ventrally (Figs. 4 and 5). The processus muscularis quadrati is visible most clearly in lateral view (Fig. 5). It is relatively narrow at the tip. A commissura quadrato-orbitalis, connecting the process’s tip to the sidewall of the braincase, is absent. The tip of the processus muscularis extends anteriorly. Its posterior edge is flat, almost horizontal in orientation, whereas the anterior edge is steep and overhanging. The processus muscularis bears a prominent lateral process close to its anterior margin. This process borders the m. orbitohyoideus anteriorly (Fig. 6). The sidewall of the cavum cranii is only weakly chondrified. The processus ascendens clearly connects to the pila antotica above the center level of the foramen oculomotorii (Fig. 5), i.e., a high processus ascendens is present in the species. An arch of cartilage originates from the pila antotica anteriorly and curves ventrad to connect to the trabecula cranii and encircle the foramen oculomotorii. The sidewall does not form a foramen opticum. The nervus opticus simply passes through the membraneous lateral wall of the cavum cranii anterior to the foramen oculomotorii. The pila antotica gives rise to anterior and posterior processes dorsally. Both form the upper marginal cartilages of the sidewall. Frontoparietals have not yet formed. The lower jaw is segmented into two functional units, the cartilago meckeli laterally and the cartilago labialis inferior (or infrarostral cartilage) medially. Meckel’s cartilage is robust and conspicuously convex dorsally (Fig. 7). It forms the processes present in most anuran larvae: dorsomedial and ventromedial processes as part of the articulation with the infrarostral cartilage, and the processus retroarticularis as insertion point for the angularis-muscle group. The anterior face of the cartilago meckeli is deeply concave. The cartilago labialis inferior is U-shaped in frontal view, with a broad and flat middle part. Both infrarostral cartilages are connected by a medial symphysis.

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Fig. 2. Drawing of a cleared and double-stained specimen (study ID BrachellaM6): (a) ventral view, and (b) dorsal view of neurocranium, first visceral arch, and first three vertebrae.

The movable upper jaw, cartilagines labiales superiores (or suprarostral cartilages) are U-shaped in ventral view (Figs. 2 and 4). Right and left suprarostrals are fused in the midline but bear a clearly discernible suture (Fig. 4). There is no sign of subdivision of the cartilago labialis superior into a pars corporis and pars alaris as in many other frog species. Distally, the cartilage forms a broad and blunt posterodorsal process. It articulates syndesmotically with the adrostral cartilage (Figs. 2 and 3). The latter is a smoothly L-shaped body of cartilage positioned dorsolateral to the posterodorsal process and anterodorsal to the cartilago meckeli. Hyobranchial apparatus The hyobranchial skeleton (Fig. 7) is remarkable in many respects. The axis running from the processus anterior to the processus posterior of the ceratohyale is long, whereas the processus lateralis hyalis is short in comparison. This geometry gives the ceratohyale an

overall oblique orientation (Fig. 7). The processus lateralis hyalis is broadly expanded dorsoventrally (Fig. 5), forming an articular condylus for articulation with the palatoquadrate at its dorsal edge. Anterolateral processes are not formed at the ceratohyalia. The elongation of the basibranchiale parallels the antero-posterior extension of the ceratohyale. The basibranchiale connects by synchondroses (stained only faintly by Alcian blue) to the plana hypobranchiales posteriorly. A processus urobranchialis on the ventral side of the basibranchiale is absent. The plana hypobranchiales are fused medially (synchondrosis). The posterior, tapering end of each planum continues posteriorly in a rod of cartilage that, after a short distance, expands into spiculum IV that is flat and bears four horizontal projections (Fig. 7). Ceratobranchiale I originates from the lateral margin of the planum hypobranchiale. It forms a broad processus anterior branchialis that extends into the


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Fig. 3. Three-dimensional computer reconstruction from serial histological sections of specimen BrachellaM1. Cranium, including hyobranchial apparatus, and atlas in dorsal views. In (a) the complete cranial musculature is shown. In (b) superficial muscles are hidden to expose some deep muscles.

posterior concavity of the ceratohyale. Beyond the processus anterior branchialis the ceratobranchiale I continues laterally as a very slender rod of cartilage bent in an S-shape (Fig. 7). All four ceratobranchialia are thin and end freely distally; commissurae terminals and lateral projections (common in other species) are absent, and there is no commissura proximalis I. Ceratobranchialia II and III are fully confluent proximally, i.e., the commissura proximalis II is present. Both ceratobranchialia form a broad U-shaped arch, which is open distally. The proximal base of the arch connects to the posterior prolongation of the planum hypobranchiale (Figs. 4 and 7). At the same transverse level, a strong processus branchialis projects ventrally from the connected ceratobranchials and bends medially with its tip. The spicula are unique in several features. The proximal end of spiculum I is in touch with, but does not fuse confluently with, ceratobranchiale I. In arching posterolaterally, spiculum I gets close to ceratobranchiale II. Spiculum II is connected to ceratobranchiale II and ceratobranchiale I only by perichondrial contact. Spiculum III likely is the most anterolateral, finger-like projection from the plate extension of spiculum IV. Ceratobranchiale IV descends from the ventral side of

the spiculum IV-plate. After a short distance it bends laterally and soon meets ceratobranchiale III. Both are connected by the fibers of their perichondrium but they are not confluent. Although the condylus of the ceratohyale is located relatively far posteriorly, the long ceratohyalia fill much of the space between the partes articulares quadrati (Fig. 4). The branchial part of the hyobranchial apparatus is not the basket-shaped structure commonly found in other species; rather, it is small and flat (Figs. 4, 5 and 10). It hardly goes beyond the otic capsule contours in ventral view (Fig. 4). Haas and Richards (1998) proposed 16 landmarks to estimate the relative contribution of the ceratohyal, hypobranchial plate, and branchial basket to the buccopharyngeal floor area. In L. mjobergi, the total area is composed of 42% ceratobranchial (CH), 18% hypobranchial (HB), and 40% branchial basket (BB) area (specimen BrachellaM6). Thus, BB is relatively reduced and CH relatively enlarged in L. mjobergi (24–28% and 55–62%, respectively, in generalized Litoria tadpoles; Haas and Richards, 1998). The lever arm ratio of the ceratohyal (b/a, Fig. 8; Wassersug and Hoff, 1979) is 0.3 when measured perpendicular to the longitudinal body axis, but approx. 0.4 when measured along an oblique

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Fig. 4. Three-dimensional computer reconstruction from serial histological sections of specimen BrachellaM1. Cranium, including hyobranchial apparatus and atlas, in ventral views: (a) all muscles shown, and (b) superficial muscles made invisible.

Fig. 5. Three-dimensional computer reconstruction from serial histological sections of specimen BrachellaM1. Cranium, including hyobranchial apparatus and atlas, in lateral view.

axis (x, Fig. 8). The geometry of the ceratohyale (a ¼ 661; Fig. 8) and the oblique orientation of the m. orbitohyoideus (Fig. 6) suggest an oblique axis of rotation of the ceratohyale.

Vertebral column The notochord is the major element in the larval axial skeleton. Unlike typical pond tadpoles from other


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Fig. 6. Three-dimensional computer reconstruction from serial histological sections of specimen BrachellaM1. Cranium, including hyobranchial apparatus and atlas, in lateral views. In (a) all cranial muscles are shown, and in (b) some superficial muscles are hidden to expose deeper muscles.

species, the notochord in L. mjobergi does not project into the planum basale of the neurocranium (Figs. 3 and 4); rather, it ends within the first presacral vertebra, the atlas. The anterior tip of the notochord lies in a

prominent, ossified anterior process of the atlas. The process has similarity to the dens axis of amniotes and is considered a dens analog (Fig. 4). This dens is fastened to the planum basale by a ligamentum transversum

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height. The following more posterior vertebrae diminish in size gradually and the last one is only a faintly stained ossification at the perichordal sheath. In specimen BrachellaM6, there are 35 vertebral centra ossifications (Fig. 9). The notochord is fully encircled by the vertebral ossifications (perichordal formation).

Musculature

Fig. 7. Three-dimensional computer reconstruction from serial histological sections of specimen BrachellaM1. Lower jaw cartilages and muscles originating from it. Frontal view.

occipitalis (new term for tadpoles) that runs over its tip (not shown in figures). The atlas forms two relatively large anterolateral processes (proc. articulares; atlantal cotyles) to articulate with the arcus occipitalis of the neurocranium (Figs. 3 and 4). The faces of the processus articulares are concave anteriorly to match the convex shape of the arcus’ condyle. The dens of the atlas is the third, medial articulation of the atlas with the neurocranium. In specimen BrachellaM6, transverse processes are present on vertebrae II and III. The neural arches of the atlas arise from the dorsolateral side of the centrum, posterior to the processus articulares. The atlas’ neural arches are devoid of transverse processes. The neural arches’ dorsal ends bend abruptly posteriorly and are broad and cartilaginous. Their posteroventral corners form postzygapophysial articulations with the prezygapophysis of the subsequent vertebra. In specimen BrachellaM6 (Fig. 9), only presacral vertebrae V and VI form closed neural arch rings around the vertebral canal. All other neural arches are open dorsomedially at this stage. Vertebral centra vary in size along the antero-posterior axis. The atlas is shortest and vertebral length increases in the subsequent vertebrae (Fig. 9). As in most other frogs, vertebra IX is the prospective postmetamorphic sacral vertebra. Its neural arch has a prezygapophysis to articulate with vertebra VIII but no postzygapophysis to vertebra X. From vertebra IX on, ten large centra follow that are notably increased in

Jaw muscles The m. mandibulolabialis comprises superior and inferior parts, which extend into the anterior and posterior parts of the funnel-shaped oral disk, respectively. Keratodonts are absent, thus the muscle’s fibers do not insert in keratodont ridges; rather, fibers run toward the marginal area of the oral disk. Distally, the fibers are single layered and slightly spaced (reconstructed as a closed sheath in the 3D model for simplicity; Figs. 4 and 6). The m. mandibulolabialis inferior originates with bundles of fibers from two sites: the posterior side of the processus ventromedialis and the anterior face concavity of Meckel’s cartilage, lateral to the cartilago labialis inferior. The m. mandibulolabialis superior originates lateral to the inferior part, somewhat dorsal on the anterior face of cartilago meckeli (Figs. 6 and 7). A m. submentalis is absent. The m. intermandibularis originates relatively far laterally from the anterior face of cartilago meckeli, immediately ventral to the origin of the m. mandibulolabialis superior. The bundle of fibers curves around cartilago meckeli ventrally. The muscle flattens out distally, approximately ventral to the pars reuniens (Fig. 4). The m. intermandibularis meets its counterpart from the other side in a median raphe of connective tissue. The m. levator mandibulae internus arises from the anteroventral face of the otic capsule and adjacent planum basale (parachordal) (Figs. 3, 6 and 10). It runs anteriorly over the fenestra subocularis. It crosses the pars articularis quadrati dorsally but is ventral to all other muscles of the levator series. It inserts on the most lateral prominence of cartilago meckeli (Fig. 6). The m. levator mandibulae longus superficialis is located in an unusual medial position. Three heads originate posteriorly, unite more anteriorly, and finally insert via a short tendon on the processus dorsomedialis meckeli (Fig. 6). The origins are: (1) lateral side of trabeculae cranii (posterior to level of pila antotica); (2) pila antotica, processus ascendens and cupula anterior of otic capsule; and (3) connective tissue dorsolateral to otic capsule and fascia of m. levator mandibulae longus profundus. The m. levator mandibulae longus profundus is the largest of the jaw levators (Fig. 3). Its superficial fibers are almost confluent posteriorly with the paravertebral


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Fig. 8. Hyobranchial apparatus in dorsal view. Drawing of a cleared and double-stained specimen (study ID BrachellaM6). The ceratohyalia and basibranchiale are particularly long in the anterior–posterior axis. The ceratobranchialia are thin curved rods of cartilage without any lateral projections. The ceratobranchialia do not form commissurae terminales at their ends (see text for details). Lever arm ratio: b/a.

Fig. 9. Vertebral column. Drawing of a cleared and double-stained specimen (study ID BrachellaM6). Note the number and sizes of vertebrae. Arrow points to future sacral vertebra (IX). Bone stippled, cartilage shaded.

system. The longissimus tract and the m. lev. mand. profundus are separated only by a tendinous inscription. Deep bundles of the profundus originate from the otic capsule and the posterior parts of the palatoquadrate, including the processus ascendens. The internal fiber architecture of the profundus is characterized by loosely spaced bundles of fibers (Fig. 10). This does not seem to be a histological artifact as the feature is present in all specimens sectioned, both paraffin and plastic resin embedded, as well as in different larval sizes. The fleshy part of the profundus ends approximately at the level of the jaw joint from where it continues as a long tendon (Fig. 6) that runs over the cartilago meckeli, bends ventromediad, posterior to the adrostral, and passes medial to the m. mandibulolabialis to attach at the posteroventral margin of the cartilago labialis inferior. The m. levator mandibulae articularis is a short and thin muscle that originates from the dorsal side of the

palatoquadrate at the base of the processus muscularis (Fig. 3). It attaches to the lateral part of cartilago meckeli, dorsal to the insertion of the m. lev. mand. internus (Fig. 6) and crosses the latter. The m. levator mandibulae externus is short and thin. It originates from the medial side of the processus muscularis quadrati (Fig. 3). From there it extends anteriorly for a short distance to merge with the tendon of the m. levator mandibulae longus profundus. The m. levator mandibulae lateralis is absent. The angularis group is part of the depressor mandibulae group (cranial nerve VII innervation). Three muscles of different lengths and orientations belong to this group; all of them insert on the processus retroarticularis meckeli (Figs. 4 and 6). The m. quadratoangularis is the most ventromedial of the three. It originates from the ventral side of the pars articularis quadrati (Fig. 4). It originates immediately anterior to

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Fig. 10. Transverse section through the anterior otic region. The section illustrates several mentioned features: the spaced fibers of the m. levator mandibulae superficialis, dermal layer thickness of the skin, flat branchial apparatus, buccal floor and buccal roof papillae, lack of filter plates.

the quadrato-hyal articulation. The insertion is fleshy, without a notable tendon, at the ventromedial side of the processus retroarticularis. The m. hyoangularis is the strongest muscle of the group as estimated from crosssectional area. Several more or less separate tracts of fibers originating from the ceratohyale belong to this muscle (Fig. 4). The m. suspensorioangularis has its fleshy origin along the lateral side of the processus muscularis quadrati (Fig. 6). In lateral view it is covered by the mm. orbitohyoideus and suspensoriohyoideus. The insertion of the suspensorioangularis is mediated by a long tendon that merges with the tendon of the m. hyoangularis and inserts ventrally at the processus retroarticularis of cartilago meckeli. Hyobranchial muscles Despite belonging to the depressor mandibulae group by innervation (cranial nerve VII) and metamorphic fate, the mm. orbitohyoideus and suspensoriohyoideus (Fig. 6) act exclusively on the ceratohyale in larvae. Both originate laterally from the processus muscularis quadrati; the m. orbitohyoideus arises broadly from the tip area of the process, whereas the suspensoriohyoideus

arises from the posterior margin of the process and the adjacent arcus subocularis quadrati. The m. suspensoriohyoideus is medial to the orbitohyoideus (Fig. 6). The m. interhyoideus connects the processus laterales hyalis ventrally in a straight line (Fig. 4). Right and left muscle parts meet in a medial tendinous inscription. The m. interhyoideus posterior is present as a veil of extremely fine, scattered and spaced fibers underlying the epithelium of the posterolateral and posterior wall of the cavum peribranchiale. Due to its small fiber diameter and scattered fibers it was not possible to reconstruct the muscle reliably from histological serial sections. It is not shown in the figures. Muscles associated with the branchial arches are weakly developed. The branchial levators are small. The mm. levatores arcuum branchialium I+II are merely loosely scattered fibers in a connective tissue sheath (it is hardly possible to reconstruct those fibers precisely from paraffin sections; Figs. 3, 4 and 6 give only an approximation of size, number, and orientation). The muscle fibers of branchial levators III+IV are much more organized in a bundle and attach to the terminal tips of ceratobranchiale III and IV, respectively (Fig. 6).


Branchial levator III originates from the otic capsule dorsal and posterior to the foramen ovale. Levator IV originates from connective tissue ventral to the cupula posterior of the otic capsule. The m. transversus ventralis IV originates from the same strand of connective tissue more medially and expands mediad and anteriad as a horizontal fan of loose fibers (reconstructed as closed surface in the 3D reconstruction). The short m. dilatator laryngis is embedded in the latter muscle (Fig. 4). Three mm. constrictores branchiales are present (II–IV) in the branchial septa. Constrictor I (sensu Haas, 1997) is absent. All three constrictors insert on the arch formed by ceratobranchialia II and III in reversed order, i.e., constrictor II inserts most posteriorly, constrictor IV most anteriorly (Fig. 4). Constrictor II originates from the distal end of ceratobranchiale II. Constrictores III and IV both take origin from the tip of ceratobranchiale III. The processus branchialis is the attachment site for the mm. subarcualis rectus I, subarc. rectus II–IV, subarc. rectus accessorius, and subarcualis obliquus II (Fig. 4). The m. subarcualis rectus I has, unlike in most other known tadpoles, only one head. The site of origin is at the lateral side of the processus posterior hyalis. Conversely, the m. subarcualis rectus II–IV is short. It connects the processus branchialis to the distal ceratobranchiale IV. The m. subarcualis rectus accessorius originates from the ventral side of the spiculum IV plate. It inserts ventral to the m. subarc. rectus IV on the processus branchialis. Finally, the m. subarcualis obliquus II originates from the processus branchialis and reaches anteromedially to the mid-sagittal plane where it forms a weak tendon. The tendon runs rostrad in the mediosagittal plane and attaches to the flat ventral side of the posterior end of the basibranchiale (a processus urobranchialis is not formed). In most tadpoles, the m. diaphragmatobranchialis attaches to ceratobranchiale III. In L. mjobergi, the muscle is detached from the branchial apparatus and instead inserts on the lateral wall of the otic capsule (canalis semi-circularis lateralis). The muscle takes origin from the connective tissue of the abdominal septum. The muscle consists of two columns tightly bundled along most of their length, but diverging at both ends (Figs. 4 and 6). The m. rectus cervicis originates from the same abdominal septum tissue and runs between the columns of m. diaphragmatobranchialis (Fig. 6). The m. rectus cervicis is a thick muscle, however, internally composed of loosely spaced bundles of fibers. The m. rectus cervicis inserts far anteriorly on the ventral side of the ceratohyale (processus anterior). Another longitudinal muscle runs along the internal wall of the operculum (i.e., ventral lining of cavum peribranchiale). The muscle likely is an anterior extension of the m. rectus abdominis system. It is a flat muscle

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band composed of few fiber tracts. Anteriorly it attaches to a protuberance, the ventromedial face of the pars articularis quadrati (Fig. 4). The moderately thick m. geniohyoideus connects the posterior margin of the cartilago labialis inferior (near its symphysis) to the ventral side of the planum hypobranchiale (Fig. 4). Postcranial muscles Here we give only a preliminary account of those postcranial muscles that relate to the cranium. Most evidently the paraxial longissimus tract extends far anteriorly on the otic capsule. The muscle’s almost seamless transition into the m. levator mandibulae longus profundus (Figs. 3 and 6) has already been noted above and is a unique feature in L. mjobergi. The m. intertransversarius capitis inferior (Fig. 4) connects the processus transversum of vertebra II (not shown) to the ventral parts of the posterior otic capsule. One bundle of fibers attaches to the otic capsule just ventrolaterally to the foramen perilymphaticum inferius; the second bundle attaches nearby but more medially at the cartilage connecting the otic capsule and the planum basale. The m. intertransversarius capitis superior also originates from processus transversum II but inserts at the arcus occipitalis and adjacent otic capsule cartilage (sulcus occipitalis, Fig. 3). Immediately posterior to the m. diaphragmatobranchialis are two muscles, both part of the body wall musculature. Pending further verification, based on more extensive developmental series, these muscles are tentatively assigned to the m. obliquus. The anterior one, the pars scapularis (Gaupp, 1896) of the m. obliquus originates from the fascia of the paravertebral longissimus system and inserts on connective tissue ventrally where m. rectus cervicis, m. rectus abdominis, and m. diaphragmatobranchialis meet. The posterior portion, the m. obliquus proper, has dorsal and ventral connections to the skin. Ventromedially, the fibers of the pars scapularis could hardly be distinguished in histological sections from the lateral fibers of the m. rectus abdominis; both were reconstructed as one body (Fig. 4). An alternative interpretation would be that what we tentatively call pars scapularis here could be a lateral expansion of the rectus abdominis group.

Discussion Natural history The vermiform tadpole of L. mjobergi lives in the gravel beds of small forest streams. We never collected these larvae from other microhabitats in the same stream, such as leaf litter accumulations (where the

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related M. nasuta is common) or sandy stretches of the stream. In an aquarium filled with gravel from the collecting site (Fig. 1), we observed that large specimens of L. mjobergi are not only able to use existing crevices, but will also push their way into spaces smaller than the body diameter. Thus, L. mjobergi is an active burrowing tadpole. Locomotion is eel-like and characterized by unusual mobility of the head and trunk (Fig. 1). We assume that interstitial spaces are used opportunistically, particularly by small larvae lacking the force to push aside gravel. Larger individuals are able to push aside superficial gravel, and might just burrow deep enough to conceal themselves. Small tadpoles of early larval stages may be able to get deeper into the sediment just by using interstices. The burrow use and burrowing behaviors of L. mjobergi are impossible to observe in the field and need further investigation under laboratory conditions. In preliminary aquarium experiments, L. mjobergi tadpoles did not burrow when offered sand substratum. Active burrowing by tadpoles is rare but has been reported in the South American microhylid Otophryne pyburni (Wassersug and Pyburn, 1987) and in various neotropical centrolenid tadpoles. Similar to L. mjobergi, the larvae of O. pyburni live shallowly buried at the bottom of clear streams; however, the two species prefer different sediments: sand in the case of O. pyburni and gravel in the case of L. mjobergi. Neotropical centrolenid tadpoles live in accumulated leaf litter and stream sediments, and are sometimes found in moist plant debris at some distance from the stream (Villa and Valerio, 1982; Lynch et al., 1983; Mijares-Urrutia, 1990; Hoff et al., 1999). Some were reported to burrow as deep as 200 mm into benthic debris (Savage, 2002). Mijares-Urrutia (1990) found larvae of Centrolenella andina in quiet pools with slow currents. Tadpoles hide superficially in the sediment and among rocks and fallen leaves and escape with bursts of free swimming when disturbed. Mijares-Urrutia did not give information about the grain size of the sediment. In contrast, we found L. mjobergi only in fast flowing, riffle sections of the streams in gravel sediments. We never saw any L. mjobergi tadpole resting exposed or swimming in escape.

Morphology With the exception of Wassersug and Pyburn’s (1987) description of O. pyburni, anatomical accounts of burrowing or fossorial tadpoles are scarce in the literature; none of the centrolenid larvae has been analyzed in great detail (except some character states for Cochranella granulosa in Haas, 2003). Superficially, centrolenid tadpoles are quite similar to the larvae of Leptobrachella in body shape (Starrett, 1960; Mijares-

Urrutia, 1990). Centrolenid tadpoles have long tails with low tailfins and thick muscular parts. Their eyes are rudimentary and pigmentation may be reduced (Villa and Valerio, 1982; Altig and McDiarmid, 1999b). Long and slender tadpoles living in leaf litter have evolved in the southeast Asian Staurois (Inger and Wassersug, 1990; Malkmus et al., 1999, pers. obs.). A more detailed comparison can be made between the tadpoles of L. mjobergi and O. pyburni. Various features of both taxa, particularly of the head, can be hypothesized as adaptations to the respective sediment used. In O. pyburni, the skull is dorsoventrally flattened and broad cartilagines labiales superiores and cornua trabeculae support the snout; both have been interpreted as adaptations to sand penetration (Wassersug and Pyburn, 1987). In contrast, the head of L. mjobergi is narrow and rounded, followed by an almost cylindrical trunk. This confers an eel-like body shape that facilitates entering crevices of unpredictable shape and orientation. The neurocranial and jaw cartilages of L. mjobergi are robust but lack the hypertrophy of snout cartilages and snout skin of O. pyburni. The epidermis of the entire body is supported by a firm and thick dermal tissue layer of collagen fiber in L. mjobergi (Fig. 10), which likely protects the tadpole among the rough-edged gravel (Fig. 1). Various soft tissue structures of O. pyburni that apparently serve to block or trap sand grains and prevent sand from entering the pharynx and gills (e.g., expanded ventral velum; see Wassersug and Pyburn, 1987) are absent in L. mjobergi. Differences prevail, but L. mjobergi and O. pyburni also have some common features. The muscular part of the tail is high at the body–tail junction and the tail fins are low. The tails are used for propulsion in the substrate as well as swimming outside the substrate. The eyes are small and non-protruding. These features also apply to the fossorial tadpoles of centrolenids and Staurois. In L. mjobergi and O. pyburni, the dorsal and ventral trunk muscles extend further rostrally than in other tadpoles and allow for flexion and extension of the cranium. These muscle characters are unknown for centrolenids and Staurois. From the anatomical evidence as well as observations of living animals (Fig. 1), the maneuverability of the head and the anterior vertebral column in L. mjobergi is unusual for anuran larvae. Flexion of the body axis in the sagittal plane has been reported before in other species, but without indication that bending took place between the head and first vertebra (Wassersug, 1992). We present anatomical and photographic (Fig. 1) evidence that bending can occur between head and vertebral column. In six lateral view images we measured the angles between a line connecting the nostril to the center of the eye and a line in parallel with the anterior trunk dorsal contour, including extension


(Fig. 1a), flexion (Fig. 1c), and intermediate resting positions. The angles ranged from 1381 to 1701 giving an approximate range of dorso-ventral head mobility of 321. In Fig. 1a hyperextension seems to stem from both head extension plus a smooth upward arching along all of the presacral vertebral column. In contrast, the ventral flexion of the head in Fig. 1c appears to be restricted to the occipital joint. Lateral bending could not be determined from the existing photographic evidence. We consider two primary features to account for head maneuverability in L. mjobergi: the exclusion of the notochord from the planum basale and the design of the atlanto-occipital joint. Similar joints were reported in salamanders and a fossil caecilian with a median process called tuberculum interglenoideum or odontoid process (Wake and Lawson, 1973; Duellman and Trueb, 1992; Jenkins and Walsh, 1993). We use the term dens atlantis in L. mjobergi to stress the analogy, not homology, of these structures in amphibians. Apart from some amphibians, the similarities of the L. mjobergi occipital joint to the design of the atlantoaxial articulation in mammals, including humans (Leonhardt et al., 1987), are obvious. These involve two inclined lateral articular faces and a medial dens that establishes a third articulation. Both in the L. mjobergi occipital joint and in the mammalian atlanto-axial joint the dens is retained by a transverse ligament. Based on structural analogies we assume that similar axes of rotation are realized in the atlantooccipital articulation of L. mjobergi and the atlantoaxial articulation in mammals (e.g., for humans Leonhardt et al., 1987). We are unaware of any data on rotation in the occipital joint of salamanders. Similar to the dens axis in humans (Leonhardt et al., 1987), in L. mjobergi rotation in the occipital joint around the body axis could happen in the longitudinal axis of the dens atlantis, and lateral and dorsoventral movements by translocation in the atlantal cotyles. In L. mjobergi we assume that the curvatures of the atlantal cotyles and occipital arches define the movement of the atlas on a circular radius in sagittal and frontal planes, respectively. The centers of such dorso-ventral and lateral movements must be more anteriorly than the articulation itself in a virtual center of rotation approximately in the hypophyseal region, depending on the radius of the joint curvature. Our functional understanding of tadpole musculature relies largely on anatomical relationships. Few studies have tested functional hypotheses for anuran tadpoles by electromyography (Gradwell, 1972b; Larson and Reilly, 2003). Activity of most of the thin branchial muscles has never been recorded with EMG in tadpoles. Although EMG measurements have not been made in L. mjobergi, several muscles likely drive cranial movements: the paravertebral longissimus system reaches far

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rostrally and presumably serves for dorsal extension of the head (assisted by the m. levator mandibulae longus profundus; Fig. 6). The mm. rectus cervicis et rectus abdominis also reach far rostrally, ventral to the rotational axis, and therefore could cause ventral flexion of the head relative to the trunk. Asymmetric contractions of the paravertebral longissimus system, the m. rectus cervicis and/or the mm. intertransversarii capitis could generate lateral bending of the head. The m. obliquus (pars scapularis) and m. diaphragmatobranchialis might rotate the head around the longitudinal axis of the dens atlantis. The fact that the m. diaphragmatobranchialis has no connection to the ceratobranchiale III, where it inserts in most species (Gradwell, 1972a; Haas, 1997; Haas and Richards, 1998), corroborates that this muscle has assumed a new function in head movements of L. mjobergi larvae. In other tadpoles it appears to lower the ceratobranchials (Gradwell, 1972b). Kenny (1969a) and Gradwell (1972b) speculated about the function of the branchial muscles in tadpoles. As judged by anatomy alone, several muscles likely are involved in depressing the branchial apparatus (m. diaphragmatobranchialis, m. rectus cervicis), others in elevating it (mm. levatores arcuus branchialium I–IV). It is unclear whether this branchial pump is recruited in normal cyclic irrigation (Larson and Reilly, 2003). In L. mjobergi, the branchial basket is reduced and branchial muscles are feeble. Furthermore, the m. diaphragmatobranchialis is not available for branchial lowering. The overall weak structural condition suggests that branchial arch movements do not contribute significantly to normal irrigation cycles, in terms of a forceful branchial pump supporting the buccal pumping mechanism. Rather, the branchial apparatus probably follows the movements of the ceratohyale passively. The feeble branchial muscles may still play a role in occasional hyper-inspiration or hyper-expiration. Another unusual feature of L. mjobergi is the shape of the parasphenoid. In the stages examined it is a long oval ring with a posterior rectangular plate. The parasphenoid abuts the medial edges of the trabeculae cranii. To our knowledge such a shape of the parasphenoid has not been reported in anuran larvae before.

Trophic structures Currently we can only speculate about the feeding habits of L. mjobergi. Buccal volume, lever–arm ratio, and buccal area can be indicative of macrophagous, generalized, or microphagous feeding preferences in a given species (Wassersug and Hoff, 1979). In L. mjobergi the branchial area (40% of the bucco-pharyngeal floor area) is relatively reduced and the ceratohyal area (42%) relatively enlarged (55–62% and 24–28%, respectively,

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in generalized Litoria tadpoles; Haas and Richards, 1998). The lever–arm ratios of the ceratohyale (0.29–0.5) fall into the groups of benthic generalized tadpoles on the one end and macrophagous tadpoles on the other end of the range (Wassersug and Hoff, 1979), depending on the axis of measurement. The measurements do not allow a clear assignment of L. mjobergi to the classification of feeding modes based on hyobranchial geometry as in Wassersug and Hoff (1979). In L. mjobergi the branchial basket barely extends beyond the outlines of the upper skull parts, the ceratobranchials are very thin and terminally not fused (commissurae terminales absent). The branchial basket is very flat and filter plates are absent (Inger, 1983). Enlarged ceratobranchials and relatively reduced branchial baskets occur in suctorial rheophilous tadpoles (Wassersug and Hoff, 1979; Haas and Richards, 1998). However, even more reduced branchial baskets and larger ceratohyalia were reported for macrophagous tadpoles (Hyla nana, Lavilla, 1990; Vera Candioti et al., 2004; Lepidobatrachus laevis, Ruibal and Thomas, 1988; Anotheca spinosa, Wassersug and Hoff, 1979; pers. obs.). L. mjobergi lacks the enormous development of the m. orbitohyoideus found in macrophagous Hyla nana (Vera Candioti et al., 2004) or ovophagous Anotheca spinosa (pers. obs.). In these species, this muscle generates the forces for suction. However, the special insertion pattern of the m. rectus cervicis in L. mjobergi suggests that this muscle might act synergistically with the moderately developed m. orbitohyoideus to generate larger forces. Histological sections of the gut, so far examined, do not support our initial hypothesis that L. mjobergi was a macrophagous tadpole (see, for example, worm feeding reported in Hyla nana; Vera Candioti et al., 2004). Gut contents did not contain large objects such as nematodes or oligochaete worms. Rather, we found predominantly heterogeneous organic debris without identifiable parts of animals or plants. However, L. mjobergi lacks those structures identified as indicative of efficient microphagous suspension feeding (and found in the fossorial O. pyburni): high filter plates and filter ruffles. As striking as the lack of elaborate filter plates (Fig. 10) are the numerous papillae (some long, but most of them pustules) on the buccal floor and buccal roof arenas (Inger, 1983). The buccal floor and roof pustules contain secretory cells. Strings of mucus could entrap particles in the center of the buccal cavity and transport particles directly to the esophagus; rather than mucous entrapment above the filter plates in most tadpoles (Kenny, 1969a, b). The oral disk has a characteristic cup- or funnelshaped structure and is well equipped with muscle fibers of the m. mandibulolabialis, though its function remains obscure. However, the shape of this derived oral disk and the lack of keratodonts means that a substrate-

scraping mode of feeding, typical of most streamassociated tadpoles (Taylor et al., 1996), is unlikely. The skeletal structures of the jaws give no further clues with respect to feeding. The jaw cartilages are reminiscent of those of other pelobatids (e.g., Rocˇek, 1981), and differ only in proportion. In sum, some features of the feeding apparatus of L. mjobergi are unique and do not compare readily to other known feeding types in tadpoles. Larger specimen samples and evaluation of their gut contents will be necessary to reveal resource use in L. mjobergi.

Phylogeny L. mjobergi belongs to the Megophryidae (Frost, 2004), a southeast Asian group of ground dwelling frogs, formerly included in the Pelobatidae. Megophryidae form a monophyletic group with other Old World pelobatoids (Haas, 2003; Garcı´ a-Parı´ s et al., 2003; Pu´gener et al., 2003; Hoegg et al., 2004; Roelants and Bossuyt, 2005; San Mauro et al., 2005). Lathrop (1997) reviewed megophyid taxonomy. Generic relationships were addressed by Zheng et al. (2004), however, without considering Leptobrachella. Ramaswami (1943) described the rostral cartilages in Megophrys. Despite the vermiform body shape and the derived anatomical features in the musculo-skeletal apparatus of L. mjobergi, a number of larval morphological characters show its phylogenetic relationships: the excessive number of larval tail vertebrae has been reported before in Megophrys major (Griffiths, 1963) and is present in M. nasuta as well (pers. obs.). The large and elongate adrostral cartilage, presence of a m. mandibulolabialis superior, and insertion of m. subarcualis rectus II–IV on ceratobranchiale III are derived character states of the Eurasian megophryid and pelobatid clade in Haas (2003) and are shared by L. mjobergi indicating its megophryid relatedness. The only species known to possess the m. subarcualis rectus accessorius are the megophryids L. mjobergi, Leptobrachium hasselti and Megophrys montana, and the non-pelobatid South African Heleophryne natalensis (Haas, 2003). L. mjobergi differs from M. montana and L. hasseltii (Haas, 2003) in having a high suspensorium (low in the two other species).

Acknowledgements We wish to thank the Sarawak Forest Department, in particular Datuk Cheong Ek Choon, Director, and Bolhan Budeng, for issuing collecting permits (NPW.907.4-36); the Economic Planning Unit, The Prime Minister’s Department, Malaysia, and especially Mrs. Munirah Abd. Manan, for issuing research permit


No. 1168 to A. Haas. K. Felbel and E. Gretscher skillfully prepared serial sections of the specimens. We thank Greg Handrigan and Richard Wassersug for thoughtful criticism and valuable comments on an early draft of this work. Finally, we are grateful to Volkswagen-Stiftung, Germany, who supported the study with Grant I/79 405.

Appendix A. Supplementary data The online version of this article contains additional supplementary data. Please search for doi:10.1016/ j.zool.2005.09.008 at http://dx.doi.org.

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