A 3D Interactive Model and Atlas of the Jaw Musculature of Alligator mississippiensis

Modern imaging and dissemination methods enable morphologists to share complex, three-dimensional (3D) data in ways not previously possible. Here we present a 3D interactive model of the jaw musculature of the American Alligator (Alligator mississippiensis). Alligator and crocodylian jaw musculature is notoriously challenging to inspect and interpret because of the derived nature of the feeding apparatus. Using Iodine-contrast enhanced microCT imaging, a segmented model of jaw muscles, trigeminal nerve, brain and skull are presented as a cross-sectional atlas and 3D, interactive pdf of the rendered model. Modern 3D dissemination methods like this 3D Alligator hold great potential for morphologists to share anatomical information to scientists, educators, and the public in an easily downloadable format.


Introduction
Modern technology has afforded morphologists and evolutionary biologists the ability to visualize and share the anatomy of species using a variety of imaging modalities such as serial histological reconstruction, computed tomography (CT) scanning, magnetic resonance imaging (MRI), confocal microscopy, and laser scanning among others [1,2]. Recently, contrast techniques, such as Iodine Potassium Iodide (I 2 KI) and Phosphotungstic acid (PTA), coupled with CT imaging have enabled the visualization of soft tissues in ways not previously possible [3][4][5]. These techniques are particularly beneficial to studying musculoskeletal anatomy because the contrast techniques clarify soft tissue anatomy and organization more vividly than MRI. Segmentation analysis of these serial images then facilitates the creation of threedimensional (3D) models that can be easily rendered into highresolution, interactive 3D pdfs or even printed models using rapid prototyping methods [6]. Finally, the recent surge in biomechanical analyses, such as finite element modeling and multi-body dynamics analysis, has made disseminating accurate, threedimensionally accurate complex anatomy a necessity as collaborative projects investigating the functional morphology of animal systems mature [6][7][8].
Accurate three-dimensional representations of jaw muscles, like those of Alligator are beneficial to many researchers including those interested in crocodylian natural history and feeding ecology, evolutionary morphology and vertebrate paleontology, and biomechanics. Interpretations from an early version of this particular model have already been applied to one investigation into the soft tissue anatomy of Alligator [9,10]. But also, these smallsized, web-hosted, easily downloadable, interactive files are easily accessible by the general public, undergraduate students, and K-12 students interested in modern anatomical studies as well as charismatic extant archosaurs. Examples of shared, web-based vertebrate imaging resources can be found at Digimorph (http:// www.Digimorph.org/); Aves3D (www.Aves3D.org); Paleoview (http://paleoview3d.marshall.edu/); and the 3D Alligator (web. missouri.edu/,hollidayca/3DAnatomy/Alligator3D; http:// www.oucom.ohiou.edu/dbms-witmer/3D_gator.htm). This contribution follows in the footsteps of these cutting edge, shared anatomical resources.
Here we employed I 2 KI contrast staining and microCT imaging to develop the most anatomically accurate segmented threedimensional model and interactive 3D pdf of the jaw muscles, nervous tissues, and skull of the American alligator (Alligator mississippiensis) (Figs. 1-3). Alligator, and crocodylian jaw musculature has been notoriously challenging to study for several reasons. First, the characteristically flat skull makes the adductor muscles, or jaw-closing muscles logistically difficult to access deep to the temporal bars, large quadrate, and robust mandible. Second, individual crocodylian muscles are generally poorlydefined compared to those of lizards, birds and mammals making their individual identification challenging [11,12]. Third, this hypertrophied amalgam of jaw musculature has resulted in conflicting interpretations of muscle homology and evolution [13][14][15][16] (see Holliday and Witmer [2007] [11] for a review). In addition to the jaw musculature, we include the brain and trigeminal nerve divisions as they pass among the musculature. The relative positions of the jaw muscles and the divisions of the trigeminal nerve are classic topological characters used to interpret anatomy and homology of vertebrate jaw muscles [13]. We intend this contribution to serve as a visual atlas rather than a text-based description as the jaw muscle anatomy of Alligator and crocodylians have been described in numerous other papers [11][12][13][14][15][16].

Results and Discussion
Measurements of the skull and muscle volumes, as well as commonly used abbreviations are found in Table 1. Abbreviations for muscles are also provided in the text following their first use.

Ventral view
In the transverse sections (Fig. 4), beginning at the dorsalmost section, one can see the origins of many of the vertical adductor muscles including m. adductor mandibulae externus profundus(mAMEP), m. adductor externus medialis(mA-MEM), m. adductor externus superficialis(mAMES), and m. pseudotemporalis superficialis(mPSTs) as they pass ventrally  from the dorsotemporal fossa and lateral surfaces of the quadrate and laterosphenoid. Moving ventrally, the section passes through the trigeminal ganglion and the ophthalmic and maxillary nerves as the pass rostrally towards the face. The adductor muscles begin to expand in cross-section as they continue towards their mandibular attachments. The attachments of m. pterygoideus dorsalis(mPTd) can be seen on the cartilaginous interorbital septum. Caudally, m. depressor mandibulae passes from the occipital surface of the skull towards the retroarticular process. In the next section, the

Rostral view
In the axial sections (Fig. 5), beginning at the rostralmost section, m. pterygoideus dorsalis and the maxillary nerve are visible passing ventral to the orbit. Ventrally, the mandibular nerve is visible passing dorsal to m. intramandibularis within the Meckelian fossa. In the next section caudally, many of the adductor muscles are visible as they pass from the lateral wall of the braincase towards the mandible. The cartilago transiliens is visible linking bellies of m. adductor mandiblue externus and m. pseudotemporalis superficialis with m. intramandibularis as it wraps laterally around the pterygoid buttress. Medially, the ophthalmic and maxillary nerves are separated by the laterosphenoid lateral bridge. In the next section, the laminar pattern of the adductor muscles is easily appreciated with m. adductor mandibulae externus superficialis located laterally followed medially by the other vertical adductors and finally m. pseudotemporalis profundus and m. pterygoideus dorsalis. The next section occurs in a plane through the trigeminal ganglion and the mandibular nerve. Ventral to the nerve are m. pterygoideus dorsalis and the beginnings of m. pterygoideus ventralis as it comes off of the caudal edge of the pterygoid buttress. The next section caudally shows m. adductor mandibulae externus superficialis attaching to the dorsal surface of the surangular, m. adductor mandibulae posterior passing from the surface of the quadrate to the mandibular fossa and then mm. pterygoideus dorsalis and ventralis passing caudally towards the caudal end of the mandible. Finally, the caudalmost section passes through the neck and retroarticular process. The majority of m. depressor mandibulae is visible as it passes from the caudal surface of the paroccipital process to the dorsal surface of the retroarticular process. Ventrally the fibers of m. pterygoideus ventralis are visible passing underneath the mandible.

Lateral view
In the parasagittal sections (Fig. 6), beginning at the medialmost section, the trigeminal ganglion, maxillary nerve and majority of the origin of m. pterygoideus dorsalis are visible. In the next section lateral, the mandibular nerve passes ventrolaterally towards the mandible and is surrounded by

Conclusions
Alligator jaw musculature, like that of other vertebrates, is three-dimensionally complex and difficult to illustrate in twodimensional media. Here we provided the first volumetric model of Alligator, and crocodylian musculature based on raw imaging data. This downloadable, 3D model of Alligator jaw muscles, skull and nervous tissues shows the potential the next generation of anatomical tools have for morphologists, biologists, and the general public.

Materials and Methods
One 12-month-old fresh-frozen cadaveric Alligator mississippiensis was obtained from Rockefeller State Refuge, Grand Chenier, Louisiana, US and accessioned into the University of Missouri Vertebrate Collections as MU AL031. The head was removed, fixed in 10% neutral buffered formalin and stored in 70% ethanol. The head was then immersed in 10% Iodine Potassium Iodide (i.e., I 2 KI; Lugol's Iodine) for 5 weeks with 2 intervening CT scans to check on diffusion success. The final scan was conducted on a Siemens Inveon MicroCT scanner using 80 kV, 500 mA, and slice thickness of 83 microns at the University of Missouri Biomolecular Imaging Center. Scan data were imported as DICOM files into Amira v 5.2 (Visage Imaging) for segmentation. Anatomical structures were segmented manually using both magic wand and paintbrush tools by the authors. A defect in the scanner resulted in an obvious stitching artifact along the z-axis of the scan visible passing through the junction of the face and braincase. This did not significantly impact the interpretation of the data. Individual segmented structures were saved as STL files, smoothed and cleaned in Geomagic 12, and then imported into Adobe Acrobat 3D v.8 Toolkit, further reduced, and organized into a 3D model. This file was then imported into Adobe 8 3D and organized into its final format. Screen captures of specific slices of the segmented model were taken from Amira to show the outlines of muscles and soft tissues.  Figure S1. 3D interactive model of the jaw musculature of Alligator mississippiensis as modeled from I2KI staining and CT-scanning. (PDF)