You are on page 1of 25

JOURNAL OF MORPHOLOGY 269:398–422 (2008)

Development of the Dermal Skeleton in Alligator

mississippiensis (Archosauria, Crocodylia) With
Comments on the Homology of Osteoderms
Matthew K. Vickaryous* and Brian K. Hall

Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J1

ABSTRACT The dermal skeleton (5 exoskeleton) has Among craniates there are two phylogenetically
long been recognized as a major determinant of vertebrate independent and developmentally distinct skeletal
morphology. Until recently however, details of tissue devel- systems: a relatively deep endoskeleton that pre-
opment and diversity, particularly among amniotes, have forms in cartilage; and a more superficial dermal
been lacking. This investigation explores the development skeleton (5 exoskeleton, dermoskeleton) that does
of the dermatocranium, gastralia, and osteoderms in the not (Patterson, 1977; Smith and Hall, 1990, 1993).
American alligator, Alligator mississippiensis. With the
Whereas the dermal skeleton was once the predom-
exception of osteoderms, elements of the dermal skeleton
develop early during skeletogenesis, with most initiating inant skeletal system, as witnessed by the all-
ossification prior to mineralization of the endoskeleton. encasing mineralized armour of many stem gna-
Characteristically, circumoral elements of the dermatocra- thostomes (structural-grade ostracoderms), in most
nium, including the pterygoid and dentigerous elements, modern forms it has undergone extensive reduction
are among the first to form. Unlike other axially arranged and modification (Romer, 1956; Moss, 1964; Zylber-
bones, gastralia develop in a caudolateral to craniomedial berg et al., 1992; Smith and Hall, 1993; Sire and
sequence. Osteoderms demonstrate a delayed onset of de- Huysseune, 2003). For tetrapods, the most obvious
velopment compared with the rest of the skeleton, not remnants include the craniofacial skeleton (derma-
appearing until well after hatching. Osteoderm develop- tocranium or desmocranium), dental tissues, and
ment is asynchronous across the body, first forming dor- one or more elements of the pectoral apparatus.
sally adjacent to the cervical vertebrae; the majority of suc- Although less common—particularly among mam-
cessive elements appear in caudal and lateral positions. mals and lissamphibians—the dermal skeleton also
Exclusive of osteoderms, the dermal skeleton initiates
includes bones developing within the eye (scleral
osteogenesis via intramembranous ossification. Following
the establishment of skeletal condensations, some preossi-
ossicles), eyelid (palpebral), integument (osteo-
fied spicules become engorged with many closely packed derms), and across the abdomen (gastralia). Among
clusters of chondrocyte-like cells in a bone-like matrix. This modern taxa, one of the most diverse collections of
combination of features is characteristic of chondroid bone, dermal elements is found in crocodylians.
a tissue otherwise unreported among nonavian reptiles. No All 23 species of extant crocodylians are charac-
secondary cartilage was identified in any of the specimens terized by a well-developed bony dermal skeleton,
examined. With continued growth, dermal bone (including which while lacking scleral ossicles and clavicles,
chondroid bone) and osteoid are resorbed by multi- does include an interclavicle, one or more pairs of
nucleated osteoclasts. However, there is no evidence that palpebrals, numerous parasagittally arranged post-
these cells contribute to the rugose pattern of bony orna- cranial osteoderms, an articulating set of gastralia,
mentation characteristic of the crocodylian dermato- and a robust dermatocranium defining the face,
cranium. Instead, ornamentation develops as a result of orbit, palate, temporal-vault region, and mandible
localized concentrations of bone deposited by osteoblasts. (Table 1; see below). As in most vertebrates, the
Osteoderms develop in the absence of osteoblastic cells,
dermatocranium is the primary determinant of
osteoid, and periosteum; bone develops via the direct trans-
formation of the preexisting dense irregular connective tis-
skull morphology, and hence a major source of phy-
sue. This mode of bone formation is identified as metapla-
sia. Importantly, it is also demonstrated that osteoderms Contract grant sponsors: Natural Sciences and Engineering
are not histologically uniform but involve a range of tissues Research Council of Canada, The Jurassic Foundation, Dalhousie
including calcified and uncalcified dense irregular connec- University Department of Biology Lett Fund.
tive tissue. Between taxa, not all osteoderms develop by
homologous processes. However, it is concluded that all *Correspondence to: Matthew K. Vickaryous, Department of Bio-
logical Sciences, University of Calgary, 2500 University Drive, N.W.,
osteoderms may share a deep homology, connected by Calgary, Alberta, Canada T2N 1N4.
the structural and skeletogenic properties of the dermis. E-mail:
J. Morphol. 269:398–422, 2008. Ó 2007 Wiley-Liss, Inc.
Published online 25 October 2007 in
KEY WORDS: alligator; osteoderm; intramembranous Wiley InterScience (
ossification; chondroid bone; metaplasia DOI: 10.1002/jmor.10575


TABLE 1. Comparison of earliest onset of osteogenesis for dermatocranial elements of Alligator mississippiensis

Region/element FS, this study FS, Rieppel (1993b) FS, other

Premaxilla 18 19 18 (Westergaard and Ferguson, 1990)
Maxilla 17 18 18 (Westergaard and Ferguson, 1990)
Nasal 21 22 20 (Klembara, 1991)
Prefrontal 18 20
Lacrimal 20 20
Postorbital 18 20
Jugal 18 20
Frontal 18 20
Parietal 22 23
Squamosal 19 21
Quadratojugal 19 21
Vomer 18 21
Palatine 18 21
Pterygoid 17 18
Ectopterygoid 21 21 20 (Klembara, 1991)
Dentary 17 19 17 (Westergaard and Ferguson, 1990)
Splenial 18 21
Angular 17 18
Surangular 19 19
Coronoid 17 21

The postpareital is excluded as it was not observed. The results from this study as well as the work of Westergaard and Ferguson
(1990) were obtained from serial histology; data from Klembara (1991) and Rieppel (1993b) were obtained from whole-mount prepa-
rations. See text for details. FS, Ferguson (1985, 1987) Normal Table of Development stage.

logenetic data (Rieppel, 1993a). In a recent phyloge- asphenoid (Klembara, 1993) and postparietal (Klem-
netic evaluation of living and fossil alligatoroid bara, 2001), have been described elsewhere, the
crocodylians (alligators and caiman), 58.5% (n 5 present study concentrates on documenting the
96) of 164 characters employed, at least in part, the remaining dermatocranial elements (Table 1), along
dermatocranium (Brochu, 1999). with gastralia and osteoderms; development of the
Appropriate to the long-standing interest in croc- interclavicle is the subject of another study to be
odylian anatomy, as the closest living relatives of described elsewhere. The purpose of this investiga-
birds and a convenient structural/biomechanical tion is to analyze, in detail, the pattern, sequence,
analogue of many fossil tetrapods, development and and mode of dermal skeletogenesis in A. mississip-
ontogeny of the skeleton has been the subject of piensis, as a representative nonavian reptile, from
numerous investigations (e.g., Parker, 1883; Meek, the embryonic period to posthatching.
1911; Shiino, 1914; Mook, 1921a; de Beer, 1937;
Deraniyagala, 1939; Kesteven, 1957; Müller, 1967;
Iordansky, 1973; Dodson, 1975; Bellairs and Kamal,
1981; Ferguson, 1981, 1984, 1985; Westergaard and METHODS AND MATERIALS
Ferguson, 1986, 1987, 1990; Müller and Alberch,
The majority of Alligator mississippiensis specimens employed
1990; Klembara, 1991, 2001, 2004, 2005; Rieppel, in this study were collected from the Rockefeller Wildlife Refuge,
1993b). To date, however, this work has primarily Grand Chenier, Louisiana, during the summer of 2002. Several
focused on aspects of the endoskeleton, viz., the additional embryos, also from the Rockefeller Wildlife Refuge
chondrocranium, splanchnocranium, and limbs. Stud- (collected in the summers of 1987 and 1988), were kindly donated
by Dr. G. B. Müller (University of Vienna). Eggs were collected
ies of the dermal skeleton have been limited to from the nests of wild populations and incubated in boxes con-
either the pattern and sequence of bone formation taining nesting media at 308C and 90–95% humidity. All embryos
(e.g., Parker, 1883; Rieppel, 1993b) or targeted ac- were morphologically staged according to the Normal Table of
counts of particular elements (e.g., postparietal: Development of Ferguson (1985, 1987). For A. mississippiensis,
Klembara, 2001). As for most nonavian reptiles, as for many reptiles (including birds), oviposition occurs when
eggs are at a relatively advanced stage of embryogenesis (Billett
comparatively few details are known about crocody- et al., 1985). Furthermore, rate of development is highly depend-
lian dermal skeletogenesis and posthatching skele- ent on ambient temperature and environmental conditions (Fer-
tal tissue ontogeny (de Ricqlès et al., 1991). guson, 1985; Hall and Miyake, 2000). Hence, staging embryos
This investigation explores dermal skeleton de- permits more accurate comparisons between individuals than
would be achieved by the use of absolute time alone. In addition
velopment in Alligator mississippiensis, the Ameri- to embryos, several hatchlings and subadults were dissected and
can alligator. As development of the dentition select elements were processed for whole-mount histochemistry
(Westergaard and Ferguson, 1986, 1987, 1990), par- (Table 2). Dried skeletal materials, representing multiple suba-

Journal of Morphology
TABLE 2. Alligator mississippiensis specimens employed during this study

FS 16
Serially sectioned: head (1).
FS 17
Whole-mounted: complete embryo (2, Alizarin red; 2, Alizarin red and Alcian blue).
Serially sectioned: head (2); pectoral region (2); pelvic region (1).
FS 18
Whole-mounted: hemisectioned head (1, Alizarin red; 1, Alizarin red and Alcian blue); pectoral region (1); pelvic region (1).
Serially sectioned: head (2); pectoral region (1); pelvic region (1).
FS 19
Whole-mounted: hemisectioned head (2, Alizarin red; 2, Alizarin red and Alcian blue); pectoral region (1); pelvic region (1).
Serially sectioned: head (3); pectoral region (1); pelvic region (1).
FS 20
Whole-mounted: complete head (1, Alizarin red); pectoral region (1); pelvic region (1).
Serially sectioned: head (2); pectoral region (1); pelvic region (1).
FS 21
Whole-mounted: complete head (1, Alizarin red; 3, Alizarin red/Alcian blue); pectoral region (2); pelvic region (1).
Serially sectioned: head (3); pectoral region (1); pelvic region (1).
FS 22
Whole-mounted: complete embryo (1, Alizarin red; 1, Alizarin red/Alcian blue); complete head (1; Alizarin red); pectoral region
(1); pelvic region (1).
Serially sectioned: head (1); pectoral region (1); pelvic region (1).
FS 23
Whole-mounted: complete head (1, Alizarin red); hemisectioned head (1, Alizarin red); pectoral region (1); pelvic region (2).
Serially sectioned: head (1); pectoral region (1); pelvic region (1).
Subadult, snout vent length (SVL) 5 121 mm.
Whole-mounted: palpebral (1, Alizarin red).
Serially sectioned: palpebral (1); osteoderms from the nuchal shield.
Subadult, SVL 5 175 mm.
Whole-mounted: palpebral (1, Alizarin red).
Serially sectioned: palpebral (1); osteoderms from the nuchal shield.
Subadult SVL 5 195 mm.
Whole-mounted: palpebral (1, Alizarin red); complete gastralial system (1).
Serially sectioned: portions of the head including the maxilla, pterygoid, and dentary; palpebral (1); osteoderms from the nuchal
shield; gastralia (2).
Serially sectioned: osteoderms (2); gastralia (2).
Skulls consulted: maximum cranial lengths of 330, 290, 270, and 120 mm.

Number in brackets indicates how many specimens were prepared. Note that additional materials from the collection of Prof. M.
W. J. Ferguson (University of Manchester) were also consulted. See text for details. FS, Ferguson (1985, 1987) Normal table of de-
velopment stage.

dults and adults were also examined. In addition, staged and embryos, cranial and postcranial materials including gastralia,
serially sectioned embryos from the collection of Prof. M. W. J. osteoderms, palpebrals, and portions of the skull, from several
Ferguson (University of Manchester) were consulted (Ferguson, subadults were also processed for whole-mount histology to eval-
1981; see also Westergaard and Ferguson, 1986). uate the progression of mineralization during posthatching de-
Anatomical terminology follows Brochu (1999; dermatocra- velopment.
nium), Claessens (2004; gastralia), Klembara (1991; chondrocra- Various histological techniques (Table 3) were employed to
nium). Taxonomic nomenclature (e.g., Crocodylia) follows Brochu identify the tissue and cellular composition of the developing der-
(2003). mal skeleton from all embryonic stages and select subadult and
adult materials. Preparation of serial sections included decalcifi-
cation in Tris-buffered 10% EDTA (pH 7.0) for 51 days (up to 4
Histology and Histochemistry weeks for subadult material), dehydration to 100% ethanol,
clearing in CitriSolv (Fisher No. 22-143975), and embedding in
All embryos were fixed in 10% neutral buffered formalin and low melting paraffin at 548C (Paraplast X-tra, Fisher No. 23-021-
stored in 70% ethanol prior to histological analysis. Multiple 401). Sections were cut at 6–7 lm and mounted on Haupt’s or 3-
individuals representing Ferguson (1985, 1987) stages (FS) 16– aminopropytriethoxy-silane coated slides. Following the staining
23 (Table 2) were processed for whole-mount clearing and stain- protocols (see below), sections were mounted with DPX (Fluka
ing with Alizarin red for calcified structures (mineralized with No. 144581).
calcium salts; most commonly bone), alone or in combination The most common connective tissue stain procedures em-
with Alcian blue for tissues rich in glycosaminoglycans (GAGs; ployed were the Hall-Brunt Quadruple (HBQ) stain (Hall, 1986;
characteristic of the extracellular matrix [ECM] of cartilage, and Witten and Hall, 2003) and Mallory’s trichrome stain, carried out
to a lesser extent of osteoid), following the protocol of Klymkow- as follows: once hydrated, deparaffinized sections were mor-
sky and Hanken (1991). Whole-mount preparations included danted in saturated aqueous mercuric chloride (HgCl2) for 10
complete (albeit eviscerated) embryos, isolated heads, pectoral min and then rinsed in distilled water. Sections were briefly
and pelvic regions, and hemisectioned heads. For each hemisec- stained with acid fuchsin (15 s), differentiated in distilled water,
tioned head, the left half was single-stained with Alizarin red then placed in phosphomolybdic acid (1 min), and rinsed again in
while the right half was double stained with Alizarin red and distilled water. The sections were then stained in an aqueous so-
Alcian blue. This permitted multiple views of the developing lution of 4 parts orange G: 4 parts oxalic acid: 1 part aniline blue
skeleton to be visualized in three-dimensions. In addition to for 1 min to 75 s, before being rinsed again in distilled water, and

Journal of Morphology

dermis; lines of resorption

dehydrated with 90% and then twice with 100% ethanol (10 s
each, total of 30 s). Additional protocols include AC pentachrome

No elastin fibers found in

(developed by A. Cole; Cole and Hall, 2004), Toluidine blue
(Witten and Hall, 2003), Masson’s trichrome stain, the Gridley

of resorption black

Lines of resorption
method for reticulin, periodic acid-Schiff (PAS) technique (with
and without removing glycogen with diatase of malt), and Ver-
hoeff elastin stain (Presnell and Schreibman, 1997). Methods

fibers black
deep blue
employed for the Prof. M.W.J. Ferguson embryo and histology
collection are discussed in Ferguson (1981; see also Westergaard

and Ferguson, 1986).

Weakly pink
Olive green-

Grey-brown RESULTS

Light blue

Adult Dermal Skeleton Morphology

TABLE 3. Histology and histochemistry of skeletal tissues and the dermis of Alligator mississippiensis


Small fibers, colourless; Blue

Detailed descriptions of adult crocodylian skeletal

morphology are available elsewhere (e.g., Huxley,
1860; Miall, 1878; Mook, 1921a,b; Iordansky, 1973;

For the purpose of comparision with the staining properties of osteoid, data from the histology/histochemistry of cartilage included.
Dark blue (red around

Cong et al., 1998); the following is presented as a

large fibers, pink

Green (red around

Green (red around

brief summary of relevant bony dermal skeleton fea-


tures with emphasis on Alligator mississippiensis.

Reticular fibers

Weakly pink to



Among crocodylians, the distinctive dermatocra-

Weakly blue

are black

nium is elongate, dorsoventrally depressed (platy-

rostral), and akinetic (except at the quadrate-artic-
Grey with black mottles; Grey

ular jaw joint), with undulating tooth rows, a perva-

sive, externally embossing pitted ornamentation
(see below), and a robust osseous secondary (hard)
capsular matrix black
Red; small amounts of
Red; small amounts of

Weakly blue; capsular

palate (Miall, 1878; Iordansky, 1973; Busbey, 1995;

grey-green mottles

Cong et al., 1998) (Fig. 1A-D). The osseous second-


Red-orange with

matrix purple

green mottles

ary palate is composed of bilateral laminae from

blue mottles

Greyish black
Reddish pink

the premaxilla, maxilla, palatine, and pterygoid

Weakly pink

(Fig. 1C); in most crocodylians, including Alligator

mississippiensis, the vomer is not visible along the
palate. Paranasal sinus cavities branch off the
lengthy nasal cavity to invade the rostrum, includ-
Dermatocranium, gastralium

ing (unique to Alligator spp.) the prefrontal bone

Blue (with purple streaks);

(Witmer, 1995; Brochu, 1999). The external nares

capsular matrix purple
Red with green mottles

are subterminal and dorsally oriented, and the in-

Red with grey mottles

ternal (secondary) choanae are nested exclusively

mottles; capsular
Red with dark blue

matrix dark blue

within the fused pterygoids (Parsons, 1970; Witmer,

1995). In Alligator spp., the external nares are sub-

divided by a process of the nasal bone, the interna-


rial bar (Fig. 1A).

In addition to framing the internal choanae, the
Periodic acid-Schiff (PAS) Weakly pink to pink Pink

fused pterygoids are characterized by an enormous,

transversely oriented, caudoventrally-inclined lat-
eral flange. Although it is frequently reported that
the lateral margin of this flange is capped with
Weakly purple

cartilage (e.g., Parker, 1883; de Beer, 1937; Schu-

Weakly blue

Dark blue

macher, 1973), this has yet to be verified histologi-



Across the superficial (external) surface of the


dermatocranium (and osteoderms; see below), croc-

odylians develop a conspicuous pattern of bony
Toluidine blue (pH 4.5)

ornamentation (osteodermic relief; Iordansky, 1973)

Hall-Brunt Quadruple

Gridley reticulin stain

stain (Alcian blue

consisting of large numbers of pits and grooves

Masson’s trichrome
Mallorys trichrome
Histological stains

(Coldiron, 1974; de Buffrénil, 1982) (Fig. 1A,B).

AC pentachrome

Verhoeff elastin

This rugose sculpturing is restricted to those ele-

ments interfacing with the dermis, including the
pH 2.5)

premaxilla, maxilla, nasal, lacrimal, prefrontal,

postorbital, jugal, frontal, parietal, postparietal,
squamosal, quadratojugal, dentary, angular, and

Journal of Morphology
Ventrally bracing the abdomen are multiple, ob-
liquely oriented rows of rod-like gastralia (Fig. 1E,F).
Each row comprises two medial and two lateral ele-
ments arranged as parasagittal pairs. Combined, the
four elements articulate to form a chevron with the
apex directed cranially. Each gastralium has a round
or ovoid cross-section and tapers both proximally and
distally. Whereas most are relatively thin and grac-
ile, the caudalmost gastralia are noticeably larger
and more robust. In crocodylians, the number of gas-
tralial rows is usually eight, although some variation
has been reported (e.g., Cong et al. [1998] identified
seven rows in Alligator sinensis; see also Chiasson,
1962; Claessens, 2004). Not uncommonly, gastralia
may fuse, develop abnormal morphologies, or even
fail to form (e.g., Claessens, 2004) (Fig. 1F). As in
Sphenodon punctatus (the tuatara), crocodylian gas-
tralia are embedded within the superficial layers of
the M. rectus abdominis (Claessens, 2004); the last
two rows also receive slips from the M. truncocauda-
lis and M. ischiotruncus.
Along the dorsal and dorsolateral body surfaces,
from immediately caudal to the skull, past the sac-
ral region and the base of the tail, crocodylian
integument is invested with large numbers of osteo-
derms (Fig. 1G,H). With the exception of the palpe-
bral (see below), osteoderms do not develop within
the integument covering the skull. In Alligator
mississippiensis, unlike other alligatorids (e.g.,
A. sinensis; Cong et al., 1998; Brochu, 1999) and
many species of Crocodylus, osteoderms do not de-
velop across the abdomen. Similar to gastralia,
osteoderms are arranged in transverse rows. The
Fig. 1. Alligator mississippiensis adult and subadult dermal medialmost (parasagittal) elements often articulate
skeleton morphology. Adult cranium in dorsal (A), left lateral at a suture (but do not fuse), whereas more laterally
(B), and ventral (palatal) (C) views, and corresponding mandi- positioned osteoderms are increasingly distant.
ble in dorsal view (D). Dorsal (A) and ventral (C) views depict- Among sequentially adjacent rows, cranially suc-
ing parasagittal halves the cranium only. Scale bars for A-D 5
50 mm. E: Alizarin red whole-mounted gastralial system from a
cessive osteoderms may imbricate on their caudal
subadult (snout vent length 5 175 mm) in ventral view, demon- counterparts.
strating seven of the eight articulating rows. F: Same specimen In general, crocodylian osteoderms are disc-like
as E illustrating the relationship of between adjacent lateral plates of bone, varying in size and dorsal/ventral
and medial elements of each row. Note the absence of a lateral profile (circular to ovoid and square) depending on
gastralium in the row marked with an asterisk (*). Scale bar
for E 5 20 mm. G: Adult osteoderms representing elements their topographic position. Characteristically, the
from precaudal osteoderm row (PC) 21. Scale bar for G 5 30 superficial (external) surface of each osteoderm
mm. H: Dorsal view of the cervical integument from the same demonstrates a similar pattern of ornamentation as
specimen as (E), illustrating the position of PC 16 to PC 23. I: previously noted for dermatocranial elements
Adult palpebral in dorsal view. Note the presence of the pitted
ornamentation similar to that of the osteoderms in G. Scale
(numerous round pits), plus a point and/or keel of
bars for H, I 5 10 mm. varying prominence (Fig. 1G). The deep surface is
relatively unmarked except for several small foram-
ina for passage of blood vessels and nerves. Among
surangular. The pattern of ornamentation is dy- osteoderms that imbricate, the area of articulation
namic, becomes emphasized with growth (Mook, has reduced ornamentation and instead develops
1921a; de Buffrénil, 1982), and is reminiscent of the longitudinally oriented striae suggestive of a synar-
embossed superficial surfaces of the dermatocra- throsis. Superimposing each osteoderm is a single
nium, osteoderms, and even the clavicle and inter- keratinous scale derived from the epidermis. These
clavicle of many basal tetrapods (e.g., Acanthostega scales substantially augment the profile of the
gunnari, temnospondyls; Coldiron, 1974; de Buffré- points and keels, particularly among the postoccipi-
nil, 1982; Coates, 1996; Castanet et al., 2003). In tal and nuchal elements.
crocodylians, the interclavicle and gastralia are The pattern of osteoderm distribution, parti-
unornamented. cularly along the cervical region, is taxonomically

Journal of Morphology
informative (Huxley, 1860; Ross and Mayer, 1983). Müller and Alberch, 1990), are well-formed and
For Alligator mississippiensis, the normal configu- stain positively for Alcian blue in whole-mount
ration includes two or three transverse rows of preparations.
postoccipital osteoderms immediately caudal to the FS 17 (22-23d). None of the dermal elements
occiput, a distinct cluster of large articulating ele- are positive for Alizarin red at FS 17. However, the
ments dorsal to the cervicals (the nuchal shield), angular and dentary are recognizable in whole-
followed by a near contiguous distribution caudal to mount preparations as textured, weakly opaque
the cervical–thoracic vertebral transition. Work by splint-like condensations occupying positions adja-
Ross and Mayer (1983) demonstrated that most cent to Meckel’s cartilage (caudoventrolaterally and
osteoderms caudal to the cervical region demon- rostrolaterally, respectively; Fig. 2A,B). In addition,
strate a one-to-one relationship with the underlying the presence of the pterygoid (Fig. 2C) and coronoid
vertebrae. On the basis of this correlation, Ross and (Fig. 2D) is confirmed in serial sections with the
Mayer established a scheme for numbering trans- pterygoid positioned alongside the medial surface of
verse rows of osteoderms, beginning with precaudal the processus pterygoideus of the palatoquadrate
osteoderm row 1 (PC 1), the caudalmost transverse (of the chondrocranium) and the coronoid lying cau-
row situated dorsal to (and musculotendinously domediolateral to Meckel’s cartilage (medially adja-
linked with) the caudalmost sacral vertebra. In cent to the angular). All four presumptive elements
practice, PC 1 can be identified by either palpation are readily identifiable as discrete condensations of
(it overlies the caudal edge of the iliac blades) osteoblasts and osteoid within the otherwise loose
or dissection. Transverse osteoderm rows cranial to mesenchyme. A small condensation of osteoprogeni-
PC 1 are numbered sequentially, with the largest tor cells in the initial stages of synthesizing col-
nuchal shield osteoderms as PC 20 and 21 lagen marks the earliest evidence of the maxilla
(Fig. 1H). (Fig. 2E). Unlike the angular, dentary, coronoid,
In many crocodylians, one or more small bony ele- and pterygoid, the maxillary condensation is achon-
ments develop within the upper eyelid, the palpe- dral and not positioned adjacent to any cartilagi-
bral(s) (supraciliary/-ies) (Deraniyagala, 1939; Bro- nous element. Instead the maxilla is located in close
chu, 1999). Similar to postcranial osteoderms, the proximity to the maxillary branch of the trigeminal
palpebral is overlain by a single scale, and while nerve, various blood vessels, and the developing
the deep (orbital) surface is relatively unmarked, dentition.
the superficial surface demonstrates an obvious pit- FS 18 (24-26d). In FS 18 whole-mount prepara-
ted ornamentation (Fig. 1I). In Alligator mississip- tions, the most prominent Alizarin red-positive ele-
piensis, the palpebral is a single element with a ments are the calcified endolymphatic ducts, visible
suboval dorsal/ventral profile and, similar to other within the occipital region of the cranium. Less
crocodylians, articulates via fibrous connective tis- manifest are the white, opaque to weakly Alizarin
sue with the prefrontal bone. red-positive condensations corresponding to the
premaxilla, maxilla, pterygoid, angular, dentary,
and coronoid. Additional elements of the orbit and
Skeletal Development Part I: Pattern
mandibular series are visible in serial section (see
and Sequence
For the sake of convenience, the description of Each premaxilla is a small quadrangular conden-
dermal skeleton development is split into two major sation, restricted to the rostroventrolateral margin
sections. In the first, the pattern and sequence of of the upper jaw adjacent to the lamina transversa-
skeletogenesis are described. The second investi- lis rostralis of the nasal capsule. At this stage, the
gates the mode of skeletal development at the level premaxilla does not underlie the primordium of the
of histology. Within each section, each of the derma- caruncle (keratinous egg tooth) nor is it directly
tocranium, gastralia, and osteoderms are discussed associated with any teeth and/or dental placodes.
separately. A summary of the earliest onset of each The dental arcade continues caudally (i.e., towards
dermatocranial element (as determined using serial the jaw joint) as the maxilla. Thin and tapering in
histology) is presented in Table 1. whole-mount preparations, the maxilla is beginning
Dermatocranium. Preceding the development to expand transversely when observed in section,
of dermal bone, precartilaginous condensations rep- corresponding with the closure of the (soft tissue)
resenting the chondrocranium and splanchnocra- secondary palatal shelves (Ferguson, 1985). The
nium are reportedly visible beginning at FS 14, con- maxilla remains closely associated with the maxil-
current with the formation of epithelial thickenings lary branch of the trigeminal and the developing
or dental placodes representing the earliest evi- dentition at the oral epithelium. The nasal bone
dence of the (nonfunctional) dentition (Westergaard has yet to form.
and Ferguson, 1986, 1990). By FS 16, much of the Among the elements bounding the orbital cavity,
chondrocranium, splanchnocranium, sclerotic carti- condensations representing each of the (presump-
lages, as well as parts of the postcranium (e.g., sty- tive) prefrontal, frontal, postorbital, and jugal are
lopodium, zeugopodium, some of the autopodium; identifiable in section by late FS18, and in whole-

Journal of Morphology
mount preparations by early FS 19. At FS 18, these graphic relationship to the orbital cavity and scleral
elements lack characteristic morphological features cartilage.
and their identity is based solely on their topo- The largest and most complex dermal element to
have developed thus far is the pterygoid (Fig. 2F).
Compared with other dermal bones, growth of the
pterygoid is relatively rapid, resulting in the forma-
tion of a lengthy, tapering palatine process rostrally
and at the caudal margin, the prominent lateral
pterygoid flange. Continued growth of the lateral
pterygoid flange has ventrally enclosed the distal
end of the (cartilaginous) processus pterygoideus of
the palatoquadrate. As a result, cartilage at the
lateral margin of this flange is becoming rapidly
overgrown. Late during FS 18, condensations repre-
senting the presumptive vomer and palatine bones
develop, with the vomer nested against the ventro-
lateral margin of the interorbital septum and the
palatine forming along the dorsolateral border of
the nasopharynx.
Among the mandibular elements, the most promi-
nent is the dentary, nested parachondrally along
the rostrolateral border of Meckel’s cartilage. At the
opposite end of the presumptive mandible, the
angular continues to develop within a distinctive,
caudoventrally positioned cell-rich band. Both the
dentary and angular demonstrate multiple centers
of ossification. Growth of the coronoid corresponds
with increased skeletogenesis of the dentary and
angular. By late FS 18, the long, blade-like splenial
condensation is present along the medial mandibu-
lar margin. The surangular is present as a conden-
sation dorsal to the presumptive Meckelian fossa.
FS 19-20 (27-30d). By FS 19, the majority of
elements from the facial, orbital, palatal, and

Fig. 2. Early development of the Alligator mississippiensis

dermatocranium. All serial sections (A-E, K, L) cut transversely
with dorsal towards the top of the image; whole-mounts (G-I) in
left lateral view with dorsal towards top of image; whole-mounts
(F, J) in medial (parasagittal) view with dorsal towards top of
image. A–F. A: Ferguson stage (FS) 17 angular condensation ven-
trolateral to Meckel’s cartilage, stained with Mallory’s trichrome.
B: FS 17 dentary condensation lateral to Meckel’s cartilage,
stained with AC pentachrome. C: FS 17 pterygoid condensation
caudally adjacent to the processus pterygoideus of the palatoqua-
drate, stained with Mallory’s trichrome. D: FS 17 coronoid con-
densation medial to Meckel’s cartilage, stained with Mallory’s tri-
chrome. E: FS 17 maxilla early condensation, stained with AC
pentachrome. Scale bars for A-E 5 50 lm. F: FS 18 pterygoid,
rostral to the right of the page, Alizarin red single-stained whole-
mount. Scale bar 5 1 mm. G-L. G-J: FS 19 Alizarin red and
Alcian blue double stained whole-mounts of developing skull in
lateral view (G), with closer views of the rostrum and mandible
(H), temporal region and jaw joint (I), and a medial view of the
same cranium (J). Scale bars for G-J 5 5 mm. K: FS 19 dentary
condensation and adjacent developing tooth, stained with Mal-
lory’s trichrome. L: FS 19 splenial condensation laterally adjacent
to Meckel’s cartilage, stained with Mallory’s trichrome. Scale
bars for K, L 5 50 lm. an, angular; co, coronoid; dt, dentary; fr,
frontal; ju, jugal; la, lacrimal; m, Meckel’s cartilage; mx, maxilla;
pl, palatine; pm, premaxilla; po, postorbital; pp, processus ptery-
goideus of the palatoquadrate; pt, pterygoid; qj, quadratojugal;
sa, surangular; sp, splenial; sq, squamosal; t, tooth; vo, vomer.

Journal of Morphology
mandibular series are present. In whole-mount, process, situated medial and parallel to the pala-
most of these condensations are identifiable as tine, partially roofs the nasopharyngeal duct and
white textured or weakly Alizarin red-positive con- secondary choanae. The large and well-ossified lat-
densations, although many demonstrate a loosely eral pterygoid flange has encircled the (cartilagi-
consolidated appearance (Fig. 2G-J). The premax- nous) processus pterygoideus of the palatoquadrate
illa remains restricted to the rostroventrolateral and is directed caudoventrolaterally towards the
margin of the nasal capsule but has formed a con- coronoid and angular of the mandibular series. The
spicuous and well-mineralized ventral (oral) margin palatine has initiated skeletogenesis forming an
(Fig. 2H). The opposite border, the dorsolateral elongate shallowly concave (ventrally) condensa-
(ascending; Iordansky, 1973) process, is weakly tion, partially encircling the dorsolateral margins of
mineralized and composed of radiating spicules of the nasal cavity proper. The rostral process of the
osteoid. The maxilla is primarily visible as a dense palatine ventrally underlaps the weakly developed
sliver of osteoid with multiple centers of mineraliza- blade-like vomer. In section, the vomerine conden-
tion, spanning the majority of the distance between sations remain closely associated with ventrolateral
the premaxilla to the jugal (Fig. 2H). In addition to margin of the interorbital septum.
the maxillary branch of the trigeminal nerve, the With the exception of the frontal bone (Fig. 2J),
rostralmost process of the maxilla lies adjacent to elements of the temporal-vault series are poorly
the nasal capsule; otherwise the maxilla remains developed. The frontal bone forms a narrow arch
distant from elements of the chondrocranium. Dor- across the dorsomedial margin of orbit, adjacent to
solateral growth of the maxilla continues to be neg- the taenia marginalis, thus initiating the future
ative for Alizarin red, but is weakly visible as an link between the prefrontal and the postorbital.
osteoid sheet in both serial section and whole- The squamosal is present as a flat, fenestrated con-
mount. Neither the premaxilla nor the maxilla has densation, partially overlying the lateral margin of
developed palatal laminae, nor is there any indica- the otic process of the palatoquadrate and the
tion of the nasal bone. (future) external ear (Fig. 2I). The quadratojugal is
Among the orbital series of elements, each of the weakly visible as a thin splint situated along the
prefrontal, postorbital, and jugal bones is visible in rostral margin of the cartilaginous quadrate. Nei-
whole-mounts by FS 19 (Fig. 2I); the lacrimal does ther the parietal nor the postparietal are present.
not appear until FS 20. The prefrontal and postorbi- Among the mandibular series, all the dermato-
tal bones frame the orbital cavity at the rostro- cranial elements can be identified as textured, opa-
medial and caudomedial corners, respectively. In que condensations; mineralization of this region
section, the prefrontal directs a deep wedge-like remains poor. The dentary is well-formed, particu-
medial process towards the frontal bone, although larly along the alveolar margin (Fig. 2H,K). In
the two elements do not articulate at this stage of transverse serial sectioned preparations, the medial
development. The postorbital forms a prominent counterpart to the dentary, the splenial, forms a
triangular aggregation, with an embayment along thin plate (Fig. 2L). Caudal to the splenial along
the rostral margin corresponding with the orbit, the medial surface of the mandible is a large,
and three distinct apices directed towards the fron- trough-like opening providing passage for the M.
tal (rostrodorsally), jugal (rostroventrally) and intramandibularis, the Meckelian (mandibular
squamosal (caudoventrally) (Fig. 2I). Contact be- adductor) fossa. The Meckelian fossa is framed by
tween the postorbital and jugal (the postorbital bar) rod-shaped condensations of the surangular (dor-
is incomplete and unossified. The jugal is first sally), coronoid (cranially), and angular (ventrally).
detected in serial section at FS 19 as a weakly Along with the development of the dermatocra-
defined condensation. By late FS 20, the jugal has nium, FS 19 marks the earliest appearance of the
formed a conspicuous triradiate span, defining the gastralia (see below) and interclavicle. Although it
ventral border of the orbital cavity (Fig. 2I). Char- will be detailed elsewhere, it is worth noting that
acteristically, the rostral process of the jugal, the the interclavicle first appears as a bilateral pair of
maxillary ramus, is relatively deep (dorsoventrally) condensations nested midventrally at the cranial
with a tongue-like morphology. The two caudally end of the pectoral apparatus.
directed processes—the jugal contribution to the FS 21 (31-35d). Similar to previous stages, dur-
postorbital bar (caudaodorsally) and quadratojual ing early FS 21 (31-32d) elements of the dermal
process (caudoventrally)—are considerably shorter skeleton are visible in whole-mount preparations as
and taper rapidly. The lacrimal is the last element textured white opaque to weakly Alizarin-positive
of the orbital series to form (FS 20), initially condensations (Fig. 3A-C). However, by late FS 21
appearing as a delicate, web-like condensation (34-35d) most dermal bones, including gastralia
alongside the postconcha. In section, the lacrimal is (see below) and the interclavicle, are easily recog-
observed to develop adjacent to and encircling the nizable in whole-mounted specimens. For the der-
(pre-existing) nasolacrimal canal. matocranium, elements encircling the oral and or-
The palatal series of elements remains dominated bital cavities demonstrate the most robust degree of
by the pterygoid (Fig. 2J). Rostrally, the palatal development while elements of the temporal-vault

Journal of Morphology
forcement of the facial (dorsolateral) process (Fig.
3A). Palatal shelves are also in the early (i.e., non-
ossified) stages of development, and in serial section
are observed to underlap the rostral end of the pala-
tine condensation. Caudally the maxilla tapers as a
lengthy rod of bone and osteoid towards the jugal.
FS 21 marks the first appearance of multinucleated
osteoclasts associated with dermal bone (Fig. 3D).
Within each of the premaxilla and maxilla, these
large cells are frequently nested alongside the bone
matrix adjacent to the developing dentition and
dental lamina. Osteoclasts are also found on spi-
cules located within the canal carrying the maxil-
lary branch of the trigeminal nerve through the
maxilla. Although there is no sign of the nasal bone
in early FS 21 (31-32d) whole-mounts, it is pres-
ent in section as an arched lamina of osteoid several
cells thick, roofing the dorsolateral border of the
nasal capsule (the tectum nasi). Laterally, the nasal
is overlapped by the maxilla (Fig. 3E). By late FS
21 (34-35d), the nasal bone is present in whole-
mount specimens as a thin Alizarin red-positive
plate dorsal to the rostral end of the nasal capsule.
As for other scarf joints of the dermatocranium, the
future zone of articulation between the maxilla and
nasal is filled with a relatively dense, cell-rich, con-
nective tissue.
Similar to the facial series, the orbital series of
elements are also increasingly well-developed. By
the end of FS 21, the lacrimal, prefrontal, jugal,
and postorbital are all distinctly present as conden-
Fig. 3. Developing Alligator mississippiensis dermatocra- sations of ossified and/or osteoid spicules along the
nium at Ferguson stage (FS) 21. (A-C) Alizarin red single- margins of the orbital cavity (Fig. 3A,B). The pre-
stained whole-mount in right lateral view (A), dorsal view of or- frontal is the best developed element of the orbital
bital cavity (eyeball in situ) (B), and ventral (palatal) view of
palate (C). Scale bar for A 5 5 mm. Serial sections (D-E) cut series, forming a prominent Alizarin red-positive
transversely with dorsal towards the top of the image. D: Multi- mass rostroventromedial to the eye. In transverse
nucleated osteoclast (black arrowhead) resorbing osteoid spicule section, the prefrontal abuts the dorsal border of
adjacent to the developing dentition. E: Developing scarf joint the lacrimal, overlaps the developing frontal, and
between the nasal and maxilla (E). Scale bar for E 5 50 lm.
an, angular; co, coronoid; dt, dentary; ec, ectopterygoid; ey, eye-
pinches out caudomedial to the orbital cavity. The
ball; fr, frontal; ju, jugal; la, lacrimal; mx, maxilla; na, nasal; pf, postorbital superimposes the area between the car-
prefrontal; pl, palatine; pm, premaxilla; po, postorbital; ps, par- tilaginous sclera of the eyeball and the dorsal pro-
asphenoid; pt, pterygoid; qj, quadratojugal; sa, surangular; sp, cess of the palatoquadrate. Contact between the
splenial; sq, squamosal; vo, vomer. jugal and squamosal, combined as the postorbital
bar, is incomplete. Spanning the ventral margin of
series are the most weakly represented. Many of the orbital cavity, the trifurcate jugal has initiated
the earliest established elements (e.g., the premax- ossification within the postorbital process and
illa, maxilla, prefrontal, pterygoid) now have a tra- quadratojugal ramus. The rostral contact with the
becular-like appearance in section: each element maxilla is observed to form a scarf joint in section
appears as a distinctive network of interconnecting (jugal overlapping maxilla). Skeletogenesis of the
bony and/or osteoid and chondroid bone spicules lacrimal bone trails that of all the other orbital se-
(see below). ries elements; it does not become Alizarin-positive
The premaxilla and maxilla have each advanced until late FS 21 (Fig. 3A). In serial section, numer-
both in terms of the degree of mineralization and ous osteoclasts are associated with spicules lining
overall size (Fig. 3A), but remain separate from one the nasolacrimal duct.
another and their contralateral counterparts. With Among the temporal-vault series, the frontal
continued growth, each premaxilla partially under- bone is the most advanced, and by early FS 21 it
lies the recently consolidated caruncle. In whole- forms a narrow, medially deflected, and dorsoven-
mount preparations, the rostral end of the maxilla trally arch that bridges the gap between the pre-
has developed numerous mineralized spicules radi- frontal and postorbital condensations (Fig. 3B).
ating dorsally to provide the earliest skeletal rein- Although eventually forming the dorsal rim of the

Journal of Morphology
orbital cavity, at this stage the frontal remains
obscured from lateral view by the relatively large
eye. Rostrally, the frontal bone forms a thin laminar
sheet, roofing the caudal portion of the nasal cap-
sule and underlapping the prefrontal. In a caudal
direction, approaching the eye, the frontal bone
tapers in cross-section to form a wedge, with well-
defined (uninterrupted) medial and lateral borders
adjacent to the orbit and nasal capsule, and a fenes-
trated dorsal surface. Both the squamosal (laterally
capping the otic process of the palatoquadrate) and
quadratojugal (positioned along the rostral border
of the palatoquadrate) bones are present, although
neither is heavily ossified. The parietal and post-
parietal remain undetected.
The rostral process of the pterygoid has increased
in length, and when viewed in lateral profile, the
distal tip of the lateral pterygoid flange lies in the
same horizontal plane as the external mandibular
fenestra of the mandible. In section, each of the pal-
atine and vomer has expanded transversely. The
palatine occupies a position along the lateral border
of the nasal cavity, while the vomer contributes to
the roof (Fig. 3C). FS 21 marks the first appearance
of the ectopterygoid, partially bridging the gap
between the pterygoid and jugal. The ectopterygoid
has an irregular shape with a weakly ossified ven-
tromedial process directed towards the lateral
flange of the pterygoid and a spade-shaped cranio-
lateral process forming the caudal margin of the Fig. 4. Developing Alligator mississippiensis dermatocra-
nium at Ferguson Stage early 22 (A-B) and early 23 (C-F). Aliz-
suborbital fenestra. A third, dorsal (ascending) arin red and Alcian blue double-stained whole-mounts illustrat-
process, contributing to the postorbital bar, is pres- ing an oblique, close-up view of the rostrum (A) and temporal-
ently unossified. vault (B) regions. Alizarin red single-stained whole-mounts
Corresponding with development of the facial illustrating a rostrolateral view (C) and dorsal view (D) of the
and palatal series, by FS 21 the mandibular series skull, a medial view (E) of the mandible, and a ventral (palatal)
view of the palate (F). Scale bar for D 5 5 mm. an, angular; co,
of elements are also at a relatively advanced stage coronoid; dt, dentary; eb, epibranchial; ec, ectopterygoid; fr,
of osteogenesis (Fig. 3A). The dentary continues to frontal; ju, jugal; la, lacrimal; mx, maxilla; na, nasal; pf, pre-
demonstrate multiple centers of ossification, with frontal; pl, palatine; pm, premaxilla; po, postorbital; ps, para-
more intense staining rostrally. Contralateral den- sphenoid; pt, pterygoid; qj, quadratojugal; qu, quadrate; sa, sur-
angular; sp, splenial; sq, squamosal; vo, vomer.
taries remain separated at the mandibular symphy-
sis. For identification of the remaining elements,
the Meckelian fossa provides a convenient land- (Fig. 4C-F), caudolateral growth of the premaxilla
mark. The cranial margin of this opening is well- effectively isolates the external nares from the max-
defined by the inferior (caudoventral) process of the illa. The premaxilla overlaps both the nasal and
coronoid; at this stage the superior (caudodorsal) maxilla bones to form scarf joints. In ventral (pal-
process is unmineralized. The dorsal margin of the atal) view, the dental arcade of each premaxilla is
fossa is approximated by the position of the ossify- associated with the adult complement of five teeth
ing surangular, while the ventral border is defined (at FS 23), and the palatal process has begun to os-
by the dense, rod-like angular (Fig. 3A). The sple- sify the rostral border of the foramen incisivum
nial is positioned cranial to the coronoid as a long, (Fig. 4F). Immediately caudal, the palatal shelves
thin vertically-oriented plate. of the maxilla have also begun to ossify, and by FS
FS 22-23 (36-45d). At FS 22 the morphology of 23 they have nearly eliminated the large palatal
the dermatocranium is rapidly approaching the fontanelle once continuous with the foramen incisi-
hatchling condition (Fig. 4A,B), and the superficial vum. The future alveolar margin of the maxilla has
ornamentation is increasingly well-developed. All of an undulating appearance in lateral view, and, at
the facial series elements, including the nasal, have FS 23, is associated with as many as 19 teeth. Simi-
become ossified and are visible in whole-mount lar to posthatchlings, the first four teeth increase in
specimens. Dorsolateral growth and continued min- size with tooth number four as the largest. The
eralization of the premaxilla has begun to define nasal bone is increasingly ossified, roofing the ma-
the rostral margin of the external naris. By FS 23 jority of the tectum nasi, contributing to the caudal

Journal of Morphology
border of the external naris, and has initiated for- any of the specimens examined (representing FS
mation of the internarial process (Fig. 4D). Ulti- 15–23 and various posthatching stages; see Table
mately, the internarial processes of the nasal will 2). Reportedly, it is the last dermatocranial ele-
join with counterparts from the premaxilla (as of ments to form and is visible at FS 27 (Klembara,
yet undeveloped) to subdivide the paired external 2001), demarcating the caudalmost border of the
nares. In the sagittal plane the paired nasals all dermatocranium.
but articulate, effectively isolating a large fonta- In ventral (palatal) view, the vomer remains visi-
nelle at the proximal end of the rostrum from the ble at FS 22, but is obscured by growth of the pala-
external nares. Ventrolaterally, the nasal forms a tines and merging of the maxillary palatal lamina
scarf joint, underlapping the maxilla, while cau- by FS 23 (Fig. 4F). Similarly, at FS 23 the ptery-
dally it receives the rostral process of the prefrontal goids have fused. Continued ossification of the
within a tongue and groove contact. ectopterygoid results in a contiguous ossified mar-
The prefrontal bone remains the most robust ele- gin of the suborbital fenestra.
ment of the orbital series. As seen dorsolaterally, it Overall, by FS 22–23 elements of the mandibular
forms the rostromedial border of the orbital cavity series conform to the posthatching morphology
before tapering caudally to overlap the frontal bone (Fig. 4C), except the caudal margins of the dentary
(Fig. 4D). At this stage, the prefrontal bone meets and splenial (Fig. 4E), which are incompletely ossi-
the lacrimal bone along a butt joint. The lacrimal fied. Each dentary has 19 teeth, and joins with its
bone is becoming increasingly well-ossified, nested contralateral counterpart at the mandibular sym-
firmly between the prefrontal and the overlapping physis. The coronoid is chrevon-shaped in FS 22
maxilla bones (Fig. 4A,C). The medial wall of the Alizarin red whole-mounts, indicating that both the
canal for the nasolacrimal duct is the first to ossify superior and inferior processes are ossified. By FS
by FS 22; the lateral wall is Alizarin red-positive at 23, all of the angular, surangular, and coronoid are
FS 23. Similar to early stages, osteoclasts are fre- in articulation (Fig. 4E).
quently associated with bone and osteoid lining this Gastralia. Prior to the development of gastralia,
passage. At FS 22, two of the three jugular pro- the postcranial endoskeleton, including dorsal ver-
cesses have attained the posthatching morphology: tebrae, pelvis, and hindlimbs, is already well-
the maxillary process (rostrally) and the postorbi- formed in cartilage. Gastralia develop within a
tal process (contributing to the postorbital bar) loose mesenchyme similar to that described for the
(Fig. 4B). The quadratojugal process of the jugal dermatocranial elements, externally adjacent to
does not ossify until FS 23 (Fig. 4C). The postorbital hypaxial (body wall) musculature. The abdominal
bone articulates with the squamosal via a tongue- region is dominated by an elongate spindle-shaped
like process beginning in FS 22. Medially, this pro- cleft, the umbilicus, through which the umbilical
cess creates the rostrolateral border of the supra- stalk, containing coils of the intestine, associated
temporal fenestra. blood vessels, and the allantois, passes.
As for all the dermal elements defining the or- FS 19 (27-28d). The first gastralia to form are
bital cavity, the orbital margin of the frontal is the paired lateral elements from the caudalmost
smooth and well-ossified; distal to the orbit, ossifi- row, followed shortly thereafter by lateral elements
cation of the frontal is comparatively diffuse and of the penultimate row. These successive pairs are
incomplete (Fig. 4D). However, by FS 23 the paired visible in whole-mount preparations as textured
frontal bones have fused between the orbital cav- rudiments, opaque, or weakly positive for Alcian
ities. Along with the nasal and prefrontal, this ros- blue, situated cranially adjacent to the distal end of
tral contribution partially roofs the tectum nasi. the (strongly alcianophilic) pubis (Fig. 5A). In cross-
Further caudal, the contralateral frontal bones fol- section, these caudolateral gastralia are condensa-
low the contour of the orbital cavity margin and tions of osteoprogenitor cells and/or osteoblasts
rapidly diverge, creating a large fontanelle over the secreting thin collagen fibers and osteoid (Fig. 5B).
brain. The quadratojugal is well-developed by FS Each gastralium is positioned along the ventrolat-
22 (Fig. 4B), lying lateral to the quadrate. At no eral margin of the hypaxial (body wall) muscula-
time (pre- or posthatching) does it form the small ture, and at least for the caudalmost elements, is
pointed spina quadratojugalis characteristic of most associated with muscle fibers originating from the
crocodylians. The plate-like squamosal has an pubis (one or both of the M. truncocaudalis and M.
embayment along its medial border, contributing to ischiotruncus; Claessens, 2004).
the margin of the supratemporal fenestra. Further FS 20-21 (29-35d). Gastralia develop in a cau-
caudal, the parietal has only just initiated ossifica- dal to cranial sequence, correlated with the relative
tion along its future lateral border at FS 22. By FS reduction in the length of the umbilicus. By late FS
23, the parietal has expanded to the point where it 20/early FS 21, the first pair of medial gastralia
defines the medial boundary of the supratemporal appears, developing in the caudalmost row. At
fenestra. However, the parietal remains paired around this time, the lateral elements from the cau-
with the bilateral elements separated by a large dalmost row begin to ossify, as demonstrated by
fontanelle. The postparietal was not identified in positive reactions for Alizarin red. By mid FS 21,

Journal of Morphology
FS 22-23 (36-45d). Gastralia development con-
tinues in the above described caudal and lateral to
cranial and medial progression. By mid FS 22, the
arrangement of the Alizarin red-positive gastralial
system (at this stage consisting of six lateral and
four medial elements) is beginning to resemble the
articulating adult morphology. In the oldest em-
bryos examined (two specimens at FS 23), only
seven rows of gastralia were present (Fig. 5D). It
remains unclear at which stage the complete com-
plement of eight rows with four elements (two
pairs) per row is developed. With the uptake of Aliz-
arin red, variability within the gastralial system
becomes apparent, including the absence of ele-
ments and skeletal malformations (Fig. 5D).
Osteoderms and palpebrals. Compared with
all other elements of the skeleton, the onset of
osteoderm and palpebral calcification is signifi-
cantly delayed; neither is present in embryos or
newly hatched individuals. The youngest (5 small-
est) individual in this study demonstrating evi-
dence of calcified postcranial osteoderms and palpe-
brals was 175-mm snout-vent length (SVL; esti-
mated at 1 year in age). For osteoderms, Alizarin
red-positive tissue first appears among the medial-
most nuchal shield elements, rows PC 20 and 21
(Fig. 5E). Concomitant with the early calcification
of the nuchal shield osteoderms is the first appear-
ance of the mineralized palpebral. When viewed
Fig. 5. Gastralium, osteoderm, and palpebral development dorsally, each palpebral has a roughly ovoid profile,
in Alligator mississippiensis. Cranial is towards the top of the and is positioned at the rostral apex of the semicir-
image for A, C-F. A: Ferguson stage (FS) 19 Alizarin red and cular eyelid (Fig. 5F), adjacent to, but separate
Alcian blue double stained-whole-mount. The first gastralium to from, the prefrontal bone.
develop is the lateral element of the caudalmost row (bottom
black arrowhead), cranially adjacent to the (alcianophilic) pubis Among increasingly larger (5 older) individuals,
(bottom right corner); subsequent elements develop cranially calcified osteoderms sequentially appear at posi-
(top black arrowhead) and medially (not shown). Note the weak tions caudal, lateral, and cranial (e.g., postoccipital
blue staining of the gastralium closest to the pubis, indicative rows PC 23–25; see Fig. 1H) to the nuchal shield.
of osteoid. Scale bar 5 1 mm. B: FS 19 serial section cut trans-
versely with dorsal towards top of image. Two sequential gas-
Consequently, osteoderm skeletonization does not
tralia (black arrowheads) demonstrating the relationship of conform to a strictly cranial to caudal sequence of
these elements to the body wall musculature. Scale bar 5 70 development, nor is it synchronous across the
lm. C: FS 21 Alizarin red single-stained whole-mount in ven- integument. As a corollary, the entire develop-
tral view demonstrating the development of the caudalmost gas- mental spectrum of osteoderm formation can be
tralial rows including both lateral and medial components. D:
FS 23 Alizarin red single-stained whole-mount in ventral view explored within a single (appropriately staged)
demonstrating the development of seven gastralial rows includ- individual.
ing both lateral and medial components. Note the unilateral de- Within each osteoderm, calcification is first ob-
velopment of a left medial gastralium in the cranialmost row served within the keel of the presumptive element.
(white arrow), the bifurcation of various elements, and the
asymmetrical position of the right gastralia identified with the
As the individual grows, the presence of Alizarin
white arrowhead. E: Subadult (snout vent length [SVL] 5 175 red-positive tissue begins to spread beyond the
mm) cervical integument, Alizarin red single-stained whole- keel, into the substance of the remainder. For the
mount in dorsal view demonstrating medially positioned (i.e., palpebral, calcification generally continues in a
immediately parasagittal) precaudal osteoderms (PC) 20, 21, caudolateral direction.
and 23. Note the pattern of calcification, beginning within the
keel and radiating outwards. F: Subadult (SVL 5 175 mm;
same specimen as E) eyelid, Alizarin red single-stained whole-
mount in ventral view demonstrating the position of the ossify-
ing palpebral. Scale bars for C-F 5 5 mm. Skeletal Development Part II:
there are six gastralial rows, with pairs of medial The nondental portion of the amniote dermal
elements present in the caudalmost five (Fig. 5C). skeleton is widely considered to develop via intra-
Corresponding with the sequential onset of appear- membranous ossification. As will be demonstrated
ance, gastralia ossification is not simultaneous. however, osteoderm development includes calcifica-

Journal of Morphology
tion of preexisting connective tissues and osteogen- rounding mesenchyme and osteoid stain dark blue
esis via metaplasia. Accordingly, within an indi- for Mallory’s trichrome, green for AC pentachrome
vidual, osteoderms—as organs—are not homogenous and Masson’s trichrome, and gray with Verhoeff
ossified elements. To highlight the diversity of hard elastin stain. Similarly, neither the mesenchyme
(stiff) tissues developed in osteoderms, the term nor osteoid stains positive for Bismarck brown (AC
skeletogenesis is employed. As for the previous sec- pentachrome). However, unlike mesenchyme, osteoid
tion, the mode of skeletal tissue development for within the condensation is weakly positive for
each of the dermatocranium, gastralia, and osteo- Alcian blue (pH 2.5) in both double stained whole-
derms are discussed separately. A summary of the mounts and in serial section (HBQ protocol), and
staining techniques employed, and histological and demonstrates weak metachromasia with Toluidine
histochemical characteristics of the dermis and blue (pH 4.5) (Fig. 6C). Prior to ossification, the
skeletal tissue is presented in Table 3. condensations are negative for Alizarin red in
Dermatocranial osteogenesis. Dermatocranial whole-mounts, but are weakly visible with oblique
osteogenesis begins early in the embryonic period, illumination as textured opaque concentrations.
in most instances before ossification of the chon- Following the establishment of the osteoid-rich
drocranium and splanchnocranium (Iordansky, 1973). condensation, the presumptive element begins to
Prior to dermal bone formation, the ECM is a loose manifest the adult morphology. Continued growth of
homogenous mesenchyme with many argyrophilic the condensation occurs within a fibroblastic and
fibers (Gridley method; Fig. 6A), forming a reticu- collagen fiber capsule, the future periosteum, defin-
lated meshwork, but no elastin (Verhoeff elastin ing what has been termed the bone territory
stain). Included within the ECM are numerous nerves (Pritchard, 1974). Spicules of osteoid are surrounded
and blood vessels. by numerous osteoblasts (Fig. 6D-F) and organized
The earliest sign of dermal bone is the formation into a ramified trabecular-like framework within
of a localized aggregation or condensation of small the bone territory. With continued growth, some
fibroblast-like osteoprogenitor cells and fine colla- (but not necessarily all) spicules within the bony ter-
gen fibers (Fig. 6B). As the population of cells ritory become engorged with clusters of cells that
within the condensation increases, centrally posi- abruptly change their morphology from plump to
tioned osteoprogenitor cells begin to differentiate hypertrophic. These large and irregularly arranged
into larger, plumper osteoblasts, synthesizing and spherical cells are tightly packed within a thin de-
secreting osteoid. In serial section, both the sur- posit of intercellular matrix (Fig. 6G,H). Morpholog-

Fig. 6. Details of the mode of ossification and histology of the dermatocranium in Alligator mississippiensis. All sections with
dorsal towards the top of the image. Serial sections (A, B, D, F, G, J, N-R) cut transversely; serial sections (C, E, H, I, K, L, M)
cut longitudinally. A-F. A: Ferguson stage (FS 16) stained with Gridley method for reticulin. Section taken from the area of the pre-
sumptive maxilla. Note the presence of many interconnected argyrophilic fibers. No dermal skeleton condensations are present at
this time. B: FS 19 early vomer condensation ventrolaterally adjacent to interorbital cartilage, stained with Mallory’s trichrome.
The condensation is characterized by a growing accumulation of osteoprogenitor cells and thin collagen fibers. C: FS 18 pterygoid
condensation caudally adjacent to the processus pterygoideus of the palatoquadrate, stained with Toluidine blue. Note the weak
metachromatic staining of the presumptive pterygoid. Scale bars for A-C 5 100 lm. D: FS 19 maxilla condensation medially adja-
cent to a blood vessel (black arrow), stained with Mallory’s trichrome. The initial condensation is beginning to develop radiating
spicules of osteoid. E: FS 19 prefrontal condensation composed of numerous radiating spicules, stained with Mallory’s trichrome.
Scale bars for D, E 5 200 lm. F: FS 20 mineralizing prefrontal spicule, stained with the HBQ stain. The tip of the spicule is
actively growing, as demonstrated by presence of numerous osteoblasts depositing (weakly alcianophilic) osteoid. Scale bar 5 100
lm. G-L. G, H: FS 21 spicule of maxilla (G) and prefrontal (H) demonstrating the presence of large numbers of hypertrophic cells,
stained with Mallory’s trichrome. This cell-rich tissue is identified as chondroid bone. Note that cell density decreases in areas that
have become mineralized (red). I: FS 22 section through ossifying postorbital, stained with AC pentachrome. J: FS 23 section
through lacrimal, stained with Gridley method for reticulin. Various argyrophilic fibers penetrate into the surrounding ECM. Scale
bars for G-J 5 200 lm. K: FS 23 section through prefrontal, stained with Mallory’s trichrome. Note the histoarchitectural polarity,
with the less interrupted ventral border (bottom of image, white arrows) and radiating spicules directed dorsally. L: FS 23 section
through premaxilla, stained with Verhoeff elastin stain. This section demonstrates the early stages of histoarchitectural polarity,
with a less interrupted deep border (right of image, white arrows) and various radiating spicules directed superficially (left of the
image). Scale bars for K, L 5 200 lm. M-R. M: FS 23 section through alveolar bone of the maxilla, stained with Mallory’s trichrome.
Multinucleated osteoclasts (black arrowheads) are often associated with remodeling the alveolar bone. Scale bar 5 100 lm. N: Sub-
adult (SVL 5 195 mm) section through nasal, stained with Toluidine blue. Note the metachromatic capsular matrix surrounding most
of the lacunae. In the cell-rich seam (middle of image), the interterritorial matrix is also weakly metachromatic. Scale bar 5 50 lm.
O: Subadult (SVL 5 195 mm; same as N) section through the suture linking the angular and dentary, stained with HBQ. Many large
collagen fibers reinforce the connection between these elements. P, Q: Subadult (SVL 5 195 mm; same as N) section through the
angular (P) and splenial (Q), stained with AC pentachrome. Note the histoarchitectural polarity of the bone, with the superficial sur-
face of the angular (P) (interfacing with the dermis; white arrowheads) demonstrating many radiating spicules, whereas the medial
surface of the splenial (Q) (interfacing with the tissues lining the oral cavity; white arrows) is smooth and unornamented. Scale bars
for O-Q 5 40 lm. R: Subadult (SVL 5 195 mm; same as N) section through alveolar bone of the maxilla, stained with Mallory’s tri-
chrome. Several large multinucleated osteoclasts are identified (black arrowheads). Scale bar for R 5 100 lm. an, angular; dt, den-
tary; ic, interorbital cartilage; pp, processus pterygoideus of the palatoquadrate; pt, pterygoid; vo, vomer.

Journal of Morphology

Figure 6

ically, this tissue closely resembles a hypertrophic As islands of osteoid and chondroid bone
cartilage undergoing interstitial growth, with within the bone territories become mineralized,
dilated cells arranged in doublets and (nonco- the growing spicules are visualized with Alizarin
lumnar) isogenous groups; furthermore cell proc- red. In whole-mounts, elements at this initial
esses/canaliculi are not readily demonstrable. How- stage of ossification typically appear as highly
ever, histochemically the intercellular matrix stains emarginated and fenestrated rudiments. In sec-
identically to osteoid, although with no selective af- tion, ossifying spicules and intercellular matrix
finity for Alcian blue. This combination of chondro- are mottled red with most connective tissue
cyte-like cells in a bone matrix is characteristic of stains (Mallory’s trichrome [Fig. 6G,H], Masson’s
chondroid bone (Huysseune and Verraes, 1986; trichrome, AC pentachrome [Fig. 6I], and HBQ),
Huysseune and Sire, 1990; Huysseune, 2000; Gillis and gray-black with Verhoeff elastin stain. Histo-
et al., 2006; see also Hall, 2005). logically, bone territories contain chondroid bone,

Journal of Morphology
osteoid, and woven bone. Woven (and chondroid) becomes increasingly robust and well-mineralized
bone is weakly PAS positive, following removal of with peripherally deposited woven bone.
glycogen and orthochromatic with Toluidine blue, In some regions of the dermatocranium, the large
except for the territorial (capsular) matrix of wo- numbers of osteoblasts and osteocytes are con-
ven bone lacunae, which are metachromatic. trasted by the presence of multinucleated osteo-
Using the Gridley stain, ossifying elements are clasts (with 2 to 251 nuclei each) (Fig. 6M).
gray to grayish-brown with black reticular fibers Multinucleated osteoclasts are found in association
passing through the osteoid and into the perios- with osteoid, woven bone, and chondroid bone, but
teum and surrounding connective tissue (Fig. unlike osteoblasts they are not observed along su-
6J). Within an individual dermatocranial bone perficial surfaces of the dermatocranium. Instead,
territory, the mesenchyme between adjacent spi- they are found at the tips of deeply (inwardly)
cules is composed of thin fibers positive for directed spicules lining passages for large nerves
Alcian blue (HBQ protocol; Fig. 6F). (e.g., the maxillary branch of the trigeminal), ducts
Shortly after mineralization begins, elements (the nasolacrimal duct), and alveolar bone (Figs.
interfacing with the presumptive dermis (particu- 3D, 6M).
larly those of the facial and orbital series) demon- Shortly after mineralization begins, presumptive
strate a distinctive histoarchitectural polarity. elements have acquired the basic posthatching mor-
While deep bone surfaces are relatively smooth and phology and are increasingly well-stained with Aliz-
uninterrupted, the superficial (integument) surface arin red. Although originally separate, expansion of
is often irregular with radiating and branching spi- adjacent bone territories ultimately leads to contact
cules (Fig. 6K,L). Macroscopically, these spicules between the outer, fibrous portion of the periostea,
combine to form the numerous pits and depressions giving rise to sutures (Pritchard, 1974). The inner-
ornamenting the dermatocranium. most cell-rich (cambial) portions of periostea remain
Continued growth occurs as the entire element distinct and independent.
undergoes appositional (centrifugal; sensu Francil- By 1-year posthatching, patches of lamellar bone
lon-Vieillot et al., 1990) ossification, with the most have been deposited adjacent to the pre-existing
obvious accumulations of osteoblasts and osteoid woven and chondroid bone, giving rise to fibrola-
associated with the tips of bony spicules; elsewhere mellar architecture. Adding to the heterogeneity
osteoblasts form a continuous layer one cell thick are numerous primary osteons, scalloped resorption
(Fig. 6F,H,I,L). The once loose reticulation of lines, and deeply nested seams of bone distinctly
osteoid-rich spicules and chondroid bone masses cell-rich and weakly metachromatic (for Toluidine

Fig. 7. Details of the mode of ossification and histology of gastralia (A-F), osteoderms, and palpebrals (G-R) in Alligator missis-
sippiensis. All sections with dorsal towards the top of the image. Serial sections (A-J, M, R) cut transversely; serial sections (K, L,
O-Q) cut longitudinally. A-F. A: Ferguson stage (FS) 19 gastralium condensation, stained with Mallory’s trichrome. Osteoprogenitor
cells are aggregating and beginning to secrete thin collagen fibers (black arrowhead) externally adjacent to the body wall muscula-
ture. B: FS 20 osteoid gastralium, stained with Toluidine blue. Note the weak metachromasia of the osteoid, particularly where
lacunae are closely spaced. C: FS 21 ossifying gastralia, stained with Mallory’s trichrome. The core of the gastralium is character-
ized by a dense accumulation of large chondrocyte-like cells. Scale bars for A-C 5 50 lm. D: FS 23 ossifying gastralium, stained
with Mallory’s trichrome. The gastralium remains cell-rich, and is now enveloped by a well-defined periosteum. Scale bar 5 100
lm. E: Subadult (snout vent length [SVL] 5 195 mm) gastralium, stained with AC pentchrome. Although primarily composed of
woven bone and poorly calcified matrix, areas of the gastralium have become eroded (white arrows) and lined with newly deposited
parallel-fibered bone. F: Subadult (SVL 5 195 mm; same specimen as E) gastralium, stained with Toluidine blue. Capsular matrix
is metachromatic, as is some of the interterritorial matrix of the cell-rich, poorly calcified core region. Scale bars for E, F 5 40 lm.
G-L. G, H: Subadult (SVL 5 175 mm) laterally positioned (i.e., not immediately parasagittal) osteoderm from precaudal row (PC)
23, stained with Masson’s trichrome. The first sign of osteoderm development begins within the superficial dermis (G; black arrow-
heads), with the formation of a dense knot of irregular connective tissue (H). Note the absence of phenotypically osteoblastic cells.
I: Subadult (SVL 5 175 mm; same as G) medially positioned (i.e., immediately parasagittal) osteoderm from PC 23, stained with
AC pentachrome. Calcification (stained here as black) begins within the keel and continues to proceed radially. Scale bars for G, I
5 1 mm. J: Subadult (SVL 5 175 mm; same as G) medially positioned osteoderm from PC 21, stained with Masson’s trichrome.
Large numbers of collagen fibers (black arrows) pass uninterrupted into the calcified matrix, firmly securing the osteoderm within
the dermis. K, L: Subadult (SVL 5 195 mm) palpebral, stained with Mallory’s trichrome (K) and HBQ stain (L). Various cells are
entrapped within the mineralized matrix, surrounded by poorly mineralized, alcianophilic, capsular matrix. Note the absence of a
cell-rich osteogenic horizon and periosteum surrounding the calcifying element. Scale bars for J, K 5 50 lm; scale bar for L 5 40
lm. M-R. M, N: Subadult (SVL 5 195 mm) medially positioned (i.e., immediately parasagittal) osteoderm from PC 21, stained with
Mallory’s trichrome. The calcified component of the osteoderm has increased in size and individual spicules are beginning to inter-
connect (M). The osteoderm is firmly embedded within the dense irregular connective tissue of the dermis, anchored by various col-
lagen fibers passing uninterrupted into the calcified matrix (N). Scale bar for M 5 1 mm; scale bar for N 5 40 lm. O: Subadult
(SVL 5 195 mm; same as K) medially positioned osteoderm from PC 21, stained with Toluidine blue. Where lacunae are closely
spaced, the interterritorial matrix is weakly metachromatic. Scale bar for O 5 50 lm. P-R: Adult (SVL 5 1.51 m) medially posi-
tioned osteoderm from PC 20, stained with Verhoeff elastin stain (P), HBQ (Q), and Mallory’s trichrome (R). The matrix is very het-
erogenous, with scalloped resorption lines (white arrows), parallel-fibered, lamellar, and woven, and dense irregular connective tis-
sue. Note the seam of unmineralized dense irregular connective tissue in R, stained here as blue. Scale bars for P-Q 5 40 lm.

Journal of Morphology

Figure 7

blue; Fig. 6N). In addition, osteocytic lacunae are adjacent to (and associated with) alveolar bone
surrounded by an unossified territorial matrix. Su- (Fig. 6R).
tural contacts between adjacent elements are rein- Gastralial osteogenesis. Development of gas-
forced by dense connective tissue (Fig. 6O) that tralia closely parallels that of the dermatocranium.
lacks both reticular fibers and elastin. The histo- Gastralia begin to develop shortly after the initia-
architectural polarity first identified early during tion of elements in the dermatocranium, concurrent
skeletogenesis remains, with parachondral surfaces with the earliest signs of interclavicle formation.
being relatively smooth and superficial borders Similar to dermatocranial elements, gastralia begin
interfacing with the dermis being rugose (Fig. 6P- as condensations of osteoprogenitor cells (Figs. 5B,
Q). Multinucleated osteoclasts are absent from the 7A) that differentiate into larger, plumper osteo-
superficial dermatocranial surfaces but are found blasts and begin secreting osteoid. Early on, the

Journal of Morphology
condensation ECM stains similar to the surround- is composed of large diameter, tightly packed colla-
ing mesenchyme (dark blue for Mallory’s trichrome, gen fibers, organized to form highly stratified or-
green for Masson’s trichrome), is weakly PAS posi- thogonal (‘‘herringbone’’) horizons. Moving distally
tive, and negative for Bismarck brown (AC pentach- towards the epidermis, the deep dermis grades into
rome). However, unlike mesenchyme (but similar to the looser, more poorly stratified superficial dermis.
the prebone matrix of dermatocranial elements) In contrast to the deep dermis, the superficial der-
gastralial osteoid is also metachromatic (with Tolui- mis is characterized by thinner fibers of collagen
dine blue; Fig. 7B) and weakly positive for Alcian that appear to lack a common pattern of orien-
blue in both serial section (with HBQ) and double tation. In addition, the superficial component of
stained whole-mount preparations. the dermis is more cellular than deeper horizons.
With continued synthesis, the osteoid-rich con- The entire dermis stains well with various connec-
densations are concentrated into long, splint-like tive tissue stains, although there is little evidence
morphologies, predictive of the future bony element of elastin or reticular fibers. Blood vessels are
(see Fig. 5A). Similar to the bone territories of der- observed throughout the dermis but glands are
matocranial elements, each presumptive gastra- absent.
lium is sheathed in a fibroblastic-collagenous fiber As the results obtained for both osteoderm and
capsule. However, unlike dermatocranial elements, palpebral development are similar, they are de-
gastralia do not develop branching spicules or tra- scribed together. Early on, each presumptive osteo-
becular-like frameworks; they remain as compact derm begins as a distinct dome of connective tissue,
rods. Overall, the osteoid has many entrapped with elements immediately flanking the sagittal
osteocytes with spherical morphologies resembling plane frequently demonstrating a midline keel and
chondroid bone tissue previously described for the an acute cross-sectional profile. The first signs of
dermatocranium (Fig. 7C,D). skeletogenesis begin within the osteoderm keel or
As ossification begins, it is concentrated in the the rostromedial border of the eyelid, with the
peripheral margins of the osteoid matrix. In cross- formation of dense a knot of irregular connective
sections stained with HBQ, the cell-rich central por- tissue, the osteoderm primordium (Fig. 7G,H).
tion of the condensation retains a weak affinity for Notwithstanding its compact structure, this pri-
Alcian blue, suggesting the presence of osteoid, mordium is otherwise indistinguishable from the
whereas the remainder of the element is pink and surrounding dermis: histochemically both the osteo-
less cellular, comparable with woven bone (Fig. 7C). derm/palpebral primordium and dermis have the
Similarly, in Toluidine blue-stained sections, ossifi- same affinities for connective tissue stains. Inter-
cation is accompanied by orthochromatic staining, spersed within the dermis and osteoderm/palpeb-
except for the capsular matrix of osteocytes. In sub- ral primordium are fusiform fibroblast-like cells
adults and adults, ossified gastralia are composed (Fig. 7H). These cells are not organized into any
primarily of woven bone, with deposits of parallel- particular horizons nor do they aggregate to form
fibered bone present adjacent to neurovascular condensations. Furthermore, there is no evidence of
channels (Fig. 7E). The central core of each gastra- these cells depositing osteoid.
lium remains relatively cell-rich, and conspicuously With continued growth, the center of the primor-
the capsular and interterritorial matrix of this area dium begins to calcify. Calcification proceeds radi-
is diffusely mineralized and metachromatic with ally (centrifugally), directly incorporating collagen
Toluidine blue (Fig. 7F). There is no sign of carti- fibers from the surrounding dermis (Fig. 7I,J). As a
lage associated with any of the gastralia at any- result, the osteoderm/palpebral primordium is
time. securely anchored within the connective tissue ma-
Osteoderm skeletogenesis. Scalation begins trix of the integument. At this stage of skeletogene-
early during the embryonic period (FS 19–23; Ali- sis, the primordium is primarily a diffusely calcified
bardi and Thompson, 2000), and thus the conforma- dense irregular connective tissue, lacking the con-
tion of the integument is well established in solidated histological appearance of true bone.
advance of osteoderm formation. Prior to osteoderm However, similar to bone there are entrapped osteo-
(and palpebral) calcification, the integument (inclu- cyte-like cells within lacunae, interconnected by
ding that over the eyelid) has already matured into canaliculi (Fig. 7K) and the capsular matrix is
well structured fibrillar network, readily divisible weakly positive for Alcian blue (Fig. 7L). The boun-
into epidermal and dermal horizons. The epidermis daries of the mineralization front are discernable,
is composed of a superficial stratum corneum (of but negative for the presence of reticular fibers, and
variable thickness), and a single layer of cells form- as noted above, lack osteoid and cell-rich osteogenic
ing the regenerative stratum germinativum. The horizons. Histochemically, the osteoderm/palpebral
dermis includes a stratum superficiale (superficial primordium stains similar to diffusely calcified
dermis), stratum compactum, and stratum laxum. bone with various connective tissue stains (e.g., Ma-
The strata compactum and laxum are combined to sson’s trichrome, Mallory’s trichrome; Fig. 7J,K).
form the stratum profundum (the deep dermis). In The territorial matrix surrounding each lacuna
section, the deep dermis adjacent to the hypodermis is unossified and weakly metachromatic with

Journal of Morphology
Toluidine blue; otherwise the osteoderm primor- Interspersed within the mineralized matrix are
dium, like the dermis, is weakly orthochromatic. seams of unmineralized dense irregular connective
The PAS reaction is faint but positive, suggesting tissue (Fig. 7R). Characteristically, the cells associ-
low amounts of carbohydrates. There is no evidence ated with these seams are more rounded and
for chondroblasts/-cytes within either the dermis or densely organized than cells throughout the re-
the developing osteoderm. mainder of the element, although this tissue does
In whole-mounted specimens, Alizarin-positive not conform to the previous described histology for
tissue is observed to spread throughout the keel, chondroid bone. At the deepest surface of the osteo-
before invading the remaining substance of the derm (the base), the ventral and lateral margins
(nonmineralized) osteoderm (Fig. 5E). Numerous have many large, penetrating Sharpey fibers that
ossified spicules radiate from the original center of pass uninterrupted into the dermis. Compared with
mineralization. Similar to earlier stages of osteo- earlier stages of skeletogenesis, the dense connec-
derm skeletogenesis, development of spicules occurs tive tissue encapsulating each osteoderm is rela-
in the absence of cells phenotypically identifiable as tively thin and the large collagen fibers of the
osteoblasts. The entire element remains enshrouded dermis immediately adjacent to each osteoderm are
with a dense, irregular connective tissue that lacks in tension (staining red with Masson’s and AC pen-
argyrophilic fibers (Gridley method). Within the tachrome; Flint and Lyons, 1975). Osteoderms do
osteoderm, areas between the spicules are filled not demonstrate any signs of cartilage at anytime
with an open, loose tissue that closely resembles during their development (contra Kleister, 1992).
As the osteoderm develops further, spicules anasto-
mose and become increasingly robust, and the miner- DISCUSSION
alization front spreads deeper into the surrounding One of the principal aims of this study was to pro-
dense, irregular connective tissue (Fig. 7M,N). In sec- vide details on the pattern and sequence of crocody-
tion, the mineralization front is defined along most of lian dermal element formation. To this end, serial
the superficial surface of the osteoderm by a thin histology and multiple whole-mounted specimens
basophilic line. However, at the apex of the keel and were employed, including single-stained (Alizarin
along the deep (proximal) margins, the basophilic red), double stained (Alizarin red and Alcian blue),
line is less distinct. Merging inseparably with the cal- and hemisectioned preparations. The results herein
cified dense irregular connective tissue are patches of agree with data from other studies in demonstrat-
woven bone, identifiable as structurally consolidated ing the importance of serial histology for discrimi-
and histochemically well-ossified (e.g., red with Mal- nating early skeletal tissue (e.g., Haluska and
lory’s trichrome, AC pentachrome, and HBQ, and Albrech, 1983; Hanken and Hall, 1988; Rieppel,
gray with Verhoeff elastin stain). Throughout the 1993a,b), although it should be noted that the role
osteoderm matrix (including both calcified dense of epigenetic (environmental) and/or genetic factors
irregular connective tissue and woven bone) wher- accelerating the rate of embryonic development
ever multiple lacunae are closely-spaced, the inter- (leading to the earlier observation of dermal bones)
territorial matrix between them is mottled purple cannot be discounted (see below). The use of hemi-
with Toluidine blue (Fig. 7O); otherwise the osteo- sectioned heads permitted a controlled comparison
derm is orthochromatic. of single (Alizarin red) versus double (Alizarin red
Among skeletally mature osteoderms, the tissue and Alcian blue) whole-mount staining methods.
architecture and degree of matrix mineralization is Each method was found to have distinct advan-
not homogeneous (Fig. 7P-R). The majority of each tages: once ossification is initiated, single-stained
element is composed of an inseparable combination specimens demonstrate a greater affinity for Aliza-
of woven bone and diffusely mineralized dense rin red than double stained specimens, suggesting
irregular connective tissue. The latter demon- that omission of Alcian blue, which includes glacial
strates variegated staining: Mallory’s, red with blue acetic acid as a solvent and fixative, removes a
mottles; AC pentachrome, red with green-gray mot- source of decalcification. However, prior to mineral-
tles; Verhoeff, gray with black mottles (Fig. 7P). In ization, opaque (Alizarin red-negative) condensa-
some areas (most commonly along the superficial tions are easier to visualize against the Alcian blue-
surfaces of the osteoderm distal to the apex), this positive endoskeleton of double stained materials.
tissue combination has undergone resorption, as In addition, double-stained specimens provide topo-
evident from the scalloped lines, and is replaced by graphic details (i.e., the relationship of dermal
lamellar bone (Fig. 7P). In addition, lamellar bone elements to the endoskeleton) unavailable on sin-
is also deposited around vascular channels of the gle-stained specimens.
primary (and secondary) osteons. Cells in lacunae
are entrenched throughout the fibrolamellar bone–
calcified connective tissue matrix (Fig. 7P,Q). Not- Pattern of Dermal Skeletogenesis
withstanding the presence of scalloped lines, no Dermatocranium. Among components of the
multinucleated osteoclasts are observed. dermal skeleton, the dermatocranium is the best

Journal of Morphology
known and most widely studied. The basic pattern results presented here, Westergaard and Ferguson
and sequence of dermatocranial element formation (1990) also detected the dentary at FS 17.
reported in this study corresponds well with previ- As a group, the temporal-vault series includes
ous efforts (e.g., de Beer, 1937; Bellairs and Kamal, many relatively late forming elements. The re-
1981; Klembara, 1991; Rieppel, 1993b). Minor dif- corded succession is: frontal (FS 18) ? squamosal
ferences, where noted, relate to the developmental and quadratojugal (FS 19) ? parietal (FS 22). A
stage at which the earliest onset is observed based comparable order (albeit with a delayed onset) was
on serial histology (Table 3). As the study of Rieppel noted by Rieppel (1993b): frontal (FS 20) ? squa-
(1993b) provides the most comprehensive overview mosal and quadratojugal (FS 21) ? parietal (FS
of staged patterns of ossification in Alligator missis- 23). The parasphenoid reportedly begins to ossify at
sippiensis, this will form the bulk of the compara- FS 20 (Klembara, 1993) while the postparietal is
tive discussion. Other comparisons will be made not present until FS 27 (Klembara, 2001).
where relevant. In common with other reptiles (e.g., avian Gallus
Among the facial series of elements, the sequen- gallus [Jollie, 1957; Murray, 1963; Hamilton, 1965];
tial pattern of development reported here, viz., squamates Elaphe obsoleta [Haluska and Alberch,
maxilla (FS 17) ? premaxilla (FS 18) ? nasal (FS 1983], Lacerta vivipara [Rieppel, 1992], and Naja
21), is the same as that recorded by Rieppel (1993b; kaouthia [Jackson, 2002]; and turtles Chelydra ser-
Table 3), although he consistently observed each pentine and Apalone spinifera [Rieppel, 1993c;
element one Ferguson developmental stage later. Sheil, 2003; Sheil and Greenbaum, 2005]), the der-
Early formation of the maxilla (at early FS 18) and matocranium initiates ossification prior to the endo-
premaxilla (at mid FS 18) was also noted by Wester- skeletal system, in most cases beginning with the
gaard and Ferguson (1990). The earliest detection pterygoid, dentary, angular, maxilla, and premax-
of the nasal bone comes from the work of Klembara illa. For Alligator mississippiensis, there is a con-
(1991) at FS 20. It is also worth noting that forma- venient relationship between the early development
tion of the internarial bar, a synapomorphy of Alli- of tooth-bearing elements (at FS 17–18) and the
gator spp., begins at FS 22; previous work had sug- previously established dentition (at FS 14; Wester-
gested that most crocodylians were nearly identical gaard and Ferguson, 1986, 1990). A similar associa-
until FS 24 (Ferguson, 1987). tion almost certainly holds true for other reptiles,
Combined with the palatal series, the premaxilla including (edentulous) avians and turtles. The
and maxilla produce the osseous secondary palate. ancestors of each of avians and turtles (whether it
The functional importance of this structure is be a member of Proganosauria or Diapsida; see
reflected in the early onset of formation noted for Vickaryous and Hall, 2006a) display a well-devel-
all the participating elements: pterygoid (early FS oped dentition. In addition, at least for avians, ex-
17) ? maxilla (late FS 17) ? premaxilla, palatine, perimental studies have demonstrated that the
and (underpinning the configuration) vomer (FS early steps in the signaling pathways for tooth de-
18). The early formation of the pterygoid noted here velopment are retained, even though true teeth are
also corresponds with observations by Müller never expressed (Chen et al., 2000). Similarly, the
(1967) on Crocodylus cataphractus. The last palatal early formation of the pterygoid may relate to the
series element to develop is the ectopterygoid, prominence of this element among basal reptiles
recorded here at FS 21, although Klembara (1991) (Rieppel, 1993a), as well as its current role as a
has detected it as early as FS 20. With the excep- major attachment point for jaw adductors. Al-
tion of the pterygoid (identified at FS 18) Rieppel though the relationship is less clear, the uncom-
(1993b) did not record other palatal series elements monly early formation of the coronoid in A. missis-
prior to FS 21. The results obtained here for the sippiensis (and the squamate L. vivipara; Rieppel,
onset of orbital series elements were also dissimilar 1992) may also relate to the musculotendinous sys-
to those of Rieppel (1993b). Whereas Rieppel tem; the coronoid is an insertion for the mandibular
(1993b) reported that all the elements developed at adductor tendon (5 stem tendon) (Iordansky, 1973).
or around FS 20, this study observed a staggered Unlike ophidians and turtles, the ectopterygoid and
onset of development: prefrontal and postorbital parietal of A. mississippiensis are some of the last
(FS 18) ? jugal (late FS 18) ? lacrimal (FS 20). elements to develop.
Matching the dentigerous elements of the upper Gastralia. With the exception of Sphenodon,
jaw, and correlating with the previous development among extant taxa the development of discrete,
of the dentition (FS 15; Westergaard and Ferguson, articulating gastralia is unique to crocodylians; the
1990), the dentary also develops early during homologue of gastralia in turtles, the hyoplastron,
embryogenesis (at FS 17). The sequence of onset for hypoplastron, xiphiplastron, are fused and nonmov-
the mandibular series is: angular, dentary, and able. Whereas it has been reported that gastralia
coronoid (FS 17) ? splenial (FS 18) ? surangular are present as early as FS 17 (Ferguson, 1987), this
(FS 19). The sequence documented by Rieppel study was unable to identify presumptive elements
(1993b) is: angular (FS 18) ? dentary and surangu- until FS 19, corresponding with the onset of various
lar (FS 19) ? splenial (FS 21). Confirming the elements of the dermatocranium (see above) and

Journal of Morphology
the regionalization of the dermis (into superficial loose mesenchyme and then differentiate into osteo-
and deep layers; Alibardi and Thompson, 2000). blasts, forming skeletal condensations. Osteoblasts
The initial gastralia are the lateral elements of the of the condensation deposit seams of osteoid that
caudalmost row, adjacent to the pubis. Skeletogene- may either interlace to form trabeculated networks
sis then continues in a cranial and (somewhat (dermatocranial elements; Fig. 6D-F) or concen-
delayed) medial sequence. This pattern of develop- trate into rod-like splints (gastralia; Fig. 5A,C).
ment appears to be correlated with the progressive Osteoid-rich condensations form within fibrously
closure of the ventral body wall between the pubis defined bone territories. The deposited osteoid ma-
and the umbilical stalk, and reduction in the rela- trix demonstrates typically affinities for connective
tive size of the umbilicus. A similar pattern of lat- tissue stains (e.g., dark blue with Mallory’s tri-
eral to medial development permitting passage of chrome; Fig. 6D,E), and is weakly positive for PAS
the umbilical stalk is witnessed in the formation of (demonstrating carbohydrates). Although, the
the turtle plastron (Gilbert et al., 2001). osteoid is always negative for Bismarck brown (AC
Corresponding with previous reports (e.g., Claes- pentachrome), early on it is weakly positive for
sens, 2004), the gastralial system was observed to Alcian blue (using HBQ [Fig. 6F] or in double-
demonstrate variation in the number of individual stained whole-mount preparations) and (weakly)
elements present in each row (Fig. 5D). This varia- metachromatic for Toluidine blue, indicating the
tion was observed in both embryos and subadults, presence of small amounts of GAGs.
and included both the absence (nonformation) of Continued ossification closely resembles the
individual gastralia and the asymmetrical distri- osteogenic modality of the avian dermatocranium
bution of elements. It remains unclear if this varia- (Lengelé et al., 1996a,b). In some spicules, plump
bility has any functional implications. It is also osteoblasts rapidly alter their cell morphology, be-
interesting to note that gastralia do not develop sec- coming hypertrophic, and distribution, becoming
ondary cartilage. As mechanically articulating der- densely packed with little intercellular matrix and
mal bones (each with its own periosteum; Fig. 7C,D), no readily identifiable canaliculi or cell processes.
gastralia appear to demonstrate all the necessary These crowded aggregations of cells with chondro-
criteria. The unexpected absence of secondary carti- cytic morphologies closely resemble isogenous groups
lage has been noted for other nonavian reptiles, in- and doublets, thus suggesting interstitial growth
cluding the dermatocranium of ophidians (Murray, prior to ossification. Conversely, the intercellular
1963; see also Hall, 1984). matrix is histochemically identical to bone (e.g.,
Osteoderms. The pattern of osteoderm forma- dark blue with Mallory’s trichrome [Fig. 6G,H],
tion observed in Alligator mississippiensis is con- weakly positive for PAS, negative for Alcian blue
sistent with previous studies on structural-grade and Bismarck brown). For avians, the development
lizards (Moss, 1969; Zylberberg and Castanet, 1985; of a cell-rich tissue histologically intermediate be-
Levrat-Calviac and Zylberberg, 1986) and armadil- tween classically defined bone and hypertrophic
los (Vickaryous and Hall, 2006b). Osteoderms de- cartilage has been identified as chondroid bone
velop late during ontogeny, and are generally among (Lengelé et al., 1996a,b). A strikingly similar tissue
the last skeletal elements to form. In A. mississip- is widespread among teleosts (Huysseune, 2000; see
piensis the earliest sign of calcification occurs 1 below). As osteogenesis in the dermatocranium con-
year after hatching, in the area of the presumptive tinues, islands of chondroid bone become encapsu-
nuchal shield (dorsal to the cervical vertebrae; lated by woven and lamellar bone matrix, ulti-
Fig. 5E). From this craniomedial position along mately persisting as cell-rich patches within the
the dorsum, consecutive osteoderms develop (in no fibrolamellar bone matrix of skeletally mature indi-
particular order) caudally, laterally, and cranially viduals.
(forming the postoccipital cluster). For each ele- For gastralia, ossification begins in the periphery
ment, mineralization is initiated first within the and continues medially towards the center. Rapid
keel and before spreading radially (centrifugally). appositional ossification confines chondroid bone to
Similar to postcranial osteoderms, palpebrals also the core, and while interterritorial matrix is gradu-
develop relatively late during ontogeny. ally deposited, this area remains distinctly cell-rich
(Fig. 7C,D). In skeletally mature gastralia, the
interterritorial matrix of this central, cell-rich por-
Comparison of Mode of Dermal tion is metachromatic (with Toluidine blue; Fig. 7F)
Skeletogenesis and only diffusely mineralized. Otherwise, the ele-
Dermatocranium and gastralia. Elements of ment is well-ossified and orthochromatic.
the dermatocranium and the gastralia undergo a Osteoderms and palpebrals. The modes of
common mode of osteogenesis that is comparable osteoderm and palpebral skeletogenesis compare
with intramembranous bone formation previously well with each other and with the description pro-
noted for dermal elements of other vertebrates vided by Moss (1969) for mineralized elements pres-
(Pritchard, 1974; Vickaryous and Hall, 2006b). ent in the integument of Heloderma horridum, the
Briefly, osteoprogenitor cells aggregate within a Gila monster. Presumptive elements begin as

Journal of Morphology
domes or thickenings of the dermis. Within the oth- An alternative hypothesis, advanced by de Buf-
erwise irregular arrangement of connective tissue, frénil (1982), proposed that the pitted and grooved
a dense knot of collagen fibers, the osteoderm (or pattern represents localized resorption by osteo-
palpebral) primordium, first appears (Fig. 7G,H). clasts. Although intuitively reasonable, de Buffrénil
At no time are any phenotypically osteoblastic cells was unable to locate osteoclasts in support of the
present, nor is there evidence of osteoid or a perios- claim. Thus the identification of multinucleated
teum. As calcification of the primordium begins, ex- osteoclasts associated with the dermatocranium
trinsic (pre-existing) collagen fibers from the sur- (see Figs. 3D,6M,R), while not unexpected, is note-
rounding connective tissue become progressively worthy. Similar to those of teleosts (Sire et al.,
incorporated (Fig. 7I,J). Subsequently, patches of 1990), in Alligator mississippiensis, multinucleated
woven bone develop, inseparably merging with the osteoclasts are capable of resorbing osteoid and
calcified dense irregular connective tissue. The ab- chondroid bone. Furthermore, it is clear that these
sence of osteoblastic cells and a discrete periosteum syncytial cells regulate the shape of individual ele-
is consistent with bone formation via metaplasia ments of the dermatocranium. However, multi-
(Beresford, 1981). Prior to skeletal maturation, por- nucleated osteoclast distribution is restricted to
tions of the mineralized matrix are eroded, and passages that penetrate elements (e.g., for neuro-
lamellar bone is deposited in place. In skeletally vascular bundles or ducts) and alveolar bone (Fig.
mature osteoderms, seams of unmineralized dense 6M,R); no multinucleated osteoclasts were observed
irregular connective tissue, having become entrapped along the superficial surfaces of any dermatocranial
within the advancing mineralization front, persist element, gastralium, osteoderm, or palpebral. While
within the otherwise mineralized osteoderm matrix. it remains possible that mononucleated osteoclasts
It should be noted that Alligator mississippiensis are present in crocodylians (and not discernable
osteoderms do differ from those of H. horridum with the histochemical methods employed herein),
(Moss, 1969) and the gecko Tarentola mauritanica in other vertebrates these cells are responsible for
(Levrat-Calviac and Zylberberg, 1986) in one major fine-scale resorption (Witten et al., 2001) and are
respect: whereas osteoderms of H. horridum and unlikely to be responsible for the deep pitting char-
T. mauritanica develop a superficial horizon of acteristic of crocodylian ornamentation.
heavily mineralized, collagen-poor tissue, those of Development of the dermatocranial ornamenta-
A. mississippiensis do not. On the basis of histology, tion begins early during the skull development,
histochemistry, and the pattern and mode of develop- well in advance of palpebral and postcranial osteo-
ment, palpebrals are identified as eyelid osteoderms. derm formation. In section, the bone architecture of
The mode of skeletogenesis described for crocody- elements interfacing with the integument is polar-
lians and structural-grade lizards osteoderms dif- ized, with a highly emarginated superficial (external)
fers considerably from that recorded for the mam- surface (Fig. 6K,L,P) and a relatively smooth and
mal Dasypus novemcinctus (Vickaryous and Hall, uninterrupted deep (internal) surface (Fig. 6K,L,Q).
2006b). For D. novemcinctus, osteoderm formation Moreover, plump osteoblasts and osteoid are typi-
begins with the formation of a condensation of cally concentrated at the tips of these outwardly
numerous plump osteoblasts secreting osteoid. With directed spicules (Fig. 6F), whereas the deep sur-
continued growth, each D. novemcinctus osteoderm face is typically associated with smaller osteoblasts
is encapsulated within a fibrous periosteum. Accord- reminiscent of bone lining cells.
ingly, the mode of osteoderm formation in D. novem- Chondroid bone. Chondroid bone is common to
cinctus is consistent with intramembranous ossifica- many vertebrates, including teleosts (Huysseune
tion, closely resembling the development of derma- and Verraes, 1986; Huysseune and Sire, 1990;
tocranial elements. Huysseune, 2000; Witten and Hall, 2003; Gillis
Development of ornamentation. Similar to et al., 2006), mammals (Beresford, 1981, 1993;
basal tetrapods, the crocodylian dermatocranium is Vinkka, 1982), and birds (Lengelé et al., 1996a,b),
characterized by an embossing pitted and grooved but hitherto considered absent from nonavian rep-
ornamentation. A comparable pattern of bony relief tiles and lissamphibians. Indirect support of this
develops along the superficial (dorsal) surface of hypothesis came from the observed correlation
osteoderms. On the basis of this observed similarity, between the development of chondroid bone and
Iordansky (1973) suggested that ornamentation secondary cartilage (Lengelé et al., 1996a). Whereas
developed as osteoderms fused with the skull sur- secondary cartilage is present in teleosts (Benja-
face (although, no explanation was offered as to min, 1990; Witten and Hall, 2003), mammals
why osteoderms had ornamentation). The current (Beresford, 1981; Tran and Hall, 1989), and birds
study finds no evidence to support this hypothesis. (Murray, 1963; Hall, 1986) (see also Hall, 2001),
During development, there is no evidence of osteo- this tissue fails to develop in nonavian reptiles and
derms, exclusive of the palpebrals, developing su- lissamphibians, even during fracture repair (Hall
perficial to the dermatocranium. Furthermore, the and Hanken, 1985; Irwin and Ferguson, 1986). In
pitted and grooved relief is already present prior to agreement with the abovementioned studies, no
(postcranial) osteoderm and palpebral formation. secondary cartilage was observed during this study.

Journal of Morphology
Perhaps unexpectedly, this included the absence of 1969). In Dasypus novemcinctus, the adult element
cartilage from the lateral margin of the lateral pter- includes a combination of woven, lamellar, and
ygoid flange, at least up to and including subadults Sharpey fiber bone (Vickaryous and Hall, 2006b). In
of 195-mm SVL (contra Parker, 1883; de Beer, 1937; the structural-grade lizard Tarentola mauritanica,
Iordansky, 1973; Schumacher, 1973; Bellairs and osteoderms are composed of two discrete horizons; a
Kamal, 1981). During embryogenesis, the pterygoid superficial horizon of heavily mineralized, collagen-
initially develops adjacent to the cartilaginous proc- poor tissue underpinned by deeper layer of bone
essus pterygoideus of the palatoquadrate. Rapidly, (Levrat-Calviac and Zylberberg, 1986). For Alliga-
however, the pterygoid overgrows its parachondral tor mississippiensis, osteoderms demonstrate a
support, thus excluding this cartilage from the dis- broad spectrum of tissues, including woven and la-
tal margin (contra de Beer, 1937). mellar bone, and calcified and uncalcified dense
In teleosts, the presence of chondroid bone is irregular connective tissue (Fig. 6P-R). In reptiles,
often correlated with rapid skeletal growth (Huys- the presence of bone in these elements is best
seune and Verraes, 1986; Huysseune, 2000; Witten explained as tissue metaplasia. However, recalling
and Hall, 2003; Gillis et al., 2006). It remains to be the presence of calcified and uncalcified dense irreg-
determined if this is the case for Alligator missis- ular connective tissues, it is inaccurate to refer to
sippiensis. Functionally however, the presence of osteoderms as strictly metaplastic bones (sensu
isogenous cell groups within chondroid bone implies Haines and Mouhuiddin, 1968): they are not (neces-
a period of interstitial growth prior to mineraliza- sarily) composed exclusively of bone and they do
tion. Furthermore, the development of chondroid not develop exclusively via metaplasia (e.g., the
bone in both Alligator mississippiensis and Gallus dense irregular connective tissues). Moss (1969)
gallus illustrates that this tissue type is widespread sought to emphasize the nonuniform histology of
among archosaurian reptiles. osteoderms by employing sclerification in lieu of
The osteoderm enigma. As previously noted, ossification. Correspondingly, he considered the
osteoderms are common to representative members term osteoderm misleading. However, while Moss’
of most reptilian lineages (excluding avians), as argument is compelling, outright rejection of the
well as some anurans and the mammalian clade term osteoderm is unnecessary and unlikely to be
Cingulata (e.g., Moss, 1969; Ruibal and Shoemaker, broadly embraced. Herein osteoderms are defined
1984; Hill, 2005, 2006; Vickaryous and Hall, as a structural category of skeletal organ. However,
2006b). Although Moss (1969) originally suggested while osteoderms are not homologous as organs
that all osteoderms are homologous, both in terms between taxa, all osteoderms develop within, and
of mode of development and as structural entities, a remain continuous with, connective tissue of the
more recent study (Hill, 2005) casts doubt on this well-structured dermis. Consequently, osteoderms
interpretation. Hill’s (2005) investigation proposed share a common origin relating to latent structural
that postcranial osteoderms arose independently and skeletogenic properties of the integument. Sim-
among at least five different amniote lineages (Par- ilar conclusions were reached by Zylberberg and
eiasauria, Squamata, Archosauria, Placodonta, and Wake (1990) and Main et al. (2005) (see also Hill,
Cingulata). In addition, postcranial osteoderms are 2006). As such, osteoderms may be an example of
also known from some turtles (exclusive of the cara- what has been termed a deep homology (Main
pace and plastron; e.g., the fossil form, Proga- et al., 2005; Hill, 2006; see Shubin et al., 1997; Gil-
nochelys quenstedti; Gaffney, 1990; see also Barrett bert and Bolker, 2001; Hall, 2003), with the dermis
et al., 2002), at least one sphenodontid (Pamizini- as the shared structural foundation and a source of
saurus tlayuaensis; Reynoso, 1997), a basal reptile nonchondrogenic skeletoprogenitor cells underlying
(Sclerosaurus armatus; von Huene, 1912), and a ba- the osteoderm phenotype.
sal synapsid (Elliotsmithia longiceps; Reisz et al.,
1998). Developmental data corroborate the phylo-
genetic pattern, at least as far as reptiles and mam-
mals are concerned, for as noted above, the modes None of this work could have been done without
of skeletogenesis for the mammal Dasypus novem- the extraordinary assistance of Dr. Ruth Elsey and
cinctus (Vickaryous and Hall, 2006b) and the the staff of the Rockefeller Wildlife Refuge, Grand
reptiles Heloderma horridum and Alligator missis- Chenier, Louisiana. Special thanks to Jimmy
sippiensis are quite dissimilar. Unlike D. novem- (James) Hartobey, Jeb Linscombe, Megan Pearson,
cinctus, during osteoderm development in reptiles Scooter Trosclair, and John Turner. Prof. Mark W.
there is no evidence of cells with an osteoblastic J. Ferguson (University of Manchester) provided
phenotype, no osteoid, and no periosteum (Fig. 7G- access to his collection of Alligator mississippiensis
R). The initiation and continued growth of reptilian slides and specimens. Additional specimens were
osteoderms occurs as the dermis both calcifies in made available by Dr. Gerd Müller (University of
situ and transforms into bone. Vienna). The first author thanks Drs. Kathleen
Regardless of development, skeletally mature Smith (Duke University), Val Lance (San Diego
osteoderms are not histologically uniform (Moss, Zoo), and Alan Pinder, Boris Kablar, and Ian Mobbs

Journal of Morphology
(all of Dalhousie University) for insightful com- Cong L, Hou L, Wu X-C, Hou J. 1998. The gross anatomy of Al-
ments and discussion. Two anonymous reviewers ligator sinensis Fauvel. Beijing: Science Press.
de Beer GR. 1937. The Development of the Vertebrate Skull.
and Dr. F.W. Harrison made significant improve- Oxford: Oxford University Press.
ments to the text. Ms. Jessica Arbour, Donna Krailo de Buffrénil V. 1982. Morphogenesis of bone ornamentation in
and Alma Cameron assisted in the laboratory. Com- extant and extinct crocodilians. Zoomorphology 99:155–166.
pletion of this manuscript and revisions were sup- de Ricqlés A, Meunier FJ, Castanet J, Francillon-Vieillot H.
1991. Comparative microstructure of bone. In: Hall BK, edi-
ported by an Alberta Ingenuity Fund Postdoctoral tor. Bone, Vol. 3: Bone Matrix and Bone Specific Products.
Fellowship to the first author. Boca Raton, Florida: CRC Press. pp 1–78.
Deraniyagala MA. 1939. The tetrapod reptiles of Ceylon, Vol. I:
Testudinates and Crocodilians. Colombo, Ceylon: Colombo
Museum of Natural History. 412 p.
LITERATURE CITED Dodson P. 1975. Functional and ecological significance of rela-
tive growth in Alligator mississippiensis. J Zool 175:315–355.
Alibardi L, Thompson MB. 2000. Scale morphogenesis and Ferguson MWJ. 1981. The value of the American alligator (Alli-
ultrastructure of dermis during embryonic development in gator mississippiensis) as a model for research in craniofacial
the alligator (Alligator mississippiensis. Crocodilia, Reptilia). development. J Craniofac Genet Dev Biol 1:123–144.
Acta Zool (Stockholm) 81:325–338. Ferguson MWJ. 1984. Craniofacial development in Alligator
Barrett PM, Clarcke J, Brinkman DB, Chapman SD, Ensom mississippienesis. In: Ferguson MWJ, editor. The Structure,
PC. 2002. Morphology, histology and identification of the ‘gra- Development and Evolution of Reptiles. London: Academic
nicones’ from the Purbeck Limestone Formation (Lower Cre- Press. pp 223–273.
taceous: Berriasian) of Dorset, southern England. Cretaceous Ferguson MWJ. 1985. Reproductive biology and embryology of
Res 23:279–295. the crocodilians. In: Gans C, Billett F, Maderson PFA, editors.
Bellairs AdA, Kamal AM. 1981. The chondrocranium and the Biology of the Reptilia, Vol. 14: Developement A. New York:
development of the skull in recent reptiles. In: Gans C, Par- Academic Press. pp 329–491.
sons TS, editors. Biology of the Reptilia, Vol. 11. New York: Ferguson MWJ. 1987. Post-laying stages of embryonic develop-
Academic Press. pp 1–263. ment for crocodilians. In: Webb GJW, Manolis C, Whitehead
Benjamin M. 1990. The cranial cartilages of teleosts and their PJ, editors. Wildlife Management: Crocodiles and Alligators.
classification. J Anat 169:153–172. Sydney: Surrey Beatty and Sons Pty Limited.
Beresford WA. 1981. Chondroid Bone, Secondary Cartilage and Flint MH, Lyons MF. 1975. The effect of heating and denatura-
Metaplasia. Munich: Urban & Schwarzenberg. 454 p. tion on the staining of collagen by the Masson trichrome pro-
Beresford WA. 1993. Cranial skeletal tissues: diversity and evo- cedure. Histochem J 7:547–555.
lutionary trends. In: Hanken J, Hall BK, editors. The Skull, Francillon-Vieillot H, de Buffrénil V, Castanet J, Geraudie J,
Vol. 2: Patterns of Structural and Systematic Diversity. Chi- Meunier F, Sire J-Y, Zylberberg L, de Ricqles A. 1990. Micro-
cago: University of Chicago Press. pp 69–130. structure and mineralization of vertebrate skeletal tissues.
Billett F, Gans C, Maderson PA. 1985. Why study reptilian de- In: Carter JG, editor. Skeletal Biomineralization; Patterns,
velopment? In: Gans C, Billett F, Maderson PA, editors. Biol- Processes and Evolutionary Trends, Vol. 1: New York: Van
ogy of the Reptilia, Vol. 14: Development A. New York: Wiley. Nostrand Reinhold. pp 471–548.
pp 3–39. Gaffney ES. 1990. The comparative osteology of the Triassic
Brochu CA. 1999. Phylogenetics, taxonomy, and historical bio- turtle Proganochelys. Bull Am Mus Nat Hist 194:1–263.
geography of Alligatoroidea. Soc Vert Paleontol Memoir 6 19: Gilbert SF, Bolker JA. 2001. Homologies of process and modular
9–100. elements of embryonic construction. J Exp Zool (Mol Dev
Brochu CA. 2003. Phylogenetic approaches toward crocodylian Evol) 291:1–12.
history. Annu Rev Earth Planetary Sci 31:357–397. Gilbert SF, Loredo GA, Brukman A, Burke AC. 2001. Morpho-
Busbey AB. 1995. The structural consequences of skull flatten- genesis of the turtle shell: The development of a novel struc-
ing in crocodilians. In: Thomason JJ, editor. Functional Mor- ture in tetrapod evolution. Evol Dev 3:47–58.
phology in Vertebrate Paleontology. Cambridge: Cambridge Gillis JA, Witten PE, Hall BK. 2006. Chondroid bone and sec-
University Press. pp 173–192. ondary cartilage contribute to apical dentary growth in juve-
Castanet J, Francillon-Vieillot H, de Ricqles A, Zylberberg L. nile Atlantic salmon. J Fish Biol 68:1133–1143.
2003. The skeletal histology of the Amphibia. In: Heatwole Haines RW, Mohuiddin A. 1968. Metaplastic bone. J Anat 103:
H, Davies M, editors. Amphibian Biology, Vol. 5: Osteology. 527–538.
Chipping Norton, Australia: Surrey Beatty & Sons. pp 1598– Hall BK. 1984. Developmental processes underlying the evolu-
1683. tion of cartilage and bone. In: Ferguson MWJ, editor. The
Chen Y, Zhang Y, Jiang T-X, Barlow AJ, St. Amand TR, Hu Y, Structure, Development and Evolution of Reptiles, Sympo-
Heaney S, Francis-West P, Chuong C-M, Maas R. 2000. Con- sium No 52 of the Zoological Society of London. London: Aca-
servation of early odontogenic signaling pathways in Aves. demic Press. pp 155–176.
Proc Natl Acad Sci USA 97:10044–10049. Hall BK. 1986. The role of movement and tissue interactions in
Chiasson RB. 1962. Laboratory Anatomy of the Alligator. Dubu- the development and growth of bone and secondary cartilage
que, Iowa: Brown.56 p. in the clavicle of the embryonic chick. J Embryol Exp Mor-
Claessens LPAM. 2004. Dinosaur gastralia; origin, morphology, phol 93:133–152.
and function. J Vert Paleontol 24:89–106. Hall BK. 2001. Development of the clavicles in birds and mam-
Coates MI. 1996. The Devonian tetrapod Acanthostega gunnari mals. J Exp Zool 289:153–161.
Jarvik: Postcranial anatomy, basal tetrapod interrelationships Hall BK. 2003. Descent with modification: the unity underlying
and patterns of skeletal evolution. Trans Roy Soc Edinburgh homology and homoplasy as seen through an analysis of de-
87:363–421. velopment and evolution. Biol Rev Camb Philos Soc 78:409–
Coldiron RW. 1974. Possible functions of ornament in labyrin- 433.
thodont amphibians. Occas Pap Mus Nat Hist Lawrence Hall BK. 2005. Bones and Cartilage: Developmental and Evolu-
33:1–19. tionary Skeletal Biology. New York: Elsevier Academic Press.
Cole AC, Hall BK. 2004. The nature and significance of inverte- 512 p.
brate cartilages revisited: Distribution and histology of carti- Hall BK, Hanken J. 1985. Repair of fractured lower jaws in the
lage and cartilage-like tissues within Metazoa. Zool 107:261– spotted salamander: do amphibians form secondary cartilage?
273. J Exp Zool 223:359–368.

Journal of Morphology
Hall BK, Miyake T. 2000. Craniofacial development of avian Lengelé B, Schowing J, Dhem A. 1996b. Embryonic origin and
and rodent embryos. In: Tuan RS, Lo CW, editors. Methods of fate of chondroid tissue and secondary cartilages in the avian
Molecular Biology, Vol. 135: Developmental Biology Protocols, skull. Anat Rec 246:377–393.
Vol. 1. Totowa, New Jersey: Humana. pp 127–137. Levrat-Calviac V, Zylberberg L. 1986. The structure of the
Haluska F, Alberch P. 1983. The cranial development of Elaphe osteoderms in the Gekko: Tarentola mauritanica. Am J Anat
obsoleta (Ophidia, Colubridae). J Morphol 178:37–55. 176:437–446.
Hamilton HL. 1965. Lillie’s Development of the Chick; an Intro- Main RP, de Ricqlés A, Horner JR, Padian K. 2005. The evolu-
duction to Embryology. New York: Holt, Rinehart and Win- tion and function of thyreophoran dinosaur scutes: Implica-
ston. tions for plate function in stegosaurs. Paleobiology 31:291–
Hanken J, Hall BK. 1988. Skull development during anuran 314.
metamorphosis. I. Early development of the first three bones Meek A. 1911. On the morphogenesis of the head of the croco-
to form—The exoccipital, the parasphenoid, and the fronto- dile. J Anat Physiol 45:357–377.
parietal. J Morphol 195:247–256. Miall LC. 1878. The Skull of the Crocodile. A Manual for Stu-
Hill RV. 2005. Integrative morphological data sets for phyloge- dents. London: MacMillan.
netic analysis of Amniota: The importance of integumentary Mook CC. 1921a. Individual and age variations in the skulls of
characters and increased taxonomic sampling. Syst Biol 54: recent Crocodilia. Bull Am Mus Nat Hist 44:51–66.
530–547. Mook CC. 1921b. Notes on the postcranial skeleton in the Croc-
Hill RV. 2006. Comparative anatomy and histology of xenar- odilia. Bull Am Mus Nat Hist 44:67–100.
thran osteoderms. J Morphol 267:1441–1460. Moss ML. 1964. The phylogeny of mineralized tissues. Intl Rev
Huxley TH. 1860. On the dermal armour of Jacare and Cai- General Exp Zool 1:298–331.
man, with some notes on the specific and generic characters Moss ML. 1969. Comparative histology of dermal sclerifications
of recent Crocodilia. J Proc Linn Soc Lond 4:1–28. in reptiles. Acta Anat 73:510–533.
Huysseune A. 2000. Skeletal system. In: Ostrander GK, editor. Müller F. 1967. Zur embryonalen Kopfentwicklung von Crocody-
Microscopic Functional Anatomy. San Diego: Academic Press. lus cataphractus Cuvier. Rev Suisse Zool 74:189–294.
pp 307–317. Müller GB, Alberch P. 1990. Ontogeny of the limb skeleton in
Huysseune A, Sire J-Y. 1990. Ultrastructural observations on Alligator mississippiensis: Developmental invariance and
chondroid bone in the teleost fish Hemichromis bimaculatus. change in the evolution of archosaur limbs. J Morphol 203:
Tissue Cell 22:371–383. 151–164.
Huysseune A, Verraes W. 1986. Chondroid bone on the upper Murray PDF. 1963. Adventitious (secondary) cartilage in the
pharyngeal jaws and neurocranial base in the adult fish Asta- chick, and the development of certain bones and articulations
totilapia elegans. Am J Anat 177:527–535. in the chick skull. Aust J Zool 11:368–430.
Iordansky NN. 1973. The skull of the Crocodilia. In: Gans C, Parker WK. 1883. On the structure and development of the
editor. Biology of the Reptilia, Vol. 4: Morphology D. New skull of the Crocodilia. Trans Zool Soc Lond 11:263–310.
York: Academic Press. pp 201–262. Parsons TS. 1970. The nose and Jacobson’s organ. In: Gans C,
Irwin CR, Ferguson MWJ. 1986. Fracture repair of reptilian Parsons TS, editors. Biology of the Reptilia, Vol. 2: Morphol-
dermal bone: Can reptiles form secondary cartilages? J Anat ogy B. New York: Academic Press. pp 99–191.
146:53–64. Patterson C. 1977. Cartilage bones, dermal bones and mem-
Jackson K. 2002. Post-ovipositional development of the mono- brane bones, or the exoskeleton versus the endoskeleton. In:
cled cobra. Naja kaouthia (Serpentes: Elapidae). Zool 105: Andrews SM, Walker AD, editors. Problems in Vertebrate
203–214. Evolution. London: Academic Press. pp 77–122.
Jollie MT. 1957. The head skeleton of the chicken and remarks Presnell JK, Schreibman MP. 1997. Humason’s Animal Tissue
on the anatomy of this region in other birds. J Morphol 100: Techniques, 5th ed. Baltimore: The Johns Hopkins University
389–436. Press. 572 p.
Kesteven HL. 1957. On the development of the crocodilian Pritchard JJ. 1974. Growth and differentiation of bone and con-
skull. Proc Linn Soc New South Wales 82:117–124. nective tissue. In: Goldspink G, editor. Differentiation and
Kleister JSE. 1992. Osteoderm development in crocodilians: Growth of Cells in Vertebrate Tissues. London: Chapman &
consequences for vertebrate phylogeny. Am Zool 32:146A. Hall. pp 101–128.
Klembara J. 1991. The cranial anatomy of early ontogenetic Reisz R, Dilkes DW, Berman DS. 1998. Anatomy and relation-
stages of Alligator mississippiensis (Daudin, 1802) and the ships of Elliotsmithia longiceps Broom, a small synapsid
significance of some of its cranial structures for the evolution (Eupelycosauria: Varanopseidae) from the late Permian of
of tetrapods. Palaeontograph Abteilung A: Paleaozoologie, South Africa. J Vert Paleontol 18:602–611.
Stratigraphie 215:103–171. Reynoso V-H. 1997. A ‘‘beaded’’ sphenodontian (Diapsida: Lepi-
Klembara J. 1993. The parasphenoid and associated dermal dosauria) from the Early Cretaceous of Central Mexico. J
structures of the parabasisphenoid of Alligator mississippien- Vert Paleontol 17:52–59.
sis (Daudin, 1802). Palaeontograph Abteilung A: Paleaozoolo- Rieppel O. 1992. Studies of skeleton formation in reptiles. III.
gie, Stratigraphie 228:143–164. Patterns of ossification in the skeleton of Lacerta vivipara
Klembara J. 2001. Postparietal and prehatching ontogeny of Jacquin (Reptilia, Squamata). Fieldiana: Zool, New Series
the supraoccipital in Alligator mississippiensis (Archosauria: 68:1–25.
Crocodylia). J Morphol 249:147–153. Rieppel O. 1993a. Patterns of diversity of the reptilian skull. In:
Klembara J. 2004. Ontogeny of the palatoquadrate and adjacent Hanken J, Hall BK, editors. The Skull; Patterns of Structural
lateral cranial wall of the endocranium in prehatching Alliga- and Systematic Diversity. Chicago: University of Chicago
tor mississippiensis (Archosauria: Crocodylia). J Morphol Press. pp 344–390.
262:644–658. Rieppel O. 1993b. Studies on skeleton formation in reptiles. V.
Klembara J. 2005. Ontogeny of the partial secondary wall of Patterns of ossification in the skeleton of Alligator mississip-
the otoccipital region of the endocranium in prehatching Alli- piensis Daudin (Reptilia, Crocodylia). Zool J Linn Soc 109:
gator mississippiensis (Archosauria. Crocodylia). J Morphol 301–325.
266:319–339. Rieppel O. 1993c. Studies on skeleton formation in reptiles: Pat-
Klymkowsky MW, Hanken J. 1991. Whole-mount staining of terns of ossification in the skeleton of Chelydra serpentina
Xenopus and other vertebrates. Methods Cell Biol 36:419– (Reptilia. Testudines). J Zool 231:487–509.
441. Romer AS. 1956. Osteology of the Reptiles. Chicago: University
Lengelé B, Schowing J, Dhem A. 1996a. Chondroid tissue in the of Chicago Press.
early facial morphogenesis of the chick embryo. Acta Embryol Ross FD, Mayer GC. 1983. On the dorsal armor of the Crocodi-
193:505–513. lia. In: Rhodin AGJ, editor. Advances in Herpetology and Evo-

Journal of Morphology
lutionary Biology. Cambridge, Massachusetts: Museum of cinctus (Mammalia, Xenarthra, Cingulata). J Morphol 267:
Comparative Zoology. pp 305–331. 1273–1283.
Ruibal R, Shoemaker V. 1984. Osteoderms in anurans. J Herpe- Vinkka H. 1982. Secondary cartilages in the facial skeleton of
tol 18:313–328. the rat. Proc Finnish Dent Soc 78 (Suppl 7):1–137.
Schumacher G-H. 1973. The head muscles and hyolaryngeal von Huene F. 1912. Die Cotylosaurier der Trias. Palaontolog-
skeleton of turtles and crocodilians. In: Gans C, Parsons TS, raphica 59:69–102.
editors. Biology of the Reptilia, Vol. 4: New York: Academic Westergaard B, Ferguson MWJ. 1986. Development of the den-
Press. pp 101–199. tition in Alligator mississippiensis. Early embryonic develop-
Sheil CA. 2003. Osteology and skeletal development of Apalone ment in the lower jaw. J Zool 210:575–597.
spinifera (Reptilia: Testudines: Trionychidae). J Morphol 256: Westergaard B, Ferguson MWJ. 1987. Development of the den-
42–78. tition in Alligator mississippiensis. Later development in the
Sheil CA, Greenbaum E. 2005. Reconsideration of skeletal de- lower jaws of embryos, hatchlings and young juveniles. J Zool
velopment of Chelydra serpentina (Reptilia: Testudinata: Che- 212:191–222.
lydridae): Evidence for intraspecific variation. J Zool 265:235– Westergaard B, Ferguson MWJ. 1990. Development of the den-
267. tition in Alligator mississippiensis: Upper jaw dental and cra-
Shiino K. 1914. Studien zue Kenntnis des Wirbeltierkopfes. I. niofacial development in embryos, hatchlings, and young
Das Chondrocranium von Crocodilus mit Berücksichtigung juveniles, with a comparison to lower jaw development. Am J
der Gehirnnerven und Kopfgefasse. Anat Hefte 50:254–381. Anat 187:393–421.
Witmer LM. 1995. Homology of facial structures in extant arch-
Shubin N, Tabin C, Carroll S. 1997. Fossil, genes and the evolu-
osaurs (birds and crocodilians), with special reference to para-
tion of animal limbs. Nature 388:639–648.
nasal pneumaticity and nasal conchae. J Morphol 225:269–
Sire J-Y, Huysseune A. 2003. Formation of dermal skeletal and
dental tissues in fish: A comparative and evolutionary
Witten PE, Hall BK. 2003. Seasonal changes in the lower jaw
approach. Biol Rev Camb Philos Soc 78:219–249.
skeleton in male Atlantic salmon (Salmo salar L.): Remodel-
Sire J-Y, Huysseune A, Meunier FJ. 1990. Osteoclasts in teleost ling and regression of the kype after spawning. J Anat 203:
fish: Light- and electron-microscopical observations. Cell Tis- 435–450.
sue Res 260:85–94. Witten PE, Hansen A, Hall BK. 2001. Features of mono- and
Smith MM, Hall BK. 1990. Developmental and evolutionary ori- multinucleated bone resorbing cells of the zebrafish Danio
gins of vertebrate skeletogenic and odontogenic tissues. Biol rerio and their contributio to skeletal development, remodel-
Rev Camb Philos Soc 65:277–374. ing, and growth. J Morphol 250:197–207.
Smith MM, Hall BK. 1993. A developmental model for evolution Zylberberg L, Castanet J. 1985. New data on the structure and
of the vertebrate exoskeleton and teeth: Role of cranial and growth of the osteoderms in the reptile Anguis fragilis L.
trunk neural crest. Evol Biol 27:387–448. (Anguidae. Squamata). J Morphol 186:327–342.
Tran S, Hall BK. 1989. Growth of the clavicle and development Zylberberg L, Geraudie J, Meunier F, Sire J-Y. 1992. Biominer-
of clavicular secondary cartilage in the embryonic mouse. alization in the integumental skeleton of the living lower ver-
Acta Anat 135:200–207. tebrates. In: Hall BK, editor. Bone, Vol. 4: Bone Metabolism
Vickaryous MK, Hall BK. 2006a. Homology of the reptilian and Mineralization. Boca Raton, FL: CRC Press. pp 171–224.
coracoid and a reappraisal of the evolution and development Zylberberg L, Wake MH. 1990. Structure of the scales of Dermo-
of the amniote pectoral apparatus. J Anat 208:263–285. phis and Microcaecilia (Amphibia: Gymnophiona) and a com-
Vickaryous MK, Hall BK. 2006b. Osteoderm morphology and parison to dermal ossifications of other vertebrates. J Mor-
development in the nine-banded armadillo Dasypus novem- phol 206:25–43.

Journal of Morphology