JOURNAL OF MORPHOLOGY 275:230–246 (2014)

Anatomy, Function, and Evolution of Jaw and Hyobranchial Muscles in Cryptobranchoid Salamander Larvae Thomas Kleinteich,1* Julia Herzen,2 Felix Beckmann,2 Masafumi Matsui,3 and Alexander Haas4 1

Functional Morphology and Biomechanics, Christian-Albrechts-Universit€ at Kiel, Am Botanischen Garten, 24118 Kiel, Germany 2 Structural Research on New Materials, Helmholtzzentrum Geesthacht, Max-Planck-Straße, 21502 Geesthacht, Germany 3 Graduate School of Human and Environmental Studies, Kyoto University, Sakyo, Kyoto 606-8501, Japan 4 Biozentrum Grindel und Zoologisches Museum, Universit€ at Hamburg, 20146 Hamburg, Germany ABSTRACT Larval salamanders (Lissamphibia: Caudata) are known to be effective suction feeders in their aquatic environments, although they will eventually transform into terrestrial tongue feeding adults during metamorphosis. Early tetrapods may have had a similar biphasic life cycle and this makes larval salamanders a particularly interesting model to study the anatomy, function, development, and evolution of the feeding apparatus in terrestrial vertebrates. Here, we provide a description of the muscles that are involved in the feeding strike in salamander larvae of the Hynobiidae and compare them to larvae of the paedomorphic Cryptobranchidae. We provide a functional and evolutionary interpretation for the observed muscle characters. The cranial muscles in larvae from species of the Hynobiidae and Cryptobranchidae are generally very similar. Most notable are the differences in the presence of the m. hyomandibularis, a muscle that connects the hyobranchial apparatus with the lower jaw. We found this muscle only in Onychodactylus japonicus (Hynobiidae) but not in other hynobiid or cryptobranchid salamanders. Interestingly, the m. hyomandibularis in O. japonicus originates from the ceratobranchial I and not the ceratohyal, and thus exhibits what was previously assumed to be the derived condition. Finally, we applied a biomechanical model to simulate suction feeding in larval salamanders. We provide evidence that a flattened shape of the hyobranchial apparatus in its resting position is beneficial for a fast and successful suction feeding strike. J. Morphol. 275:230–246, 2014. VC 2013 Wiley Periodicals, Inc. KEY WORDS: lissamphibia; caudata; cranial musculature; suction feeding; biomechanics

INTRODUCTION The biphasic lifecycle of amphibians (i.e., caecilians, salamanders, and frogs) that includes the process of metamorphosis and the profound transformations from a larval to an adult morphology provides an excellent model system in the fields of evolutionary and developmental research (Alberch et al., 1979). Especially, the contrasting specializations in the larval and adult morphologies that are C 2013 WILEY PERIODICALS, INC. V

linked by ontogenetic constraints makes amphibians important models to study the integration of anatomy, function, development, and evolution. Aquatic salamander (Caudata) larvae use suction for prey capture in water, while adult salamanders use their adhesive tongues for feeding on land (Deban and Wake, 2000; Wake and Deban, 2000). Both tasks involve the head musculature; however, the arrangement and function of particular muscles may differ before and after metamorphosis. For salamander suction feeding, models have been developed that describe the function of the skull and its muscles during the feeding strike. These models either focused on one particular species (e.g., the axolotl Ambystoma mexixanum; Lauder, 1985) or were based on a generalized salamander morphology (Deban and Wake, 2000; Deban, 2003). However, to understand the evolution of salamander larvae and how evolutionary transformations affected the feeding systems, it is important to apply a comparative approach that discriminates between different salamander groups and considers differences in the presence and attachment of cranial muscles. In previous studies, we provided hypotheses on the homologies between cranial structures in amphibian larvae and inferred hypotheses on character transformations in the larval cranial

Contract grant sponsor: Volkswagen Foundation (T.K.); Grant number: I 84/206. *Correspondence to: Thomas Kleinteich; Functional Morphology and Biomechanics, Christian-Albrechts-Universit€ at Kiel, Botanischen Garten 1-9, 24118 Kiel, Germany. E-mail: [email protected] Received 10 May 2013; Revised 22 August 2013; Accepted 6 September 2013. Published online 18 October 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jmor.20211

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TABLE 1. Specimens used in this study. TL —total length; SVL —snout vent length Species Andrias japonicus Andrias japonicus Cryptobranchus alleganiensis Cryptobranchus alleganiensis Hynobius nebulosus Hynobius nebulosus Onychodactylus japonicus Onychodactylus japonicus Dicamptodon ensatus Dicamptodon ensatus

ID

TL ([mm)]

SVL ([mm)]

Preparation

KUHE38459 KUHE460 Deban coll. NCSM74679 Hyne02 Hyne01 KUHE38445 KUHE38436 ZMH_A10055 ZMH_A10057

35 33.5 55 39.6 27 — 41 69.7 76 —

28 25.3 34 25.1 15 — 21 35.8 41 —

SRmCT Serial section SRmCT Serial section SRmCT Serial section SRmCT Serial section SRmCT Serial section

musculature that can be tested for congruence in phylogenetic analyses. A terminology that is consistent with the hypotheses on the homologies between head muscles in the different amphibian groups has been proposed (Haas, 1997, 2001; Kleinteich and Haas, 2007, 2011). Here, we apply this terminology to compare the cranial musculature in larvae of salamander species within the Hynobiidae, Cryptobranchidae, and Dicamptodontidae. The Cryptobranchidae plus Hynobiidae (i.e., the Cryptobranchoidea) have been hypothesized to be in a sister-group relationship to the remainder salamanders (Frost et al., 2006; Roelants et al., 2007; Pyron and Wiens, 2011; but see Wiens et al., 2005; Zhang and Wake, 2009). This makes the Cryptobranchoidea a key taxon to conclude on the larval cranial musculature and thus on the function of these muscles during suction feeding in the most recent common ancestor of all salamanders. The aims of this study are: 1) to provide a detailed description of the larval cranial musculature in species within the Cryptobranchoidea; 2) to conclude on the evolution of larval cranial muscles within salamanders by comparison with Dicamptodon ensatus and species within the Sirenidae, Amphiumidae, and Plethodontidae that have been examined in a previous study (Kleinteich and Haas, 2011); 3) to develop a kinematic model of the hyobranchial transformations during suction feeding; and 4) to interpret the cranial muscle function at different stages of the suction feeding strike. MATERIALS AND METHODS Specimen Preparation Table 1 contains a list of specimens that were available for this study provided by the Graduate school of Human and Environmental studies of Kyoto University (KUHE and Hyne), the collection of Dr. Stephen M. Deban (University of South Florida; Deban coll.), the North Carolina Museum of Natural Sciences (NCSM), and the Zoological Museum in Hamburg (ZMH). All specimens were formalin fixed and stored in 70% ethanol. We applied two different techniques to study the cranial musculature in these specimens: 1) synchrotron based X-ray radiation high-resolution computed micro tomography (SRmCT) and 2) histology. In preparation for SRmCT imaging, we first decapitated the specimens. The heads were then transferred to distilled water

by a stepwise decrease in the ethanol concentration (50%, 30%). Each concentration was maintained for 24 h. We then froze the specimens at 280 C for 4 h. The frozen heads were then placed in a Lyovac GT 2 freeze drying system where they were kept over night under a vacuum at approximately 230 C. Freeze drying was previously shown to increase the contrast within soft tissues for X-ray based imaging methods (Follett, 1968) and we successfully applied freeze drying in SRmCT sample preparation in previous studies on caecilian cranial muscles (Kleinteich et al., 2008a,b; Kleinteich, 2010). We performed SRmCT imaging at the German Electron Synchrotron (DESY) in Hamburg in cooperation with the Helmholtz-Zentrum Geesthacht. We used the beamlines W2 (energy ranges > 25 keV) and BW2 (energy ranges < 25 keV) of the DORIS III particle accelerator ring. The specimens were rotated by 180 and X-ray images were taken with a chargecoupled-device (CCD) sensor at each 0.25 step. After every eighth image (i.e., after 2 of rotation), the specimens were moved out of the X-ray beam to capture a reference image of the X-ray beam without the specimen. The captured intensities of the reference image were subtracted from the images that we captured with the specimen to obtain an X-ray absorption image of the specimens. SRmCT imaging resulted in 3-D volumetric datasets, which we imported into the 3-D visualization and analyzation software package Amira 5.2 (Visage Imaging). In Amira, we used the LabelsField module to assign individual voxels of the volumetric dataset to different materials, that is, the cranial muscles and the cranium. Volume renderings were generated with the Volren function in Amira. Figure 1 shows volume renderings of the segmented SRmCT datasets. In addition to SRmCT imaging, we prepared histological serial sections for comparison. For histology, we embedded the specimens in paraffin, sectioned at 10 mm slice thickness, and applied the standard Heidenhain’s Azan trichrome-staining protocol (Mulisch and Welsch, 2010).

Terminology The terms for cranial muscles in amphibian larvae are unfortunately not being used consistently in the literature and a large amount of synonyms has been introduced in the past. We carefully reviewed the terms for cranial muscles in amphibian larvae in previous studies from our group (Haas, 2001; Kleinteich and Haas, 2007, 2011) and suggested a terminology that reflects our hypotheses for the homologies among cranial muscles. Herein we build upon these previous studies and use the terminology suggested in Haas (2001) for the nervus trigeminus (cranial nerve V) innervated muscles and in Kleinteich and Haas (2007, 2011) for the remainder cranial muscles.

Modeling Hyobranchial Expansion During Suction Feeding During suction feeding, the buccal cavity increases its crosssectional area and its volume by a rotational movement of the

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Fig. 1. Volume renderings of SRmCT data of larval salamander heads. The dataset has been segmented and colored to depict different tissues within the SRmCT data. In the left column, muscles are shown in reddish brown, cartilages are light blue, and bones are yellowish white. To highlight the different groups of muscles, we applied a color coding scheme for the various muscle groups in the right column (same specimens).

hyobranchial bars (Deban, 2003). To assess the dependence of the actual buccal cavity volume on the change of position of the hyobranchial bars during the feeding strike, we prepared a schematic model of the buccal cavity as a half cylinder with an ellipsoid cross section (Fig. 2). The volume (V), respectively, the cross-sectional area (A) of this half cylinder can be calculated as:

V5p=2  x  y  z A5p=2  x  y

(1) (2)

Here, we use x as the radius of the ellipsoid half cylinder along its horizontal, y as the radius of the ellipsoid half cylinder along its vertical, and z as the length of the half cylinder (Fig. 2). In this simplified model, we focused exclusively on the position of the ceratobranchial I, as this appears to be the dominant element in the hyobranchial apparatus of salamander larvae; movements of the ceratobranchial I also affect the movements of the other ceratobranchial bars. To calculate the

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volume of the buccal cavity at different positions of the ceratobranchial I, we discriminated between a static and a kinetic part of the hyobranchial apparatus. The static part is defined by the rostral aspects of the hyobranchial apparatus, covering the basibranchial and the hypobranchial I. A bounding box that encloses the static part is considered to have the dimensions x0, y0, and z0 (Fig. 2). The kinetic part herein is considered as the volume that is defined by a bounding box, which encloses ceratobranchial I. The diagonal length of that bounding box equals the length of the ceratobranchial I (lcerato). The dorsal and lateral deflection of ceratobranchial I thus defines the volume of the kinetic part (Fig. 2); the dimensions x1, y1, and z1 depend on the angle of ceratobranchial I relative to the horizontal (a herein) and the dorsoventral axis of the specimen (ß herein):

x1 5lcerato  cos ðaÞ  sin ðbÞ

(3)

y1 5lcerato  sin ðaÞ

(4)

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Fig. 2. Model to calculate the volume and cross-sectional area of the buccal cavity based on the length (lcerato) and orientation (a and b) of the ceratobranchial I. We modeled the buccal cavity as a half cylinder which is defined by its length z and the two radiuses x and y. We calculated x, y, and z by using two boxes, one marked the more static part of the hyobranchial apparatus (x0, y0, and z0), the other one was modeled from the position of the ceratobranchial I (x1, y1, and z1). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

z1 5lcerato  cos ðaÞ  cos ðbÞ

(5)

The sums of x0 plus x1, y0 plus y1, and z0 plus z1 are the dimensions of the ellipsoid half cylinder x, y, and z. Thus the volume and cross-sectional area of the buccal cavity can be calculated as:   

V5p=2  x0 1lcerato  cos ðaÞ  sin ðbÞ  y0 1lcerato     sin ðaÞ  z0 1lcerato  cos ðaÞ  cos ðbÞ

(6)    A5p=2  x0 1lcerato  cos ðaÞ  sin ðbÞ  y0 1lcerato   sin ðaÞ (7) We used the open source computing environment R (Vers. 2.14.2; http://www.r-project.org) to simulate the movements of the ceratobranchial I along a range from 1 to 89 for the angles a and b. We further calculated the first derivatives of volume and cross-sectional area over a and b to identify the angles at which the relative increase in volume and crosssectional area is maximal. The source-code files for calculations made in R are available from the corresponding author.

RESULTS Muscles Acting on the Lower Jaw There are three groups of muscles that act on the lower jaw in salamander larvae (Fig. 1) the mm. levatores mandibulae that lift the mandible and close the mouth, 2) the m. depressor mandibulae complex that will lower the mandible, 3) the ventral jaw muscles that connect both halves of the lower jaw and in case of the m. geniohyoideus aid in jaw opening and hyobranchial compression. Mm. levatores mandibulae. The mm. levatores mandibulae (Fig. 3) comprise the m. levator mandibulae internus, the m. levator mandibulae

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longus, the m. levator mandibulae externus, and the m. levator mandibulae articularis. These muscles were found to be very similar in all larval salamander specimens examined. The m. levator mandibulae internus is the most medial of the jaw closing muscles. Its fibers emerge from the lateral edge of the caudalmost region of the frontal, the most rostral parts of the parietal, and from the lateral face of the braincase ventral to the frontoparietal suture. The m. levator mandibulae longus is situated immediately lateral to the m. levator mandibulae internus; it is much longer than the m. levator mandibulae internus. The area of origin of the m. levator mandibulae longus ranges from the lateral wall of the braincase over the dorsolateral face of the parietal and the rostral and dorsal parts of the otic capsule to the most rostral regions of the dorsal trunk musculature, dorsal to the tectum synoticum. The fibers of the m. levator mandibulae internus and the m. levator mandibulae longus converge towards their insertion site on the lingual and dorsomedial face of the lower jaw. Both muscles insert on the lingual face of the prearticular (sensu Francis, 1934) and the dorsal edge of Meckel’s cartilage, rostral to the jaw articulation. The m. levator mandibulae articularis is the smallest muscle of the jaw closing musculature. It is situated caudal and lateral to the mm. levatores mandibulae internus plus longus. The fibers of the m. levator mandibulae articularis originate from the ventrorostral edge of the otic capsule and the adjacent rostral face of the palatoquadrate. They insert to the dorsal side of Meckel’s cartilage, immediately rostral to the mandibular joint. The m. levator mandibulae externus is the most lateral muscle of the mm. levatores mandibulae. The m. levator mandibulae externus is a fleshy muscle. Its fibers originate from the lateral face of the palatoquadrate and the rostral edge of the squamosal. From all the jaw closing muscles of salamander larvae, the m. levator mandibulae externus reaches furthest rostrad; it inserts to the dorsolateral face of the dentary and with some fibers to Meckel’s cartilage along an area that reaches from ventral to the orbit to immediately rostral of the mandibular joint. M. depressor mandibulae complex. The m. depressor mandibulae complex (Fig. 4) comprises the m. depressor mandibulae, the m. depressor mandibulae posterior, and the m. hyomandibularis. Among the species examined herein, a m. hyomandibularis is only present in Onychodactylus japonicus. The m. depressor mandibulae and the m. depressor mandibulae posterior are incompletely separated, but are distinguishable as two distinct heads. The part that relates to the m. depressor mandibulae posterior can be identified as a mediocaudal layer of muscle fibers in the m. depressor mandibulae complex that slightly differs in its orientation from the rostrolateral fibers of the m. depressor mandibulae. The m. depressor mandibulae and the m. depressor mandibulae posterior originate from an Journal of Morphology

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Fig. 3. The mm. levatores mandibulae (shown in red) in the salamander species examined herein. Volume renderings of SRmCT data from the region caudal to the orbit and lateral to the otic capsule in lateral view. Left column: superficial musculature; right column: deeper layer with the m. levator mandibulae externus removed. All specimens examined herein had four mm. levatores mandibulae, that is, the m. levator mandibulae longus, the m. levator mandibulae internus, the m. levator mandibulae externus, and the m. levator mandibulae articularis. Muscles of this group close the lower jaw.

area that comprises the lateral face of the squamosal and the lateral wall of the otic capusle. In Andrias japonicus, Cryptobranchus alleganiensis, and Dicamptodon ensatus, both muscles entirely cover the lateral and dorsal side of the otic capJournal of Morphology

sule. The m. hyomandibularis in O. japonicus originates from the distal tip of ceratobranchial I. The fibers of the three muscles in the m. depressor mandibulae complex merge as they approach their insertion site at the lower jaw. In Hynobius

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Fig. 4. Muscles of the m. depressor mandibulae group (shown in orange). Oblique lateral view towards the caudolateral face of the otic capsule. This muscle group comprises three muscles which act in opening of the lower jaw and connect the lower jaw with the hyobranchial apparatus, that is, the m. depressor mandibulae, the m. depressor mandibulae posterior, and the m. hyomandibularis. Except for Onychodactylus japonicus, the m. hyomandibularis is absent in the species examined.

nebulosus and Onychodactylus japonicus, the m. depressor mandibulae complex inserts on the hyomandibular ligament that runs from immediately

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rostral to the mandibular joint along the ventral edge of the lower jaw to the ceratohyal. In Cryptobranchus alleganiensis, Andrias japonicus, and Dicamptodon ensatus, the m. depressor mandibulae complex inserts directly on the dorsal edge of the processus retroarticularis of the lower jaw, caudal to the mandibular joint. The m. depressor mandibulae posterior in Andrias japonicus and Cryptobranchus alleganiensis has, besides its insertion at the lower jaw, some fibers that attach to the distal tip and the rostral face of the ceratohyal. In Dicamptodon ensatus the insertion of m. depressor mandibulae posterior fibers on the ceratohyal is more prominent than in the two cryptobranchid species and the muscle inserts along the entire rostral face of the cartilage. In the two species of the Hynobiidae examined, the m. depressor mandibulae posterior does not insert on the ceratohyal. Ventral jaw muscles. There are two sheetlike muscles that act on the ventral side of the lower jaw, the m. submentalis and the m. intermandibularis (Fig. 5). Because these muscles are very thin and close to the skin, they are difficult to discern in CT scans of freeze dried salamander larvae. We could discriminate the two muscles in each of the specimens examined, except for the freeze dried individual of Cryptobranchus alleganiensis. By comparing to histological sections, the ventral jaw muscles could then easily be identified in C. alleganiensis. Besides the transversely oriented m. submentalis and the m. intermandibularis, a third ventral muscle, the longitudinal m. geniohyoideus, inserts on the lower jaw (Fig. 5). The m. submentalis is a very small muscle at the rostral tip of the lower jaw, spanning the mandibular symphysis. It connects the lingual faces of the two halves of the lower jaw. The two bilaterally symmetrical parts of the m. intermandibularis originate from a ventral and median raphe and then spread laterally towards their insertion. The m. intermandibularis inserts along the lingual aspect of the lower jaw, spanning almost the entire length of the mandible. The m. geniohyoideus is an elongate muscle on the ventral side of salamander larvae that runs parallel to the anterior-posterior axis of the animal. It originates from the ventral side of the fascia of the m. rectus cervicis, approximately in frontal plane with the articulation between hypobranchial I and ceratobranchial I. In Hynobius nebulosus, Onychodactylus japonicus, and Dicamptodon ensatus, the m. geniohyoideus has fibers that originate from the urohyal, in addition to the fibers that attach to the fascia of the m. rectus cervicis. The m. geniohyoideus inserts to the lingual face of the lower jaw, lateral to the jaw symphysis and dorsal to the attachment of the m. submentalis. Journal of Morphology

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Muscles Acting on the Hyobranchial Apparatus We identified four groups of hyobranchial muscles (Fig. 1, 1) the dorsal hyobranchial levator muscles, 2) internal hyobranchial muscles, which connect subsequent hyobranchial elements, 3) ventral hyobranchial muscles, and 4) laryngeal muscles. Besides, the jaw muscles m. hyomandibularis (present in Onychodactylus japonicus only) and the m. geniohyoideus also act on the hyobranchial apparatus. Dorsal hyobranchial levators. This group comprises the mm. levatores arcuum branchialium I–IV, a set of small elongated muscles that insert at the distal tips of the ceratobranchial bars (Fig. 6). The m. levator arcus branchialis I is the most rostral muscle of the group. In all species examined, except for Dicamptodon ensatus, it originates from the caudolateral surface of the otic casule, immediately dorsal to the origin of the m. depressor mandibulae and immediately ventral to the attachment of the dorsal trunk musculature to the skull. In D. ensatus, the m. levator arcus branchialis I attaches laterally on the fascia of the m. depressor mandibulae. The m. levator arcus branchialis I inserts at the distal tip of ceratobranchial I. The mm. levatores arcuum branchialium II and III share their origin in all salamanders examined with a few slight interspecific differences in the position of these two muscles. In Andrias japonicus, the mm. levatores arcuum branchialium II and III originate from the otic capsule, immediately caudal to the m. levator arcus branchialis I; in the other species, the two muscles originate from the rostrolateral face of the fascia of the dorsal trunk musculature. During the course of their fibers, the mm. levatores arcuum branchialium II and III separate and insert to the distal tips of ceratobranchial II and ceratobranchial III, respectively. The m. levator arcus branchialis IV is the most caudal muscle in the branchial levator group. It originates from the lateral face of the dorsal trunk muscle fascia. In Hynobius nebulosus and O. japonicus, the m. levator arcus branchialis IV has a few fibers that originate together with the mm. levatores arcuum branchialium II and III. In the remainder salamanders examined, this muscle is completely separate. Finally, the m. levator

Fig. .5.

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Fig. 5. Ventral jaw (muscles shown in yellow) and hyobranchial (muscles shown in purple) musculature. Volume renderings of SRmCT data in ventral view. Muscles that insert on the jaw ventrally comprise the m. submentalis, the m. intermandibularis, and the m. geniohyoideus; ventral hyobranchial muscles are the m. interhyoideus, the m. interhyoideus posterior, and the m. rectus cervicis. In the SRmCT data of Cryptobranchus alleganiensis, the ventral muscles were impossible to discern. However, we confirmed the presence of this muscle by comparing to histological serial sections.

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Fig. 6. Dorsal (shown in green), lateral (in blue), and laryngeal muscles (in pink). Volume renderings of SRmCT data in oblique lateral view, looking at the caudolateral face of the hyobranchial apparatus. The mm. levatores arcuum branchialium insert on the dorsal tips of the ceratobranchial bars. The m. branchiohyoideus externus is a voluminous muscle which covers the lateral face of the ceratobranchial I. The m. transversus verntralis IV inserts on the medial face of the ceratobranchial IV.

arcus branchialis IV inserts to the distal tip of ceratobranchial IV. Muscles that connect hyobranchial elements. This group comprises the m. branchiohyoideus externus, the m. subarcualis rectus I,

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the m. subarcualis rectus II-IV, and the mm. subarcuales obliqui II and III (Fig. 7). In all salamanders examined, the m. branchiohyoideus externus is a fleshy muscle that originates from the lateral side of the distal ceratobranchial I. It inserts along the ventral face of the proximal ceratohyal. The most rostral parts of this muscle insert immediately caudal to the articulation between hypohyal and ceratohyal except in Hynobius nebulosus, where a hypohyal is absent. In H. nebulosus, the most rostral parts of the m. branchiohyoideus externus insert close to the articulation between basihyal and ceratohyal. The m. subarcualis rectus I is an elongate muscle medial to the m. branchiohyoideus externus in salamander larvae. Its origin is restricted to a narrow region on the ventral side of ceratobranchial I, immediately caudal to the articulation between hypobranchial I and ceratobranchial I. The m. subarcualis rectus I inserts via a short tendon (except in larval Dicamptodon ensatus where the fibers are directly attached) to the ventral side of the proximal region of the ceratohyal, immediately medial to the insertion of the m. branchiohyoideus externus. In Hynobius nebulosus, where a hypohyal is absent, the insertion of the m. subarcualis rectus I on the ceratohyal follows distally immediately to the articulation between ceratohyal and basihyal; in the remainder salamander species examined, the insertion of this muscle is close to the articulation between ceratohyal and hypohyal. The mm. subarcuales obliqui II and III are two small muscles on the ventral side of the animal. Their fibers originate from the ventral and proximal tip of ceratobranchial II and III, in close proximity and distal to the articulation between hypobranchial II and ceratobranchial II (m. subarcualis obliquus II), respectively, ceratobranchial II and ceratobranchial III (m. subarcualis obliquus III). The fibers of the mm. subarcuales obliqui II and III converge and share their insertion on the urohyal process in Hynobius nebulosus and Dicamptodon ensatus, or on the lateral and ventral face of the fascia that surrounds the m. rectus cervicis. The m. subarcualis rectus II–IV stretches between the ceratobranchial bars on the ventral side of the hyobranchial apparatus. It originates from the proximal and ventral region of ceratobranchial IV. Its fibers run rostrad and insert ventrally on the proximal parts of ceratobranchials III, II, and I. In all salamanders examined, the attachments of m. subarcualis rectus II–IV fibers to the ceratobranchials are close to the proximal articulations of the cartilages. The m. subarcualis rectus II–IV reaches rostrad until immediately caudal to the origin of the m. subarcualis rectus I. Ventral hyobranchial muscles. This group comprises the sheet-like m. interhyoideus and m. interhyoideus posterior, the m. transversus Journal of Morphology

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ventralis IV, and the m. rectus cervicis (Figs. 5 and 6). Because of their thin cross-sectional area and their close proximity to the skin, it was diffi-

cult to trace the m. interhyoideus and the m. interhyoideus posterior in the CT images of freeze dried salamander larvae. For Cryptobranchus alleganiensis, we could discern the m. interhyoideus and m. interhyoideus posterior only in histological sections but not in the CT images. The m. interhyoideus is caudally adjacent to the m. intermandibularis. Like for the m. intermandibularis, the fibers of the m. interhyoideus and the m. interhyoideus posterior originate from a ventral and median raphe and then run laterally towards their insertion. The fibers of the m. interhyoideus wrap around the m. branchiohyoideus externus and attach to the ventrolateral edge of the ceratohyal. The m. interhyoideus posterior is a sheet of muscle fibers that is incompletely separated from the m. interhyoideus but that inserts on the fascia of the m. branchiohyoideus externus instead of the ceratohyal. The m. transversus ventralis IV is a thin muscle sheet in the caudal region of the hyobranchial apparatus. This muscle was identified in all specimens examined, except for a Cryptobranchus alleghaniesis specimen that was freeze dried for CT imaging. However, examination of the histology sections of a second C. alleganiensis specimen confirmed the presence of the m. transversus ventralis IV in this species. The m. transversus ventralis IV originates from the medial and ventral face of ceratobranchial IV. Its fibers run mediad and ventrad and meet with the fibers of the contralateral counterpart of this muscle in a ventromedian raphe, dorsal to the m. rectus cervicis and the pericardial sac. The m. rectus cervicis is an elongate and voluminous muscle on the ventral side of the hyobranchial apparatus. Its fibers originate from the fascia of the m. rectus abdominis, caudal to the hyobranchial apparatus and run rostrad, parallel to the anteriorposterior axis of the animal. At its insertion, the m. rectus cervicis fills the volume between the basibranchial, the hyobranchials I and II, and, in Hynobius nebulosus and Dicamptodon ensatus, the urohyal. Laryngeal musculature. The muscles that act on the larynx are the m. constrictor laryngis and the m. dilatator laryngis (Fig. 6). The lungless Onychodactylus japonicus specimens examined, have no laryngeal muscles. The m. dilatator laryngis originates from the lateral and ventral parts of the fascia that covers

Fig. .7.

Journal of Morphology

Fig. 7. Internal hyobranchial musculature (shown in blue) and hyobranchial skeletons of the salamander specimens examined herein. Volume renderings of SRmCT data in ventral view. These muscles comprise the m. branchiohyoideus externus, the m. subarcualis rectus I, the m. subarcualis rectus II–IV, and the mm. subarcuale obliqui II and III. In Hynobius nebulosus, Onychodactylus japonicus, Cryptobranchus alleganiensis, and Andrias japonicus, only the hypobranchials II are ossified; the remainder hyobranchial elements are cartilaginous. In Dicamptodon ensatus, however, the ceratohyal and the ceratobranchials, I–IV bear ossifications (shown in brownish white).

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TABLE 2. Predicted angles for maximal absolute volume and cross-sectional area and maximal increase in volume and cross sectional area of the buccal cavity in salamander larvae Angles max volume ( ) Species Andrias japonicus Cryptobranchus alleganiensis Hynobius nebulosus Onychodactylus japonicus Dicamptodon ensatus

Angles max. volume increase ( )

Angles max cross section ( )

Angles max. cross section increase ( )

Dorsal (alpha)

Lateral (beta)

Dorsal (alpha)

Lateral (beta)

Dorsal (alpha)

Lateral (beta)

Dorsal (alpha)

Lateral (beta)

45 47

57 60

1 1

1 1

50 51

89 89

1 1

1 1

45 45 44

52 53 52

1 1 1

1 1 1

51 51 48

89 89 89

1 1 1

1 1 1

the dorsal trunk muscles. Its fibers wrap around the pharynx and run dorsal and medial to the m. transversus ventralis IV. The m. dilatator laryngis inserts to the ventral and rostral tip of the arytaenoid cartilages. The m. constrictor laryngis is dorsal and caudal to the m. dilatator laryngis. The bilaterally symmetrical parts of the m. constrictor laryngis both insert on the same fascia, dorsal and ventral to the trachea. Thus, they form a ring that encompasses the caudal parts of the arytaenoid cartilages. Modeling Hyobranchial Expansion Based on the half-cylinder model of the salamander buccal cavity (Fig. 2), the volume that can be contained in the buccal cavity is predicted to be maximal at approximately 45 dorsal elevation and 52 –60 lateral deflection of the ceratobranchial I (Table 2, Fig. 8). The cross-sectional area of the buccal cavity becomes maximal when the ceratobran-

chial I is oriented at around 50 from the horizontal and perpendicular to the antero-posterior axis of the animal (Table 2, Fig. 8). For both, volume and cross-sectional area, the highest increase per degree of ceratobranchial I movement is achieved at small angles (Table 2, Fig. 8).

DISCUSSION Suction feeding in aquatic vertebrates depends on the rapid increase in volume of the buccal cavity after the jaw is opened (Lauder, 1980, 1985). Buccal cavity volume is influenced by the orientation of the elements of the hyobranchial apparatus. For salamanders, a model that discusses the function of muscles that act on the hyobranchial bars during the gape cycle was presented by Deban and Wake (2000) and Deban (2003). Here we further develop this previous model by including all jaw and hyobranchial muscles in salamander larvae and by

Fig. 8. Results of the model on buccal cavity expansion (see Fig. 2). Calculations for these graphs were based on anatomical measurements of the Onychodactylus japonicus SRmCT dataset. Left side: volume over dorsal (alpha) and lateral (beta) deflection of the ceratobranchial I. Right side: cross-sectional area of the buccal cavity over dorsal (alpha) and lateral (beta) deflection of the ceratobranchial I. The volume of the buccal cavity becomes maximal at a ceratobranchial I position of 45 (alpha) and 53 (beta); maximal cross-sectional area is achieved if the ceratobranchial I is perpendicular to the antero-posterior axis of the animal. However, for a successful suction feeding event, a rapid increase in buccal cavity volume, respectively, cross-sectional area will be more important than absolute values. The highest increase (i.e., steepest slope) of volume and cross-sectional area over different angles for the ceratobranchial I is achieved at small angles, that is, at the beginning of the feeding strike. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Fig. 9. Proposed model of cranial muscle function during suction feeding in salamander larvae based on Deban (2003). The larval salamander skull is drawn from the Dicamptodon ensatus mCT dataset. Skeletal elements shown in orange-red are moved relative to their previous position; arrows indicate direction of movements. During jaw opening, the muscles of the m. depressor mandibulae group (DM—m. depressor mandibulae, DMP—m. depressor mandibulae posterior, and HM—m. hyomandibularis) and the m. geniohyoideus (GH) cause a downward rotation of the lower jaw. Expansion of the buccal cavity is caused by rotation of the hyobranchial elements in an upright position by action of the m. branchiohyoideus externus (BHE), the m. rectus cervicis (RC), and the mm. levatores arcuum branchialium I, II, III, and IV (LAB). The m. depressor mandibulae posterior (DMP) and the m. hyomandibularis (HM) may support this rotational movement. At this part of the feeding cycle, the gill slits are hypothesized to remain closed by action of the m. subarcualis rectus II–IV (SRII-IV). Jaw closure is caused by action of the mm. levatores mandibulae, comprising the m. levator mandibulae externus (LME), the m. levator mandibulae longus (LML), the m. levator mandibulae internus (LMI), and the m. levator mandibulae articularis (not shown). The ventral muscles m. intermandibularis (IM), m. interhyoideus (IH), and m. interhyoideus posterior (IHP) compress the buccal cavity. The m. geniohyoideus (GH) and the mm. subarcuale obliqui II and III (SO) support compression of the buccal cavity by folding the hyobranchial apparatus to its resting position.

adding calculations on hyobranchial volume increase during the feeding strike. For the following discussion, we dissect the movements that are necessary for prey capture by suction feeding salamanders, in four different phases (Fig. 9): 1) jaw opening, 2) hyobranchial expansion, 3) jaw closing, and 4) hyobranchial compression. It is important to note, that during the feeding strike these phases actually might be overlapping; hyobranchial expansion for example is initiated before the lower jaw is completely opened and may not reach its maximum extent before the onset of jaw closure. Jaw Opening Rapid opening of the jaw is critical for a successful prey capture event in aquatic salamander larvae (Liem, 1978; Wainwright et al., 1989). The muscles that are in a position to increase the gape angle are the m. depressor mandibulae, the m. depressor mandibulae posterior, and the m. geniohyoideus. The m. depressor mandibulae and the m. depressor mandibulae posterior insert either immediately caudal to the mandibluar joint (Andrias japonicus, Cryptobranchus alleganiensis, and Dicamptodon ensatus) directly to a short dorsocaudal processus retroarticularis of the lower Journal of Morphology

jaw or via a tendon to the ventral edge of the lower jaw. In both cases, the effective mechanical advantage (sensu Biewener, 1991, 19891989; i.e., force at the rostral tip of the lower jaw per muscle force) of the m. depressor mandibulae and m. depressor mandibulae posterior will be very low due to a short inlever. Reciprocally, the velocity advantage of the m. depressor mandibulae and m. depressor mandibulae posterior will be very high (Westneat, 2003). The short inlever of the m. depressor mandibulae and the m. depressor mandibulae posterior will allow for fast jaw opening at the onset of a suction feeding cycle. In Andrias japonicus, Cryptobranchus alleganiensis, and Dicamptodon ensatus, we found fibers of the m. depressor mandibulae posterior that, besides their insertion on the lower jaw, attached to the distal tip of the ceratohyal. In these specimens, action of the m. depressor mandibulae posterior will rotate the ceratohyal to an upright position while the same muscle simultaneously contributes to the opening movement of the jaw. The m. geniohoideus is suggested to have a dual function. 1) With the hyobranchial apparatus held in position, action of the m. geniohyoideus will cause a downward rotation of the lower jaw. 2) While the lower jaw is held closed (by

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simultaneous action of the mm. levatores mandibulae), the m. geniohyoideus will pull the hyobranchial apparatus rostrally. Action of the m. hyomandibularis will shorten the distance between lower jaw and ceratobranchial I. This potentially can 1) contribute to the downward rotation of the lower jaw as the mouth is opened and 2) cause a rotational movement on the ceratobrachial I to move it to a more upright position. Both, the m. geniohyoideus and the m. hyomandibularis likely function to coordinate jaw and hyobranchial movements. Additionally to the muscles of the m. depressor mandibulae group, the m. geniohyoideus is in a position to aid during jaw opening. The m. submentalis was shown in frogs to cause mandibular bending when the jaw is opened (Nishikawa and Roth, 1991; Deban and Nishikawa, 1992). Although the amount of mandibular bending in salamander larvae has never been measured, its similar position in frogs and salamanders suggests a similar functionality. Hyobranchial Expansion The rapid expansion of the hyobranchial apparatus during the feeding strike is caused by dorsoventral rotation and lateral deflection of the ceratohyal and ceratobranchial bars (Deban and Wake, 2000). In the salamander larvae examined herein, the ceratobranchial bars are structured hierarchically, that is, ceratobranchial IV articulates with ceratobranchial III, ceratobranchial III plus IV articulate with ceratibranchial II, and ceratobranchial II plus III plus IV, articulate with ceratobranchial I (Fig. 7). Thus, movement of the ceratobranchial I will have an effect on all ceratobranchial cartilages, while movement of the ceratobranchial IV will most likely act only locally on the ceratobranchial IV. The appearance of the voluminous m. branchiohyoideus externus is very similar across salamander larvae. Action of this muscle will cause a rotation of the ceratobranchial I and its adjacent elements in an upright position. Based on our calculations, the highest volume of the hyobranchial apparatus is achieved when ceratobranchial I is oriented at about 50 relative to the horizontal plane. However, for successful prey capture, the absolute maximum of hyobranchial volume might be less important than rapid volume increase. The fastest volume increase is predicted to happen at low angles of the ceratobranchial I, that is, at the beginning of the hyobranchial expansion (Table 2). Especially Cryptobranchus alleganiensis seems to take advantage of the fast relative volume increase at small angles of the ceratobranchial I. The body shape of the C. alleganiensis specimens examined herein was notably dorsoventrally flattened. This suggests that the ceratobranchial I in

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its resting state is oriented almost parallel to the antero-posterior axis of the animal. This also confirms previous observations that C. alleganiensis shows the most notable hyoid depression among aquatic salamanders (Reilly and Lauder, 1992). Besides the dorsoventral rotation of the ceratobranchial I, action of the m. branchiohyoideus externus will also deflect the cartilage laterally. This will further increase the volume of the buccal cavity. Similarly to the dorsoventral rotation of ceratobranchial I, the maximum total volume of the buccal cavity is predicted for angles at around 50 –60 of lateral deflection of the ceratobranchial I. Further, the fastest increase in volume over the change in orientation of the ceratobranchial I as the cartilage is pulled laterally is expected for small angles. Based on these calculations, we predict that it is advantageous for the animals to orient the ceratobranchial bars parallel to the anteroposterior axis of the animal, at the start of the feeding strike. It was shown that the gill slits remain closed during the early stage of hyobranchial expansion (Lauder, 1985; Deban and Wake, 2000). For the suction feeding mechanism in sunfishes, it was argued that closing of the gill slits during the expansion phase prevents water influx through the gills to the buccal cavity (Lauder, 1980). Such an influx of water through the gills would be in the reverse direction to the water flow through the mouth and thus would cause poorer suction feeding performance. Closure of the gill slits could be controlled by the m. subarcualis rectus II–IV that spans the ceratobranchials IV, III, II, and I. Action of the m. rectus cervicis is likely to pull the ventral side of the hyobranchial apparatus caudally. In combination with the mm. levatores arcuum branchialium I–IV that hold the distal aspects of the ceratobranchial cartilages into place or may even pull the distal parts of the cartilages closer to the skull roof, the m. rectus cervicis also supports the rotation of the ceratobranchial cartilages into a more upright position. Jaw Closing The jaw is closed by action of the mm. levatores mandibulae. For suction feeding salamander larvae, it can be expected that the jaw closing musculature is tuned by demands for rapid movements and not necessarily for high bite forces. Rapid jaw closure will be beneficial for capturing elusive prey items (Liem, 1978) and can result in a further increase in negative pressure inside the buccal cavity by the socalled water hammer effect after the jaw is closed (Lauder, 1980). Especially the m. levator mandibuale articularis and the m. levator mandibulae externus are in a position in favor of fast jaw movements. Both these muscles have low effective mechanical advantages due to a short inlever (m. levator mandibulae Journal of Morphology

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articularis) respectively, an acute angled muscle insertion (m. levator mandibulae externus) and thus exhibit high velocity advantages. Hyobranchial Compression The last step in the gape cycle of aquatic suction feeding salamander larvae is characterized by a water flux through the gill slits by compression of the buccal cavity and relocation of the hyobranchial apparatus to its rested (folded) state. The gill slits could either be opened passively by buccal pressure or there might be an active mechanism, that is, by action of the m. transversus ventralis IV. During expansion of the buccal cavity, the ceratobranchial I was abducted by the m. branchiohyoideus externus. The m. transversus ventralis IV is in a position to potentially pull the ceratobranchial IV medially and thus strut the ceratobranchial bars to open the gill slits. The sequence of the m. intermandibularis, the m. interhyoideus and the m. interhyoideus posterior covers the ventral floor of the buccal cavity from the rostral parts of the lower jaw to the caudal aspects of the hyobranchial apparatus and contraction of these muscles will result in an overall decrease of buccal cavity volume. Folding of the hyobranchial apparatus can be achieved by action of the m. geniohyoideus that will pull the ventral side of the hyobranchial apparatus rostrally. Further, contraction of the m. subarcualis rectus I and the mm. subarcuale obliqui II and III is predicted to move the ceratobranchials I–III into a more horizontal position. Evolutionary Transformations in Salamander Cranial Muscles In our previous studies (Kleinteich and Haas, 2007, 2011), we compared caecilian, salamander, and frog larvae to conclude on the presence of the cranial muscles in larvae of the most recent common ancestor of the extant amphibians (Lissamphibia). We found many similarities in the cranial musculature of larvae within the three amphibian groups and our results herein confirm those previous results. Further, we can argue that larvae of the most recent common ancestor of the Caudata had the following cranial muscles: m. levator mandibulae internus, m. levator mandibulae longus, m. levator mandibulae externus, m. levator mandibulae articularis, m. submentalis, m. intermandibularis, m. depressor mandibulae, m. depressor mandibulae posterior, m. branchiohyoideus externus, m. interhyoideus, m. interhyoideus posterior, m. subarcualis rectus I, m. subarcualis obliquus II, m. subarcualis obliquus III, m. subarcualis rectus II–IV, m. transversus ventralis IV, mm. levatores arcuum branchialium I–IV, m. geniohyoideus, m. rectus cervicis, m. dilatator laryngis, and m. constrictor laryngis. Journal of Morphology

The most notable differences between the species examined herein were found in the presence or absence of the m. hyomandibularis. This muscle is known from salamander larvae of the Ambystomatidae (Dr€ uner, 1904; Lauder and Shaffer, 1985, 1988; described as head of the m. depressor mandibulae), Salamandridae (Dr€ uner, 1901; Edgeworth, 1935; Bauer, 1997), and Plethodontidae (Kleinteich and Haas, 2011). It was also described in the paedomorphic Sirenidae (Dr€ uner, 1904; Edgeworth, 1935; Kleinteich and Haas, 2011), Proteidae (Dr€ uner, 1901; Edgeworth, 1935; Bauer, 1997), and Amphiumidae (Erdman and Cundall, 1984; Kleinteich and Haas, 2011). Further, caecilian larvae (Kleinteich and Haas, 2007, 2011) and frog tadpoles (m. hyoangularis as proposed homolog in Kleinteich and Haas, 2011) have muscles that are considered homologous to the m. hyomandibularis in salamanders. Surprisingly, in all the species examined herein, except for Onychodactylus japonicus, the m. hyomandibualris is absent. This confirms previous descriptions of the cranial musculature of species within the Hynobiidae 1 Cryptobranchidae (5 Cryptobranchoidea) that failed to identify this muscle (Dr€ uner, 1904; Edgeworth, 1935; Eaton, 1936; Fox, 1959). However, the muscle is present in O. japonicus—the first evidence for the m. hyomandibularis in a species within the Cryptobranchoidea. Absence of this muscle in larvae of Dicamptodon ensatus was rather unexpected because of its assumed general presence in species of the Salamandroidea (comprising the Ambystomatidae, Dicamptodontidae, Salamandridae, Proteidae, Rhyacotritonidae, Amphiumidae, and Plethodontidae; sensu Zhang and Wake (2009a)). Contrary to our findings, Eaton (1936) described a m. hyomandibularis (Eaton considered this muscle a part of the m. depressor mandibulae; page 46) for Dicamptodon larvae. It cannot be excluded that we actually observed some intraspecific or ontogenetic variation in the presence of this muscle in D. ensatus herein. In the sister group to the Dicamptodontidae, that is, the Ambystomatidae (Frost et al., 2006; Roelants et al., 2007; Zhang and Wake, 2009; Pyron and Wiens, 2011), Dr€ uner (1904) failed to identify this muscle (m. ceratomandibularis in Dr€ uner; page 477) in a 14 cm TL larval specimen of Ambystoma mexicanum, while he found this muscle in the remainder specimens he investigated (ranging from 2 to 29 cm TL). However, it is evident that we failed to recognize this muscle in both specimens examined - either by histology or CT imaging. This might be more than just intraspecific variation and a more complete sampling of specimens at different sizes and ages will be needed to further explore the patterns of absence or presence of the m. hyomandibularis in the Dicamptodontidae and Ambystomatidae, respectively. In a recently published article on cranial muscle development in Ambystoma mexicanum (Ambystomatidae), Ziermann and Diogo

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Fig. 10. Two competing hypotheses on the evolution of salamanders and their implications on the required evolutionary character transformations of the m. hyomandibularis. Arrows highlight proposed evolutionary steps; boxes show proposed character states for the most recent common ancestor of a clade; colors depict different evolutionary scenarios. In caecilian and frog larvae, the m. hyomandibularis (HM; termed m. hyoangularis in anurans) originates from the ceratohyal (CH) and inserts on the lower jaw (LJ). In salamanders, however, there are various character states of the m. hyomandibularis: either being absent, present, and similar to caecilians and frogs, or present but with an origin from the ceratobranchial I (CBI). In both phylogenies, at least three evolutionary transformations are required to explain the observed pattern of variation.

(2013) also failed to identify an independent m. hyomandibuaris (called m. ceratomandibularis in Ziermann and Diogo, 2013). Further, the m. hyomandibularis differs in shape and position within salamanders and other

amphibians. In larval salamanders within the genus Siren, caecilians, and anuran larvae, the m. hyomandibularis originates from the ceratohyal while in larval salamanders other than sirenids, the muscle originates from ceratobranchial I, that Journal of Morphology

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is, it must have shifted its attachment during salamander evolution (Edgeworth, 1935; Bauer, 1997; Kleinteich and Haas, 2007, 2011). Plotting the different character states (i.e., absent, present/origin from the ceratohyal, and present/origin from the ceratobranchial I) of the m. hyomandibularis on recent salamander phylogenies (Zhang and Wake, 2009; Pyron and Wiens, 2011), shows some discrepancy on how to interpret the character transformations of the m. hyomandibularis based on the two phylogenetic hypotheses (Fig. 10). In the scenario by Zhang and Wake (2009), in which the Sirenidae are the sister taxon to the remainder salamanders, it is most parsimonious to assume presence of a m. hyomandibularis, which originates from the ceratohyal in larvae of the most recent common ancestor of all salamanders. However, it then is impossible to argue based on parsimony alone whether the m. hyomandibularis was lost in larvae of the most recent common ancestor of the Cryptobranchoidea plus Salamandroidea (i.e., the clade comprising the Ambystomatidae, Dicamptodontidae, Salamandridae, Rhyacotritonidae, Amphiumidae, and Plethodontidae; Figure 10 green scenario for Zhang and Wake) or whether it shifted its origin from the ceratohyal to the ceratobranchial I (Fig. 10 red scenario for Zhang and Wake). Both scenarios require two additional evolutionary transformations to explain the observed pattern of presence/absence of the m. hyomandibularis in salamander larvae: the m. hyomandibularis was either lost (Fig. 10 red scenario for Zhang and Wake) or gained (Fig. 10 green scenario for Zhang and Wake) two times independently. Based on the phylogeny of Pyron and Wiens (2011), in which the Cryptobranchoidea are the sister taxon to the Sirenidae plus Salamandroidea, each of the three observed character states for the m. hyomandibularis could be the ancestral state for salamanders (Fig. 10). Each of the three scenarios would then require three character transformations to explain the observed distribution of presence and shape of the m. hyomandibularis within salamanders. Scenario 1 (Fig. 10 blue scenario for Pyron and Wiens): If a m. hyomandibularis which originated from the ceratohyal was present in the most recent common ancestor of salamanders, then this muscle most likely was reduced at the root of the Cryptobranchoidea and it re-evolved in the clade leading to Onychodactylus japonicus. Further, in this scenario the m. hyomandibularis shifted its origin in the ancestry of the Salamandroidea. Scenario 2 (Fig. 10 red scenario for Pyron and Wiens): If a m. hyomandibularis which originates from the ceratobranchial I is the sym-plesiomorphic condition for todays salamanders, then the muscle was reduced two times independently in the Cryptobranchidae and within the Hynobiidae, and it shifted its insertion to the ceratohyal in the Sirenidae. Scenario 3 (Fig. 10 Journal of Morphology

green scenario for Pyron and Wiens): If the m. hyomandibularis was absent in larvae of the most recent ancestor of salamanders, then this muscle evolved independently in O. japonicus, and the Sirenidae plus Salamandroidea. Further in this scenario, the m. hyomandibularis must have shifted its origin at least once, either in the Sirenidae or the Salamandroidea. In the phylogeny by Pyron and Wiens (2011), an outgroup comparison with frogs and caecilians suggests that the first scenario is most parsimonious because presence of a m. hyomandibularis which originates from the ceratohyal is the plesiomorphic condition. Interestingly, in both phylogenies (Zhang and Wake, 2009; Pyron and Wiens, 2011), the most parsimonious explanation for the observed variation in the m. hyomandibularis between different salamander groups requires at least three evolutionary transitions (Fig. 10). This is consistent with a previous study that demonstrated how the relationships of the Cryptobranchoidea and Sirenidae close to the root of the salamander phylogeny were highly dependent on the dataset underlying the phylogeny and the chosen algorithm for phylogenetic reconstruction (Wiens et al., 2005). It is unclear whether the differences in the presence and origin of the m. hyomandibularis in larvae of different salamander species have an impact on the movements of the hyobranchial apparatus during the feeding strike. Deban and Wake (2000) and Deban (2003) pointed out that in the Cryptobranchoidea (Cryptobranchidae plus Hynobiidae) the hypobranchial II and ceratobranchial II seem to be the main load bearing components in the hyobranchial apparatus and a recent study suggests that actually the lower jaw is the driving skeletal element for suction feeding in at least adult cryptobranchids (Heiss et al., 2013). Indeed, we found in all cryptobranchids and hynobiids examined herein that the hypobranchials II are ossified while the remainder hyobranchial elements are cartilaginous (Fig. 7). In other salamanders, Dicamptodon ensatus (Dicamptodontidae) for example, the ceratohyal and ceratobranchials are ossified. One could argue that the presumably more caudal distribution of load during the feeding strike in the Cryptobranchidae and Hynobiidae altered hyobranchial movements during the feeding strike which could have effected the presence of the m. hyomandibularis, a muscle that acts in the rostral aspects (ceratohyal or ceratobranchial I) of the hyobranchial apparatus. However, presence of the m. hyomandibularis in Onychodactylus japonicus and absence in D. ensatus would not be predicted from this scenario. In Andrias japonicus, Cryptobranchus alleganiensis (both Cryptobranchidae), and Dicamptodon ensatus (Dicamptodontidae), the insertion of the m. depressor mandibulae posterior to the distal tip of the ceratohyal and the lower jaw may compensate for the absence of the m. hyomandibularis in terms

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of establishing a connection between lower jaw and hyobranchial apparatus. However, we could not find an insertion of m. depressor mandibulae posterior fibers to the ceratohyal in Hynobius nebulosus (current study), nor in Salamandrella keyserlingii (Kleinteich and Haas, 2011), although this insertion was described in a previous study (Fox, 1959). In specimens which neither have the m. hyomandibularis present, nor have fibers of the m. depressor mandibulae posterior attached to the ceratohyal, the m. geniohyoideus is the only structure to coordinate jaw opening and hyobranchial expansion. ACKNOWLEDGMENTS We would like to thank Angelika Taebel-Hellwig for preparation of the histological sections, and Anne Maria Vogt for digitizing slides. Further we wish to express our gratitude to Hartmut Greven, Stephen M. Deban, Bryan Stuart, and the North Carolina Museum of Natural Sciences for making the Cryptobranchus alleganiensis specimens available for our study. The Japan Agency for Cultural Affairs and educatory committee of the Kyoto Prefectural Government granted permission to study protected Andrias japonicus. David B. Wake’s and David Buckeley’s help to collect Dicamptodon ensatus specimens in the field is much appreciated. Stephen M. Deban and two anonymous reviewers kindly provided valuable comments on a previous version of the manuscript. Further, TK would like to thank Adam P. Summers for fruitful discussions on vertebrate functional morphology, which have been inspirational for this project. LITERATURE CITED Alberch P, Gould SJ, Oster GF, Wake DB. 1979. Size and shape in ontogeny and phylogeny. Paleobiology 5:296–317. Bauer WJ. 1997. A contribution to the morphology of visceral jaw-opening muscles of urodeles (Amphibia: Caudata). J Morphol 233:77–97. Biewener AA. 1989. Scaling body support in mammals: Limb posture and muscle mechanics. Science 245:45–48. Biewener AA. 1991. Musculoskeletal design in relation to body size. J Biomech 24:19–29. Deban SM. 2003. Constraint and convergence in the evolution of salamander feeding. In: Bels V, Gase JP, Casinos A, editors. Vertebrate Biomechanics and Evolution. Oxford: BIOS Scientific Publishers. pp 161–178. Deban SM, Nishikawa KC. 1992. The kinematics of prey capture and the mechanism of tongue protraction in the green tree frog Hyla cinerea. J Exp Biol 170:235–256. Deban SM, Wake DB. 2000. Aquatic feeding in salamanders. In: Schwenk K, editor. Feeding: Form, Function, and Evolution in Tetrapod Vertebrates. San Diego, CA: Academic Press. pp 65–94. Dr€ uner L. 1901. Studien zur Anatomie der Zungenbein-, Kiemenbogen- und Kehlkopfmuskeln der Urodelen. I. Theil. Zool Jahrb Anat Onto 15:435–622. Dr€ uner L. 1904. Studien zur Anatomie der Zungenbein-, Kiemenbogen- und Kehlkopfmuskeln der Urodelen. II. Theil. Zool Jahrb Anat Onto 19:361–690. Eaton TH. 1936. The myology of salamanders with particular reference to Dicamptodon ensatus (Eschscholtz). J Morphol 37:31–75. Edgeworth FH. 1935. The Cranial Muscles of Vertebrates. London: Cambridge University Press. p 493.

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