draft · 2019. 2. 7. · draft 2 ontogeny of the skull of melanosuchus niger (crocodylia:...

Draft

Ontogeny of the skull of Melanosuchus niger (Crocodylia: Alligatoridae)

Journal: Canadian Journal of Zoology

Manuscript ID cjz-2018-0076.R2

Manuscript Type: Article

Date Submitted by the Author: 19-Jul-2018

Complete List of Authors: VIEIRA, LUCÉLIA; Universidade Federal de GoiasSANTOS, ANDRÉ; Universidade Federal de UberlandiaHIRANO, LÍRIA; Universidade de BrasiliaMENEZES-REIS, LORENA; Universidade Federal de Uberlandia, Departamento de Anatomia HumanaMENDONÇA, JULIANA; Universidade Estadual Paulista Julio de Mesquita FilhoSebben, Antonio; Universidade de Brasilia

Is your manuscript invited for consideration in a Special

Issue?:Not applicable (regular submission)

Keyword: Embryo, Black caiman, Chondrocranium, Ossification, Melanosuchus niger

https://mc06.manuscriptcentral.com/cjz-pubs

Canadian Journal of Zoology

Draft

1


L. G. Vieira,1* A. L. Q. Santos2, L. Q. L. Hirano3, L. T. Menezes-Reis4, J. S. Mendonça5, A.

Sebben6.

1Instituto de Ciências Biológicas, Universidade Federal de Goiânia (UFG), Goiânia, Goiás,

[email protected];

2Faculdade de Medicina Veterinária, Universidade Federal de Uberlândia (UFU), Uberlândia,

Minas Gerais, [email protected];

3Faculdade de Agronomia e Medicina Veterinária, Universidade de Brasília (UnB), Brasília,

Distrito Federal, [email protected];

4Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia (UFU), Uberlândia,

Minas Gerais, [email protected];

5Instituto de Biociência, Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP), Rio

Claro, São Paulo, [email protected];

6Instituto de Ciências Biológicas, Universidade de Brasília (UnB), Brasília, Distrito Federal,

[email protected].

1 * Correspondence author: Lucélia Gonçalves Vieira, Rua Natal, 59, Qd. 07. apto 1604, Alto da Glória, Goiânia-GO, Brasil, CEP: 74.815-705. +55 62 99846 0607, [email protected]

Page 1 of 47



mailto:[email protected]:[email protected]

Draft

2


L. G. Vieira1*, A. L. Q. Santos2, L. Q. L. Hirano3, L. T. Menezes-Reis4, J. S. Mendonça, A.

Sebben1.

Abstract

We describe the formation of the chondrocranium and the ossification pattern of the skull of

Melanosuchus niger (Spix 1825). The embryos were cleared and double-stained by alizarin red

and alcian blue. Additionally, they were visualized by histological HE staining, and computed

tomography imaging. The chondrocranium of M. niger comprised the nasal capsule,

orbitotemporal and optic-occipital regions. Its development began in stage nine, with the

chondrification of the acrochordal cartilage, trabeculae, and mandibular cartilage. The optic

capsule was formed in the caudolateral portion of the chondrocranium in stage 13. The basal

plate appeared in stage 14, with foramina for the hypoglossal. The chondrocranium was

completely formed in stage 16. The first osteogenic events were noted at stage 13, in the bones,

maxilla, jugal, postorbital, and pterygoid. The quadratojugal, prefrontal, frontal, and squamosal

began their ossification in stage 14. The parietal bone began to ossify only in stage 20. The

basisphenoid began in stage 15 and the parasphenoid began in stage 16. The jaw bones ossified

between stages 13 and 16. The dermal elements started their ossification prior to the

endochondral bones.

Keywords: Embryo, Black caiman, Chondrocranium, Ossification, Melanosuchus niger.

Page 2 of 47



Draft

3

Introduction

The chondrification and sequence of ossification are of great interest for the

comprehension of the evolution of morphological systems. Mabee and Trendler (1996), Smith

(2002), Sanchez-Villagra et al. (2007a, b; 2009) and several others have contributed towards our

understanding of the role of ontogeny in evolutionary changes based on phylogenetic analysis

and heterochrony, making the description of possible ontogenetic transformations essential to the

understanding of this type of developmental pattern (Schoch 2006). Investigations into the

formation of the skull tend to focus on chondrification and ossification, because small changes in

this multidimensional system may result in great phenotypical differences. Although the crania

of vertebrates present a great variety of architectures and functions, they also feature a high

degree of conservatism in the mechanisms of development (Francis-West et al. 1998; Hall

1999a, b).

The sequence of events during the formation and ossification of the cartilaginous and

bony elements of the skull have been studied in many taxa (Burke and Alberch 1985; Rieppel

1992, 1993a, b; Fabrezi 1993; Fabrezi and Alberch 1996; Sheil 1999, 2003; Maisano 2002;

Sánchez-Villagra et al. 2008). However, for crocodilians, few details of the skeletal development

of the skull are known. The existing reports primarily explore the pattern and sequence of

ossification of Alligator mississippiensis (Daudin, 1802) (Bellairs and Kamal 1981; Rieppel

1993b): some are restricted to the formation of the dentition (Westergaard and Ferguson 1986,

1987, 1990), the development of dermal elements (Vickaryous and Hall 2008), the parasphenoid

(Klembara 1993); and postparietal (Klembara 2001) parts, while others focus on the

development of the otoccipital portion (Parker 1883; Shiino 1914; de Beer 1937; Müller 1967;

Page 3 of 47



Draft

4

Iordansky 1973). Most of these authors have concluded that certain brain structures are

phylogenetically closer to birds compared to other living reptiles (Baird 1970).

The elucidation of the development pattern of the skull in living Crocodylia can

contribute not only to the understanding of the evolution of this structure, but also to clarify

similarities and differences between the various related taxa (Klembara 2005); the skull of fossils

is the main focus in morphological and systematic studies. In this investigation, we present the

pattern and sequence of the development of the chondrocranium and ossification of the skull of

embryos of Melanosuchus niger during the pre- and post-natal period.

Materials and Methods

Location and collection of embryos

In the Extractive Reserve Cuniã Lake in the state of Rondônia, Brazil, six nests of

Melanosuchus niger Spix, 1825, were marked and protected with metal screens after the animals

had laid their eggs, between the months of October and December 2008, under permit number

12243-1/2007 RAN/IBAMA (Center for Reptile and Amphibian Conservation and

Management). In the nests, under natural incubation conditions and starting on the tenth day of

development, two eggs were removed every 24 hours. This procedure was repeated until

hatching, resulting in an ontogenetic series with 180 samples, were sufficient for this study.

Relative developmental stages were assigned to all embryos obtained in this study according to

the external morphological criteria of Vieira et al. (2011).

The embryos were removed from their eggs using surgical scissors and euthanized with

the anaesthetic thiopental (50 mg/kg), then preserved in 10% neutral-buffered formalin. The

Page 4 of 47



Draft

5

methods of anaesthesia and euthanasia were approved by the Animal Use Ethics Committee of

the Institute of Biological Sciences of the University of Brasilia (UnBDOC 100271/2009).

Clearing and double-staining of bone and cartilage

Whole bone/cartilage of embryos and hatchlings were differentially stained following the

method of Davis and Gore (1936) and a modified version of the alizarin red/alcian blue protocol

of Dingerkus and Uhler (1977), in which the bone stains red and cartilage stains blue. The

protocol consists of fixation in 5% neutral-buffered formalin for at least 48 hours, washing with

distilled water for 72 hours with subsequent evisceration and removal of the skin when

necessary. Staining of the cartilage occurs with immersion in a mixture of 10 mg of alcian blue,

80 ml of 95% ethanol and 20 ml of glacial acetic acid for 24 to 48 hours. Following staining, the

specimens were rehydrated in a descending alcohol series and neutralized with a solution of

saturated sodium borate for 24 hours. Clearing of the musculature was accomplished with a

solution of KOH (0.5-2%), followed by staining of the bone for an hour by immersion of the

specimens in an aqueous solution of KOH (0.5-2%) plus 0.1% alizarin red S, until the bones

assumed a deep red colour. Specimens were subsequently transferred to solutions of glycerine

with KOH (0.5-2%) in a concentration series of 30%, 50%, 90%, and finally pure glycerine.

After completing these protocols, the specimens were analysed with the aid of a

stereomicroscope (Leica DM 1000, Leica Microsystems GmbH, Germany), equipped with an

image capture system (Leica Camera Twain 6.7.0 Software DFU). Some samples were

disarticulated for observation and photographed at various angles. In this paper we use the term

ossification centre for the smallest areas of ossification observed in each bone, evidenced by the

Page 5 of 47



Draft

6

dyeing with alizarin, which reveals an accumulation in calcium but not necessarily the first event

in the development of the bone.

Histological Processing

Histological processing of the skulls was also carried out in the histology laboratory of

the Federal University of Uberlândia (DBHEM/UFU). Histological serial sections (6 mm thick)

were stained with haematoxylin-eosin, according to the procedure described by Martoja and

Martoja-Pierson (1970). The sections were analysed with the aid of an Olympus BX40 binocular

microscope (Olympus Corporation, United States of America) with an attached Olympus OLY-

200 camera. The images were obtained using the 5x and 10x objectives.

Adopted terminology

For the identification and description of the skeletal structures, the terminology proposed

by Mook (1921), Romer (1956), Iordansky (1973), and Vieira et al. (2016) were used, and for

the identification of structures of the chondrocranium and pattern of ossification, we used the

terminology of Bellairs and Kamal (1981) and Rieppel (1993b).

Results

Development of the chondrocranium

The chondrocranium of Melanosuchus niger is composed of three regions: the nasal

capsule, orbitotemporal region and optic-occipital region. Each region is described in detail

below.

Page 6 of 47



Draft

7

Stage 9

In stage nine, the chondrocranium is long with only a pair of unfused trabeculae present

in its caudal portion (Figs. 1A, and B), which was more evident after staining with alcian blue. It

ends near the notochord, in the orbitotemporal region (Fig. 1A), where it is possible to see the

beginnings of acrochordal cartilage, which is perforated by the notochord and positioned

caudodorsal to the trabeculae (Figs. 1B, and C). The paired Meckel's cartilage is very evident in

the ventral portion of the chondrocranium (Figs. 1A, C, and 2A).

Stage 10

At stage 10, the orbitotemporal region consists of the pilae metoptica and the quadrate

cartilage (Figs. 1B). The pilae metoptica will delimit the orbitotemporal and optic occipital

regions. In lateral view, the pair of quadrate cartilages is present, and from it projects rostrally

the pterygoid cartilage (Fig. 1B).

Stage 11

With the start of the development of the nasal capsule (Fig. 1C) in stage 11, the rostral

portion of the chondrocranium is now continuous with the orbitotemporal region. The nasal

capsule at this stage consists of the rostral portion of the common trabeculae that forms the

narrow internasal septum (Fig. 1C). The cartilage of the nasal capsule is extremely thin and

poorly stained at this point. From it projects bilaterally an arc that forms the tectum nasal. The

quadrate cartilage occupies a large portion of the laterocaudal area (Fig 2B, and C).

Page 7 of 47



Draft

8

Stage 12

In stage 12, the nasal capsule, like the other structures of the chondrocranium, presents an

extensive cartilaginous area that is well-stained with alcian blue (Fig. 1D). Medially, this capsule

is almost completely separated by the internasal septum, which caudally joins the interorbital

septum, already formed at this stage with the reduction of the common trabeculae (Fig. 1D). The

parietotectal cartilage is visible as a condensation in the dorsorostral portion of the nasal capsule

(Fig. 1D), although it is not yet present on the side wall of the capsule, which remains open. The

dorsal surface of the parietotectal cartilage is convex and extends laterally from the internasal

septum (Fig. 1E), being continuous with the anterior transverse plate ventrally, forming the zona

annularis.

At this stage, the nasal concha is present in the cavity; present as well is the paranasal

cartilage, which forms the caudal portion of the nasal capsule along with the planum antorbitale.

The planum supraseptale is clearly visible and projects dorsolaterally, where it continues to

create the taeniae marginalis connected to the pilae antotica (Fig. 2D) and taeniae medialis,

connected to the pilae metoptica (Fig. 1D). The metoptic fenestra is formed between the pilae

antotica and metoptica (Fig. 1D).

Rostrally, the chondrocranium has a sphenoethmoidal commissure that corresponds to a

small projection of the cartilage of the planum supraseptale and is not fused at this stage to the

parietotectal cartilage (Figs. 1D, and 2D). The trabeculae are limited to a small area caudal to the

interorbital septum in the shape of a "U" (Figs. 1F, and 2D), outlining the side walls of the

hypophyseal fenestra, which is still incomplete because of the absence of a crest on the caudal

wall. The trabeculae themselves project caudally to the hypophyseal fossa, creating the infrapolar

Page 8 of 47



Draft

9

process (Fig. 1F). The optic and epioptic fenestrae are well defined, especially by the marginal

and taeniae medialis (Fig. 1F).

In dorsal view, the optic occipital region has so far developed no connection to the other

regions. The notochord no longer stains with alcian blue, but it can be discerned as piercing the

structure of the occipital condyle and going towards the hypophyseal fenestra. There is a small

free proatlas (Figs. 1D). Only the lateral portion of the basal plate (Fig. IE) is stained, with the

middle region remaining undefined. The occipital portion or basal plate represents the

beginnings of the development of the skull. The foramina for the hypoglossal nerve are present

(Fig. 1F). In the most caudal portion (slightly ventrally directed), the occipital arch is present as

two individual structures lateral to the axis of the spine (Fig. 1E).

The optic capsule is continuous with the lateral margin of the occipital arch, but in the

ventral portion it is free because the basal plate at this stage is not distinct in its entirety. The bars

of Meckel's cartilage are fused rostrally; and caudally there is a dorsal projection that

corresponds to the retroarticular process. From the retroarticular process, the columella extends

dorsally toward the tympanic cavity, adjacent to the quadrate cartilage, which in turn is well-

defined (Fig. 2D).

Stage 13

Although there are discrete changes in the development of the chondrocranium in stage

13, its anatomy remains very similar to the previous stage. The interorbital septum, the planum

supraseptale and the basal plate have expanded to complete the walls of the cranium. The

trabeculae and the dorsum sella outline the hypophyseal fossa a little further.

Page 9 of 47



Draft

10

A great deal of progress in the development of the orbitotemporal and oticoccipital

regions have occurred at this point, culminating in its current form, described below. Also at this

stage, the nasal capsule represents a small part of the total chondrocranium anatomy. The

sphenethmoid commissure contacts the parietotectal cartilage, forming a close association

between the nasal capsule and orbitotemporal region, outlining also the orbitonasal fenestra. The

floor of the cavity is formed by the anterior transverse plate.

In lateral view, it is possible to evaluate the development of the interorbital septum

toward the planum supraseptale. There is still a small region that is not stained, indicating that

these structures are not yet fully developed. Three large fenestrae are clearly outlined from this

view: the otic, the epiotic, and the orbitonasal fenestrae. The last one is formed via the union of

the sphenethmoid commissure with the parietotectal cartilage.

Stage 14

In stage 14, the three regions of the chondrocranium are well-developed, robust and with

the cartilage structure well-defined (Figs. 1G, and H). The floor of the basal region is now

formed; the oticoccipital region occupies a large part of the chondrocranium and the nasal

capsule is narrow, giving a conical characteristic to the structure. It is also possible to identify the

presence of ossification centres in some dermal bones; these are reported in detail in the next

section.

At this stage, the nasal capsule appears more robust and the structures are well-marked,

representing approximately one quarter of the total size of the chondrocranium. The interorbital

septum appears robust and fully formed (Figs. 1H, and 2E). The planum supraseptale is broad

Page 10 of 47



Draft

11

and its wings are thin (Fig. 2E) and the caudal margin of this plate reaches the optic capsule (Fig.

1H). Each pilae antotica also projects caudally toward the optic capsule.

In ventral view of the oticoccipital region, the basicranial fenestra, although barely

evident, occupies approximately the medial portion of the basal plate, which is laterally

extensive and connected to the optic capsule. The optic capsule is joined dorsomedially to the

chondrocranium via the tectum synoticum, which corresponds to a thin, slightly convex and

dorsocaudally positioned plate (Fig. 1G). It presents a small concave excavation in the rostral

margin and laterally contacts the occipital arch. (Fig. 1G).

The quadrate cartilage presents, in its caudal margin, a crescent-shaped notch that opens

dorsoventrally to accommodate the columella (Fig. 1H). The small pterygoquadrate cartilage

extends from the rostromedial margin of each quadrate cartilage.

Ossification pattern of the skull and mandible

Premaxilla: At the end of stage 14, ossification centres marked with alizarin red were observed

in both antimeres, oriented transversely on the parietotectal plate above the nasal capsule.

Maxilla: At the end of stage 14, alizarin red was visibly retained caudal planum supraseptale

the premaxilla. The maxilla at this stage appears as a small and elongated triangular plate (Fig.

2E). In stage 15, the rostral extremity is broad, and the caudal region is elongated and barely

contacts the jugal bone (Fig. 3A). The ventral margin forms the largest portion of the upper lip

margin. In stage 22, some teeth are marked by the dye and the maxilla contacts the lacrimal and

nasal parts (Fig. 3D), and then contacts the premaxilla in stage 24 (Fig. 3E).

Jugal: At the end of stage 14, it is marked with alizarin red in a narrow triradiate bar in the

caudal portion of the orbit (Fig. 2E). In stage 15, it contacts the maxilla (Fig. 3A) and then

Page 11 of 47



Draft

12

proceeds to contact the quadratojugal and post-orbital in the next stage (Fig. 3B). Its triangular

shape is quite conspicuous in stage 18 (Fig. 4D).

Postorbital: displays an ossification centre at the end of stage 14, forming a small triangular

plate marked with alizarin red, which at stage 16 contacts the jugal (Figs. 2F, 3B, 4A-C, and 5B).

Its outline is well stained in lateral and dorsal views.

Frontal: It is present as a pair of lightly stained narrow bands at the rostral margin of the

cranium, outlining the orbits medially. In stage 16, they are still lightly stained and extend from

the pre-frontal plate to the postorbital (Fig. 3B, 4A-C, and 5B) and in stage 23, they contact the

parietal caudally. The ossification centres do not fuse in the embryonic period, although they

contact at the midline in stage 25.

Pterygoid: emerges as two elongated plates in stage 15 (Fig. 3A, and 5A). Both of these extend

laterally along the margins of the trabeculae of the chondrocranium and in stage 16, both are

separated by the opening of the secondary choana, close to the caudal margin of the palatal

fenestrae (Figs. 2F and 4C). In stage 26, the choana is well defined as is the lateral expansion of

the pterygoid. This expansion overlaps with the centre of ossification of the parasphenoid and

makes their viewing difficult.

Quadratojugal: emerges as a narrow structure in stage 15, positioned rostral to the quadrate

cartilage (Fig. 3A). In stage 16, it is well stained and presents itself in a triradiated form (Figs.

3B, 4A. and B). In stage 22, it contacts the jugal and quadrate bones (Fig. 3D). The contact with

the postorbital only occurs in stage 23.

Prefrontal: An ossification centre that was observed in stage 15 (Fig. 3A) is positioned in the

caudal portion of the supraorbital plate of the chondrocranium. In stage 24, the pillars of the

prefrontal are well-developed (Figs. 3E, and 5D).

Page 12 of 47



Draft

13

Squamosal: This appears during stage 15 caudolaterally positioned in the cranium (Fig. 3A, and

5A). It is well ossified in stage 22 and occupies the dorsocaudal portion of the otic capsule,

above the quadrate cartilage (Fig. 3D, and 5G).

Ectopterygoid: This is present in the embryo in stage 15 (Fig. 6A), although it is barely marked

with alizarin red until stage 19 (Fig. 6C), when the ossification advances and outlines its

ascending process.

Palatine: It appears barely stained in stage 15 (Fig. 6A). In stage 16, it appears as a narrow

structure, rostral to the pterygoid (Fig. 4E). In stage 18, it corresponds to a small subtriangular

plate, connected rostrally to the maxilla and caudally to the pterygoid (Fig. 4E).

Nasal: An ossification centre is evident in stage 15, appearing as two independent plates (Fig.

3A) over the parietotectal cartilage and extending caudally over the internasal septum to the

rostral portion of the interorbital septum. In stage 21, its morphology is well marked with alizarin

red (Fig. 3C). The nasal contacts the maxilla, lacrimal and premaxilla in stage 22 (Figs. 3C, and

5C).

Lacrimal: At stage 15, it is stained considerably by alizarin red and appears triangular (Fig. 3A).

In stage 16, it begins to develop a pointed process that is directed caudally toward the

articulation between the maxilla and the jugal bone (Figs. 3B, 4A, and B).

Vomer: In stage 22, it appears stained for the first time as a small and elongated plate,

positioned ventrally to the interorbital septum and rostrally to the palatine bone. Its morphology

does not undergo major changes during the prenatal development. At end of the incubation

period, it contacts the premaxilla and maxilla ventrally.

Page 13 of 47



Draft

14

Supraoccipital: forms from a small ossification centre located medial to the epiotic bones in the

synoptic roof. It stains for the first time in stage 19 (Fig. 5E). In stage 23, it fuses to the epiotic

bones and presents, in stage 25, a morphology similar to that of an adult.

Parietal: This bone poorly retains alizarin red in stage 20 (Fig. 5F). It is one of the last elements

to ossify and corresponds to a thin structure positioned laterally in the cranial roof and caudal to

the postorbital bone. It is still poorly stained in stage 21. During development, it expands via two

ossification centres that eventually fuse. In stage 24, the parietal is well marked and in stage 26,

the ossification centres expand through the caudal and medial portions, where they contact their

counterpart (Fig. 5H).

Palpebral: develops with an ossification centre for each plate, which stains for the first time in

stage 20. It develops rapidly and is located in the upper eyelid, not articulating with any other

bony element of the skull (Figs. 3E, F, 5D, H, and I).

Dentary: shows incipient retention of the dye alizarin red in stage 15 (Fig. 3A, and 6A). The

ossification centre is elongated and ventrolaterally follows the rostral margin of Meckel's

cartilage. In stage 16, the dentary covers two thirds of the rostral portion of the lateral surface of

Meckel's cartilage and extends caudally along its ventral margin. In stage 22, it develops and

articulates in the rostromedial portion with its counterpart.

Surangular: The ossification centre first appears in stage 12 (Fig. 3A); it is large and located

close to the caudal portion of Meckel's cartilage, near the articular fossa of the mandible (Fig.

2D). In stage 18, it contacts the dentary (Fig. 4D), and then contacts the angular in stage 19

Angular: forms a long and very narrow ossification centre in stage 15 (Fig. 3A), positioned

ventromedially in the caudal portion of Meckel's cartilage (Fig. 2D). It contacts the dentary bone

in stage 18 (Fig. 4D) and the surangular in stage 19.

Page 14 of 47



Draft

15

Coronoid: shows a small ossification centre in stage 15, caudal to the dentary (Fig. 3A). It is

well-developed in the embryo in stage 19 and forms a small, slightly triangular plate in the

caudal margin of the dentary.

Splenial: displays poor retention of the dye in stage 15 (Fig. 6A). Along with the dentary bone,

the splenial covers Meckel's cartilage and also extends caudally along the same direction (Figs.

6B, C, D, and E).

Quadrate: The ossification of the quadrate bone occurs by the replacement of the quadrate

cartilage, initially by a small, coloured portion that surrounds the margin of the columellar

incisure in stage 16 (Fig. 3B, 4A, B, and D). The quadrate retains a greater amount of alizarin red

at the end of this stage, but the outline of the quadrate cartilage remains during ontogeny. From

stage 24 the ossification process advances (Fig. 4G) and the quadrate contacts the pterygoid bone

(Figs. 3E, F, and 4F), outlining the caudal margin of the foramen for the trigeminal nerve.

Laterosphenoid: displays incipient retention of the dye in stage 21, and in stage 24, in the

rostral portion of the cranial cavity, a greater expansion is marked with alizarin red (Fig. 4F).

Basisphenoid + parasphenoid: The basisphenoid is visible for the first time in stage 16, as a

little pronounced plate positioned on the floor of the cranium, in the rostral portion of the basal

plate, between the ossification centres of the pterygoid (Figs. 2F, and 4C). It develops from a

single ossification centre that replaces part of the basal plate and the caudal portion of the

trabeculae of the chondrocranium. In stages 24, it is well ossified and expands laterally, when it

contacts the pterygoid (Figs. 4G). Also, in this stage, an ossification centre appears that

corresponds to parasphenoid. This emerges with the basisphenoid during ontogeny to form the

parabasisphenoid.

Page 15 of 47



Draft

16

Basioccipital: ossifies in stage 18 at the caudal portion of the basal plate of the chondrocranium.

It is elongated and positioned ventrally, but projects caudally during development to form the

occipital condyle (Figs. 5E, F, and 6C-H), which only completes its formation after hatching.

Exoccipital: first appears in stage 16 by means of distinct and elongated ossification centres in

both antimeres, positioned in the caudal portion of the cranium (Fig. 5B). The exoccipital is well

stained but does not articulate with any other element before stage 24 (Figs. 4F, 5E-G, and 6C-

H).

Prootic: is present in the embryo at the end of stage 20, from a small ossification centre in the

ventrolateral portion of the cranium, caudal to the laterosphenoid bone (Fig. 6D). It ossifies in

the optic capsule along the rostral margin of the quadrate cartilage and forms the caudal margin

of the foramen for the trigeminal nerve.

Epiotic: is also present in stage 20. It appears as a small plate that ossifies along the lateral

margin of the supraoccipital bone, with which it fuses at the beginning of stage 22.

Opisthotic: is present for the first time in the embryo in stage 18. It is formed from three

ossification centres that are well-stained in the caudal portion of the cranium. The opisthotic,

quadrate and supraoccipital bones are confluent and contact firmly in stage 26 (Fig. 4F).

Hyoid: It is well stained with alcian blue in stage 18 (Fig. 4E) but begins its ossification in stage

20. There is little retention of alizarin red, but enough to outline the adult form of this bone in

stage 24 (Figs. 4E and 6E).

Articular: The first sign of the articular bone is in stage 21, when it forms a small plate, slightly

stained and placed medial to the surangular which it contacts firmly in stage 24.

Page 16 of 47



Draft

17

Discussion

Development of the chondrocranium

The anatomy of the chondrocranium of Melanosuchus niger is similar to that of

Crocodylus (Bellairs and Kamal 1981). According to Iordansky (1973), the cartilaginous

cranium of the Crocodylia is generally very similar to other reptiles (De Beer 1937). During its

development, the structures formed can be grouped into three regions: the nasal capsule, the

orbitotemporal region, and the oticoccipital region.

Similar to the Crocodylian Alligator and Crocodylus (Bellairs and Kamal 1981), the nasal

capsule in M. niger consists mainly of the parietotectal cartilage in the lamina transversalis

anterior, and the conchae nasal that projects from the lateral wall of the capsule. The

orbitotemporal region is composed of the interorbital septum, the trabeculae, the planum

supraseptale, the pilae metoptica and the pilae antotica. In this region there are four fenestrae:

optical, epioptica, orbitonasal, and metoptic. The oticoccipital region corresponds to the posterior

portion of the cranium, where the optic capsules are more representative structures, as well as the

basal plate, the occipital arch, and the tectum synoticum.

Bellairs and Kamal (1981) reported that in the early stages of development in

Crocodylus, the back of the sella and basal plate are the first parts of the chondrocranium to

form. Shortly after, the formation of trabeculae can be seen as two separate structures, each in its

own side. In M. niger, we clearly see that the trabeculae are formed before the basal plate, as

well as the quadrate cartilage and Meckel's cartilage. The trabeculae in Ascalabotes are similar to

Lacerta (De Beer 1929), M. niger, and Sphenodon punctatus (Gray, 1831) (Howes and

Swinnerton 1901) in that they appear also as separate elements. The trabeculae are separated

caudally and hold the hypophyseal fenestra (Howes and Swinnerton 1901).

Page 17 of 47



Draft

18

In birds, Parker (1869) first described the formation of the chondrocranium in embryos of

Gallus which, according to the author, begins on the fourth day of the incubation period. Authors

such as Parker and Bettany (1877) and Sonies (1907) described the first chondrification event of

the chondrocranium at five days, with the formation of the acrochordal cartilage. These features

were also observed in Anas (De Beer and Barrington 1934) and appear to be a pattern for birds,

with exceptions in Phalacrocorax (Slaby 1951), where the chondrification of the visceral arches

precede the formation of the acrocordal cartilage; and Struthio (Frank 1954). where the

acrocordal cartilage develops after the basal plate.

In Crocodylus, the roof and floor of the nasal capsule develop and sustain the long and

narrow paraseptal cartilage; the roof is almost completely formed by the parietotectal cartilage,

which is continuous ventrally with the internasal septum: exactly as it develops in M. niger. In

both, the lateral wall of the nasal capsule is formed by the parietotectal cartilage in later stages.

The paranasal cartilage forms the caudal portion of the nasal capsule along with the planum

anterorbitale, as seen in the Crocodylia (Bellairs and Kamal 1981).

In M. niger, as reported for Crocodylus (Bellairs and Kamal 1981), the trabeculae merge

medially in the rostral region to form the internasal septum, and caudally, they merge to form the

thin interorbital septum. Beyond this, the trabeculae diverge in the hypophyseal fenestra and are

continuous to the basal plate. Dorsally, this septum is continuous with the cartilage of the planum

supraseptale.

The internasal septum in Crocodylus and M. niger extends by means of a small rostral

process of the lamina transversalis anterior, with the nasal fenestra outlined caudally by the

junction with the parietotectal cartilage and by this blade. Because of the large size of this

fenestra, the zona annularis is quite narrow (Bellairs and Kamal 1981). During the development

Page 18 of 47



Draft

19

of the nasal capsule in the Crocodylia, the nasal conchae project from the lateral wall of this

capsule (Shiino 1914).

The internasal septum, as in other reptiles, does not ossify in Crocodylia. In M. niger,

only the premaxilla, maxilla, nasal and vomer bones develop on the surface of the nasal capsule.

As for the sphenethmoid commissure in Crocodylus, Bellairs and Kamal (1981) reported

that it is incomplete and does not reach the nasal capsule, unlike Alligator and M. niger, where it

is continuous between the planum supraseptale and the nasal capsule, delimiting the orbitonasal

fenestra.

The medial and taeniae marginalis connect the planum supraseptale to the pilae metoptica

and antotica, respectively, and delimit the optic, metopic and epioptic fenestrae, which is a

characteristic of the Crocodylia (Bellairs and Kamal 1981). According to these authors, in place

of the typical basipterygoid process, the Crocodylia present a conspicuous pair of cartilaginous

plates, the infrapolar processes that develop caudal to the base of each trabecula and are

positioned below the basal plate. In M. niger the infrapolar processes develop in a manner

similar to Crocodylus, where these are caudal extensions of the trabeculae and delimit the

hypophyseal fossa.

The pilae metoptica, a plate that develops laterally, is similar in structure between

Crocodylia and living birds. In M. niger as well as in Crocodylus (Bellairs and Kamal 1981), the

pila metoptica represents a rostral projection of the acrochordal cartilage and develops in the

early stages. On the other hand, the pila antotica ossifies and produces the large laterosphenoid

bone in A. mississippiensis (Rieppel 1993b), Crocodylus (Bellairs and Kamal 1981) and M.

niger.

Page 19 of 47



Draft

20

During the formation of the chondrocranium in M. niger, the basicranial fenestra is not

very evident, as reported by Bellairs and Kamal (1981) for Crocodylus. In this same region, the

notochord passes through the occipital condyle and this fenestra, positioning itself over the basal

plate. The basicranial fenestra is delimited laterally by the occipital arch, which fuses dorsally to

the tectum synoticum. For M. niger, only the tectum synoticum was present in the dorsocaudal

region, contributing to the formation of the foramen magnum in the ossified cranium.

According to Bellairs and Kamal (1981), the optic capsule of Crocodylus presents

divisions similar to those of other reptiles. In M. niger, no subdivision of this region was

observed, although it shares with other Archosauria the connection of this bone to the caudal

portion of the chondrocranium by means of the tectum synoticum.

Pattern and sequence of ossification

In comparison to the existing descriptions of the formation sequence of the cranium in

Crocodylia, the ossification process in M. niger starts before it does in the others, for example, A.

mississippiensis (Rieppel 1993b; Vickaryous and Hall 2008) (Table 1).

In animals in general, the sequence of events in the formation of the skeleton reflects

functional demands. In anurans, for example, the sequence meets the needs of the elements

involved with respiration, while in teleost fish, it meets the demands of feeding, including in

species with peculiar feeding strategies such as Danio rerio (Mabee et al. 2000). Thereafter,

skeleton development involves structures associated with nutrition and afterwards, protection

(Weisel 1967; Gaudin 1978; Adriaens and Verraes 1998). A similar pattern was verified in M.

niger, which begins its ossification with most of the bones involved in the catching and chewing

of food, such as the elements of the mandible, maxilla, premaxilla, palate, nasal and jugal.

Page 20 of 47



Draft

21

The ossification of the elements of the mandible occurs earlier in groups of animals that

possess teeth and later in edentulous tetrapods and may illustrate a cascade of shared

development that controls the initial formation of the brain (Schoch 2006). Also, according to

this author, this feature is a characteristic of the evolutionary process of vertebrates, possibly

representing a response to pressure from the development of the skull. According to Schlosser

and Wagner (2004) these bones form modules that might represent functional units, such as

dermal bones of the mandible, palate, orbit, cranial vault, and floor. In M. niger, there is a trend

that involves such groups, as evidenced by the simultaneous or sequential ossification of the

bones that compose them, such as the bones of the rostral portion of the cranium.

The palatine, vomer, pterygoid, premaxilla, and maxilla bones form the skeletal structure

of the secondary palate. Its functional importance is reflected in the ossification of its bony

apparatus, which occurs in early stages. The formation of the pterygoid also occurs early in A.

mississippiensis (Rieppel 1993b; Vickaryous and Hall 2008) and Crocodylus cataphractus

(Cuvier, 1825) (Müller 1967). With the exception of the pterygoid, Rieppel (1993b) did not

report any other palatal element in A. mississippiensis before stage 21. This structure is of great

importance in this group in order for them to function in their habitat, as it acts to separate the

respiratory and oral cavities, allowing them to eat and breathe at the same time.

The ossification of the pterygoid seems to be a standard also for the crocodilians A.

mississippiensis (Rieppel 1993b), C. cataphractus (Müller 1967), Caiman yacare (Daudin, 1802)

(Lima et al. 2011) and M. niger. Its initial formation speaks to the importance of this element

among the basal reptiles (Rieppel 1993a) as well as its role as the anchor point for large adductor

muscles of the maxilla. Although the relationship is less clear, the rare early formation of the

coronoid in A. mississippiensis (Rieppel 1993b) may also be related to the musculotendinous

Page 21 of 47



Draft

22

system, having seen that this is a point of insertion for the mandibular tendon of the adductor

muscle (Iordansky 1973).

In Crocodylus palustris (Lesson, 1831), ossification centres appear simultaneously in

some bones of the skull (Bellairs and Kamal 1981). The sequence presents variations between

the Crocodylia. Particularly in A. mississippiensis (Rieppel 1993b), C. yacare (Lima et al. 2011)

and M. niger, the parietal ossifies in later stages, however most of the elements of the cranium

present ossification centres between stages 14 and 16, many of them simultaneously.

Interestingly, the parietal develops from two ossification centres. Only after hatching do these

elements fuse. The presence of a completely fused parietal is a synapomorphy used in the

analysis of the phylogeny of Crocodylomorpha.

According to Bellairs and Kamal (1981), just as it occurs in M. niger and A.

mississippiensis (Klembara 1991), the basisphenoid ossifies in the anterior portion of the basal

plate, which projects rostrally between laterosphenoids. Below the hypophyseal fenestra emerge

the parasphenoid, which fuses dorsally with the basisphenoid and forms the parabasisphenoid. In

C. porosus, the parasphenoid ossifies through three ossification centres (Bellairs and Kamal

1981) but there is only one in M. niger. The fusion of the basisphenoid with the parasphenoid is a

characteristic of the Crocodylia (Iordansky 1973). Müller (1967) reported that these plates are

ossifications of the infrapolar portion and not dermal elements, as observed for M. niger.

According to Andrade et al. (2006) and similar to M. niger, the development of the

secondary palate in living Crocodylia parallels the evolution of this structure in basal forms such

as Geosaurus (Crocodyliformes) and Notosuchus (Mesoeucrocodylia), which possess choanae

completely enclosed in the fused pterygoids; this is one of the cranial characteristics present in

the cranium that defines the Mesoeucrocodylia clade. In M. niger, it was noted that the choana

Page 22 of 47



Draft

23

gradually migrates in the pterygoid until it reaches its caudal portion, where it remains fully

bound by the bone during adulthood. Similar information had already been reported by Kälin

(1933), Langston (1973), Ferguson (1985) and Monteiro and Soares (1997).

The existence of bony plates positioned on the eyelids, named eyelid or supraorbital

bones (Bellairs and Kamal 1981), was not described here as an element of the cranium in M.

niger because this is not an ossification of the dermis above the eyes. In accordance with Romer

(1956) and Vickaryous and Hall (2008), we consider this element as a deeply embedded

osteoderm. The presence of these eyelid bones, of which there may be one or two pieces, is quite

common among Crocodylomorpha. In M. niger and other living forms, this bone appears as a

small plate on the dorsal rostral margin of the orbits. In living forms and in some fossil forms

such as Mariliasuchus amarali (Carvalho and Bertini 1999) and Sebecus icaeorhinus (Simpson,

1937) (Colbert 1946), this element is loosely attached to the cranium. In other forms, however,

this supraorbital coverage can develop to form a solid plate, firmly attached to the surrounding

cranial bones, as occurs in Stratiotosuchus and Lomasuchus palpebrosus (Gasparini et al. 1991).

Overall, our results suggest that the development of the skull of M. niger is similar to

other reptiles. The sequence of skeletal formation events reflects functional demands.

Ossification begins in the bones involved in the capture and chewing the food, such as of

elements of the jaw, maxilla, premaxilla, palatine, pterygoid, nasal and jugal. Another interesting

feature is the formation of the parietal via two ossification centres that fuse during ontogeny.

Acknowledgments

The authors thank Douglas Riff and Sacha Braun Chaves for their useful suggestions to

improve this manuscript and to Ruy, Fabrício and Karla for their assistance in the Histology

Page 23 of 47



Draft

24

Laboratory of UFU. The specimens were kindly donated by the National Center for Research

and Conservation of Reptiles and Amphibians (RAN), of the Chico Mendes Institute for

Biodiversity Conservation (ICMBio).

Adriaens, D., and Verraes, W. 1998. Ontogeny of the osteocranium in the African catfish,

Clarias gariepinus Burchell (1822) (Siluriformes: Clariidae): Ossification sequence as a

response to functional demands. J. Morphol. 235:183-237. doi.org/10.1002/(SICI)1097-

4687(199803)235:33.0.CO;2-8

Andrade, M.B., Bertini, R.J., and Pinheiro, A.E.P. 2006. Observations on the palate and choanae

structures in Mesoeucrocodylia (Archosauria, Crocodylomorpha): Phylogenetic implications.

Rev. Bras. Paleo. 9(3): 323-332. doi.org/10.4072/rbp.2006.3.07

Arthur, W. 2002. The emerging conceptual framework of evolutionary developmental biology.

Nature, 415: 757–764. doi.org/10.1038/415757a

Page 24 of 47



Draft

25

Baird, I.L. 1970. The anatomy of the reptilian ear. In Biology of the Reptilia. Edited by C. Gans

and T.S. Parsons. Academic Press, New York, pp. 193-275. vol. 2

Bellairs, Ad’A., and Kamal, A.M. 1981. The chondrocranium and the development of the skull

in recent reptiles. In Biology of the Reptilia. Edited by C. Gans and T.S. Parsons. Academic

Press, London, pp. 1-263. vol.11.

Benton, M.J., and Clark, J.M. 1988. Archosaur phylogeny and. relationships of the Crocodylia.

In Amphibians, Reptiles, Birds. Systematics Association Special. The Phylogeny. Edited by M.J.

Benton. Clarendon Press, Oxford, pp. 295-338. vol. 1.

Brock, G.T. 1929. On the development of the skull of Leptodeira hotamboia. Q. J. Microsc. Sci.

73: 289-331.

Burke, A.C., and Alberch, P. 1985. The development and homology of the chelonian carpus and

tarsus. J. Morphol. 186: 119–131. doi.org/10.1002/jmor.1051860111

Carvalho, I.S., and Bertini, R.J. 1999. Mariliasuchus: um novo Crocodylomorpha (Notosuchia)

do Cretáceo da Bacia Bauru, Brasil. Geo. Col. 24:83-105.

Colbert, E.H. 1946. Sebecus, representative of a peculiar suborder of fossil crocodilian from

Patagonia. Bull. Am. Mus. Nat. Hist. 87:217-270.

Page 25 of 47



Draft

26

Davis, D.D., and Gore, U.R. 1936. Clearing and staining skeletons of small vertebrates. Field

Museum of Natural History, Chicago. vol.4.

De Beer, G.R. 1929. The development of the skull of the shrew. Philos. Trans. R. Soc. Lond. B

Biol. Sci. No. 217:411-480. doi.org/10.1098/rstb.1929.0009

De Beer, G.R. 1930. The early development of the chondrocranium of the lizard. Q. J. Microsc.

Sci. 73:707-739.

De Beer, G.R. 1937. The Development of the Vertebrate Skull. Oxford, UK: Clarendon. 760 p.

De Beer, G.R., and Barrington, E.J.W. 1934. The segmentation and chondrification of the skull.

Philos. Trans. R. Soc. Lond. B Biol. Sci. No. 223:411-467.

Dingerkus, G., and Uhler, L.D. 1977. Enzyme clearing of alcian blue stained whole small

vertebrates for demonstration of cartilage, Stain Technol. 52:229-232.

doi.org/10.3109/10520297709116780

Fabrezi, M. 1993. The anuran tarsus. Alytes, 11: 47–63

Fabrezi, M., and Alberch, P. 1996. The carpal elements of anurans. Herpetol. 52: 188–204.

Page 26 of 47



Draft

27

Ferguson, M.W.J. 1985. Reproductive biology and embryology of the crocodilians. In Biology of

the Reptilia. Edited by C. Gans, F. Billett and P.F.A. Maderson. Academic Press, New York, pp. 329–

491. vol. 14.

Francis-West, P., Ladher, R., Barlow, A., and Graveson, A. 1998. Signalling interactions during

facial development. Mech. Dev. 75: 3–28. doi.org/10.1016/S0925-4773(98)00082-3

Frank, G.H. 1954. The development of chondrocranium of the ostrich. Ann. Univ. Stellenbosch,

30A:179-248.

Gasparini, Z., Chiappe, L.M., and Fernandez, M. 1991. A new senonian peirosaurid

(Crocodylomorpha) from Argentina and a synopsis of the South American

Cretaceouscrocodilians. J. Vert. Paleontol. 11:316-333.

doi.org/10.1080/02724634.1991.10011401

Gaudin, A.J. 1978. The sequence of cranial ossification in the California toad, Bufo boreas

(Amphibia, Anura, Bufonidae). J. Herpetol. 12:309-318. doi.org/10.2307/1563611

Hall, B.K. 1999a. Evolutionary Developmental Biology. 2nd Ed. Kluwer, Dordrecht, 491pp.

doi.org/10.1007/978-94-011-3961-8

Hall, B.K. 1999b. The Neural Crest in Development and Evolution. Springer, New York, 313pp.

doi.org/10.1007/978-1-4757-3064-7

Page 27 of 47



Draft

28

Hall, B.K. 2002. Palaeontology and evolutionary developmental biology: a science of the

nineteenth and twenty-first centuries. Palaeont. 45: 647–669. doi.org/10.1111/1475-4983.00253

Haluska, F., and Alberch, P. 1983. The cranial development of Elaphe obsoleta (Ophidia,

Colubridae). J. Morphol. 178:37-55. doi.org/10.1002/jmor.1051780104

Hamilton, H.L. 1965. Lillie’s Development of the Chick; an Introduction to Embryology. New

York: Holt, Rinehart and Winston.

Howes, G.B., and Swinnerton, H.H. 1901. On the development of the skeleton of the Tuatara,

Sphenodon punctatus; with remarks on the egg, on the hatching, and on hatched young. Trans.

Zool. Soc. Lond. 16(1)1-86. doi.org/10.1111/j.1096-3642.1901.tb00026.x

Hutchinson, J.R. 2006. The evolution of locomotion in archosaurs. C. R. Palevol. 5(3-4):519-

530. doi.org/10.1016/j.crpv.2005.09.002

Iordansky, N.N. 1973. The skull of the crocodilian. In Biology of the Reptilia. Edited by C. Gans

and T.S. Parsons. Academic Press, New York, pp. 201-264. vol. 4

Jackson, K. 2002. Post-ovipositional development of the monocled cobra, Naja kaouthia

(Serpentes: Elapidae). Zoology, 105 (2002): 203–214. doi.org/10.1078/0944-2006-00077

Page 28 of 47



Draft

29

Jollie, M.T. 1957. The head skeleton of the chicken and remarks on the anatomy of this region in

other birds. J. Morphol. 100:389-436. doi.org/10.1002/jmor.1051000302

Kälin, J.A. 1933. Beiträge zur vergleichenden Osteologe des Crocodilienschädels. Zoolog. Jahrb.

57:535-714.

Klembara, J. 1991. The cranial anatomy of early ontogenetic stages of Alligator mississippiensis

(Daudin, 1802) and the significance of some of its cranial structures for the evolution of

tetrapods. Palaeontograph Abteilung A: Paleaozool. 215:103–171.

Klembara, J. 1993. The parasphenoid and associated dermal structures of the parabasisphenoid

of Alligator mississippiensis (Daudin, 1802). Palaeontograph Abteilung A: Paleaozool. 228:143–

164.

Klembara, J. 2001. Postparietal and prehatching ontogeny of the supraoccipital in Alligator

mississippiensis (Archosauria: Crocodylia). J. Morphol. 249:147–153.

doi.org/10.1002/jmor.1046

Klembara, J. 2005. Ontogeny of the partial secondary wall of the otoccipital region of the

endocranium in prehatching Alligator mississippiensis (Archosauria. Crocodylia). J. Morphol.

266:319–339. doi.org/10.1002/jmor.10380

Page 29 of 47



Draft

30

Langston, W. 1973. The crocodilian skull in historical perspective. In Biology of the Reptilia.

Edited by C. Gans, and T.S. Parsons. Academic Press, New York, pp. 265-284. vol. 4

Lima, F.C., Santos, A.L.Q., Vieira, L.G., and Coutinho, M.E. 2011. Sequência de

desenvolvimento do sincrânio e hióide em embriões de Caiman yacare. Iheringia, Sér. Zool.

101(3) 161-172. doi.org/10.1590/S0073-47212011000200003

Mabee, P.M., Otmstead, K.L., and Cubbage, C.C. 2000. An experimental study of intraspecific

variation, developmental, timing, and heterochrony in fishes. Evolution, 45:2091-2106.

doi.org/10.1111/j.0014-3820.2000.tb01252.x

Mabee, P.M., and Trendler, T.A. 1996. Development of the cranium and paired fins in Betta

splendens (Teleostei: Percomorpha): Intraspecific variation and interspecific comparations. J.

Morphol. v. 227, p. 249-287. doi.org/10.1002/(SICI)1097-4687(199603)227:33.0.CO;2-1

Maisano, J.A. 2002. The potential utility of postnatal skeletal developmental patterns in

squamate phylogenetics. Zool. J. Linn. Soc. 136: 277–313. doi.org/10.1046/j.1096-

3642.2002.00033.x

Martoja, R., and Martoja-Pierson, M. 1970. Técnicas de Histología Animal. Barcelona: Toray

Masson S.A.

Page 30 of 47



Draft

31

May, W. 1962. Die morphologie des Chondrocraniums und Osteocraniums eines

Waldkauzembryos (Strix aluco L.). Z. Wiss. Zool. 166:133-202.

Monteiro, L.R., and Soares, M. 1997. Allometric analysis of the ontogenetic variation and

evolution of the skull in Caiman Spix 1825 (Crocodylia: Alligatoridae). Herpetol. 53:62-69.

Mook, C.C. 1921. Notes on the postcranial skeleton in the Crocodilia. Bull. Am. Mus. Nat. Hist.

44:67-100.

Müller, W. 1961. Die morphologie und Chondrocraniums und Osteocraniums eines Waldkauz

embryos (Strix aluxo). Z. Wiss. Zool. 166:133-202.

Müller, F. 1967. Zur embryonalen Kopfentwicklung von Crocodylus cataphractus Cuvier. Rev.

Suisse Zool. 74:189–294. doi.org/10.5962/bhl.part.75851

Müller, G.B., and Alberch, P. 1990. Ontogeny of the limb skeleton in Alligator mississippiensis:

Developmental invariance and change in the evolution of archosaur limbs. J. Morphol. 203: 151–

164. doi.org/10.1002/jmor.1052030204

Murray, P.D.F. 1963. Adventitious (secondary) cartilage in the chick, and the development of

certain bones and articulations in the chick skull. Aust. J. Zool. 11:368-430.

doi.org/10.1071/ZO9630368

Page 31 of 47



Draft

32

Parker, W.K., and Bettany, G.T. 1877. The morphology of the skull. London: Macmillan.

Parker, W.K. 1869. On the structure and development of the skull of the common fowl (Gallus

domesticus). Philos. Trans. R. Soc. Lond. 159:755-807. doi.org/10.1098/rstl.1869.0032

Parker, W.K. 1883. On the structure and development of the skull of the Crocodilia. Trans. Zool.

Soc. Lond. 11:263–310. doi.org/10.1111/j.1096-3642.1883.tb00361.x

Patterson, C. 1977. Cartilage bones, dermal bones and membrane bones, or the exoskeleton

versus the endoskeleton. In Problems in Vertebrate Evolution. Edited by S.M. Andrews and A.D.

Walker. Academic Press London, pp 77–122.

Pough, F.H., Janis, C.M., and Heiser, J.B. 2003. A vida dos vertebrados. São Paulo: Atheneu.

Raff, E.C., and Raff, R.A. 2000. Dissociability, modularity, evolvability. Evol. Dev. 2:235–238.

doi.org/10.1046/j.1525-142x.2000.00069.x

Rieppel, O. 1992. Studies of formation in reptiles III. Patterns of ossification on the skeleton of

Lacerta vivipara Jacquin (Reptilia, Squamata). Fieldiana Zool. 68:1-25.

Rieppel, O. 1993a. Studies on skeleton formation in reptiles II. Chamaeleo hoehnelli (Squamata:

Chamaeleoninae), with comments on the homology of carpal and tarsal bones. Herpetol. 49:66-

78.

Page 32 of 47



Draft

33

Rieppel, O. 1993b. Studies on skeleton formation in reptiles. V. Patterns on ossification in the

skeleton of Alligator mississippiensis Daudin (Reptilia, Crocodylia). J. Zool. (Lond.) 109: 301-

351. doi.org/10.1111/j.1096-3642.1993.tb02537.x

Rieppel, O. 1994. Studies of formation in reptiles. Patterns of ossification in the skeleton of

Lacerta agilis exigua Eichwald (Reptilia, Squamata). J. Herpetol. 28:145-153.

doi.org/10.2307/1564613

Romer, A.S. 1956. Osteology of the Reptiles. Chicago: University of Chicago Press.

Sánchez-Villagra, M.R., Mitgutsch, C., Hagashima, H., and Kuratani, S. 2007a. Autopodial

development in the sea turtles Chelonia mydas and Caretta caretta. Zool. Sci. 24: 257–263.

doi.org/10.2108/zsj.24.257

Sánchez-Villagra, M.R., Winkler, J.D. and Wurst, L. 2007b. Autopodial skeleton evolution in

side-necked turtles (Pleurodira). Acta. Zool. Stockh. 88: 199–209. doi.org/10.1111/j.1463-

6395.2007.00267.x

Sánchez-Villagra, M.R., Müller, H., Sheil, C.A., Scheyer, T.M., Nagashima, H., and Kuratani, S.

2009. Skeletal development in the Chinese soft-shelled turtle Pelodiscus sinensis (Testudines:

Trionychidae). J. Morphol. 270:1381–1399. doi.org/10.1002/jmor.10766

Page 33 of 47



Draft

34

Sánchez-Villagra, M.R., Ziermann, J.M., and Olsson, L. 2008. Limb chondrogenesis in

Graptemys nigrinoda (Emydidae), with comments on the primary axis and the digital arch in

turtles. Amphibia-Reptilia, 29: 85–92. doi.org/10.1163/156853808783431505

Schlosser, G., and Wagner, G. 2004. Modules in Development and Evolution. Chicago:

University of Chicago Press.

Schoch, R.R. 2006. Skull ontogeny: Developmental patterns of fishes conserved across major

tetrapod clades. Evol. Dev. 8:524-536. doi.org/10.1111/j.1525-142X.2006.00125.x

Sheil, C.A. 1999. Osteology and skeletal development of Pyxicephalus adspersus (Anura:

Ranidae: Raninae). J. Morphol. 240:49–75. doi.org/10.1002/(SICI)1097-

4687(199904)240:13.0.CO;2-Z

Sheil, C.A. 2003. Osteology and skeletal development of Apalone spinifera (Reptilia:

Testudines: Trionychidae). J. Morphol. 256:42–78. doi.org/10.1002/jmor.10074

Sheil, C.A., and Greenbaum, E. 2005. Reconsideration of skeletal development of Chelydra

serpentina (Reptilia: Testudinata: Chelydridae): evidence for intraspecific variation. J. Zool.

(Lond.) 265:235-267. doi.org/10.1017/S0952836904006296

Page 34 of 47



Draft

35

Shiino, K. 1914. Studien zue Kenntnis des Wirbeltierkopfes. I. Das Chondrocranium von

Crocodilus mit Beru¨ cksichtigung der Gehirnnerven und Kopfgefasse. Anat. Hefte. 50:254–381.

Slaby, O. 1951. Le développement du chondrocrâne du cormorant (Phalacrocorax carbo L.) au

point de vue de l’évolution. Bull. Inter. Acad. Tchèque., Sci. 52:1-47.

Smith, M.M., and Hall, B.K. 1990. Developmental and evolutionary origins of vertebrate

skeletogenic and odontogenic tissues. Biol. Rev. Camb. Philos. Soc. 65:277–374.

doi.org/10.1111/j.1469-185X.1990.tb01427.x

Smith, M.M., and Hall, B.K. 1993. A developmental model for evolution of the vertebrate

exoskeleton and teeth: Role of cranial and trunk neural crest. Evol. Biol. 27:387–448.

doi.org/10.1007/978-1-4615-2878-4_10

Smith, K.K. 2002. Sequence heterochrony and the evolution of development. J. Morphol.

252:82–97. doi.org/10.1002/jmor.10014

Sonies, F. 1907. Über die Entwicklung dês Chondrocraniums und der knorpeligen Wirbelsäule

bei den Vögeln. Petrus. Camper. 4:395-486.

Tokita, M., Chaeychomsri, W., and Siruntawineti, J. 2013. Skeletal gene expression in the

temporal region of the reptilian embryos: implications for the evolution of reptilian skull

morphology. SpringerPlus, 2:336. doi.org/10.1186/2193-1801-2-336

Page 35 of 47



Draft

36

Toerien, M.J. 1971. The developmental morphology of the chondrocranium of Podiceps

cristatus. Annale van die Uniwersiteit van Stellenbosch. Reeks A, vol. 46, no. 3. University of

Stellenbosch, South Africa. 128 pp.

Vickaryous, M.K., and Hall, B.K. 2008. Development of the dermal skeleton in Alligator

mississippiensis (Archosauria, Crocodylia) with comments on the homology of osteoderms. J.

Morphol. 269:398–422. doi.org/10.1002/jmor.10575

Vieira, L.G., Lima, F.C., Santos, A.L.Q., Mendonça, S.H.S.T, Moura, L.R., Iasbeck, J.R. and

Sebben, A. 2011. Description of embryonic stages in Melanosuchus niger (Spix, 1825)

(Crocodylia: Alligatoridae). J. Morphol. Sci. 28:11-22.

Vieira, L.G., Santos, A.L.Q., Lima, F.C., Mendonça, S.H.S.T., Menezes, L.T. and Sebben, A.

2016. Osteologia de Melanosuchus niger (Crocodylia: Alligatoridae) e a evidência evolutiva.

Pesq. Vet. Bras. 36:1025-1044. doi.org/10.1590/s0100-736x2016001000018

Vorster, W. 1989. The development of the chondrocranium of Gallus gallus. Adv. Anat.

Embryol. Cell. Biol. 113: 1–75. doi.org/10.1007/978-3-642-73999-6_1

Weisel, G.F. 1967. Early ossification in the skeleton of the sucker (Catostomus macrocheilus)

and the guppy (Poecilia reticulate). J. Morphol. 121:1-18. doi.org/10.1002/jmor.1051210102

Page 36 of 47



Draft

37

Westergaard, B., and Ferguson, M.W.J. 1986. Development of the dentition in Alligator

mississippiensis. Early embryonic development in the lower jaw. J. Zool. (Lond.) 210:575–597.

doi.org/10.1111/j.1469-7998.1986.tb03657.x


mississippiensis. Later development in the lower jaws of embryos, hatchlings and young

juveniles. J. Zool. (Lond.) 212:191–222. doi.org/10.1111/j.1469-7998.1987.tb05984.x


mississippiensis: Upper jaw dental and craniofacial development in embryos, hatchlings, and

young juveniles, with a comparison to lower jaw development. Am. J. Anat. 187:393–421.

doi.org/10.1002/aja.1001870407

Wilkins, A.S. 2002. The Evolution of Developmental Pathways. Sinauer, Sunderland.

Page 37 of 47



Draft

38

Table 1. Comparison of chondrocranium development and ossification of the skull elements of Melanosuchus niger.

Element This study Vickaryous and Hall (2008) Rieppel (1993)Premaxilla Stage 14 Stage 18 Stage 19Maxilla Stage 14 Stage 17 Stage 18Jugal Stage 14 Stage 18 Stage 20Nasal Stage 15 Stage 21 Stage 22Pterygoide Stage 15 Stage 17 Stage 18Quadratojugal Stage 15 Stage 19 Stage 21Prefrontal Stage 15 Stage 18 Stage 20Squamosal Stage 15 Stage 19 Stage 21Ectopterygoid Stage 15 Stage 21 Stage 21Palatine Stage 15 Stage 18 Stage 21Lacrimal Stage 15 Stage 20 Stage 20Frontal Stage 15 Stage 18 Stage 20Basioccipital Stage 16 Basisp./parasphenoid Stage 16 Exoccipital Stage 16 Opisthotic Stage 18 Supraoccipital Stage 19 Parietal Stage 20 Stage 22 Stage 23Palpebral Stage 20 Hyoid Stage 20 Prootic Stage 20 Epiotic Stage 20 Laterosphenoid Stage 21 Vomer Stage 22 Stage 18 Stage 21Dentary Stage 15 Stage 17 Stage 19Surangular Stage 15 Stage 19 Stage 19Angular Stage 15 Stage 17 Stage 18Coronoid Stage 15 Stage 17 Stage 21Splenial Stage 16 Stage 18 Stage 21

Page 38 of 47



Draft

39

Figure Captions:

Figure 01. Chondrocranium of Melanosuchus niger. (A) ventral view, stage 10, (B) the lateral

view, stage 10, (C) the ventral view, stage 11, (D) dorsal view, stage 12, (E) lateral view, stage

12, (F) ventral view, stage 12, (G) dorsal view, stage 14, (H) lateral view, stage 14.

Abbreviations: ac, acrochordal cartilage; bp, basal plate; col, columella; ctr, common trabeculae;

ef, epiotic fenestra; hf, hypoglossal foramen; hyf, hypophyseal fossa; ins, internasal septum; ios,

interorbital septum; ip infrapolar process; lta, lamina transversalis anterior; mc, Meckel's

cartilage; mef, metoptica fenestra; nc, nasal capsule; nac, nasal concha; no, notochord; oa,

occipital arch; oc, optic capsule; orf, orbitonasal fenestra; pia, pilae antotica; pm, pilae

metoptica; pc, pterygoquadrate cartilage; pro, proatlas, ps, planum supraseptale; ptc, parietotectal

cartilage; qc, quadrate cartilage; sc, sphenethmoid commissure; tma, taeniae marginalis; tme,

taeniae medialis; tn, tectum nasal; tr, trabeculae; ts, tectum synoticum; za, zona annularis.

Diaphanized with KOH and stained with alizarin red S and alcian blue. Scale: A, B and C 2 mm;

D, E, F, G and H 5 mm.

Figure 02. Selected cross sections of the chondrocranium Melanosuchus niger. (A) stage 10, (B-

C) stage 11, (D) stage 12, (E) stage 14, (F) stage 16. Abbreviations: an, angular; ar, articular; br,

brain; bp, basal plate; bs, basisphenoid; ctr, common trabeculae; de, dentary; hy, hyoid; ica,

internal carotid artery; ios, interorbital septum; ju, jugal; ma, maxilla; mc, Meckel's cartilage;

mn, mandibular nerve; man, maxillary nerve; nm, nictitating membrane; oc, optic capsule; pc,

pterygoquadrate cartilage; pi, pituitary; pia, pilae antotica; po, postorbital; ps, planum

Page 39 of 47



Draft

40

supraseptale; pt, pterygoid; qc, quadrate cartilage; sp, splenial; su, surangular; to, tongue; tr,

trabeculae; trg, trigeminal ganglia; II , optic nerve; III, oculomotor nerve; IV, trochlear nerve.

Stain: Haematoxylin and eosin. Scale: 3 mm.

Figure 03. Skull of Melanosuchus niger. (A) stage 15, (B) stage 16, (C) stage 21, (D) stage 22,

(E) beginning of stage 24, (F) stage 28. Lateral view. Abbreviations: an, angular; co, coronoid;

de, dentary; fr, frontal; ju, jugal; la, lacrimal; ma, maxilla; na, nasal; pfr, prefrontal; po,

postorbital; pp, palpebral; prm, premaxilla; pt, pterygoid; qj, quadratojugal; qu, quadrate; sq,

squamosal; su, surangular. Diaphanized with KOH and stained with alizarin red S and alcian

blue. Scale: 10 mm.

Figure 04. Skull of Melanosuchus niger. (A) lateral view, beginning of stage 16, (B) lateral

view, end of stage 16, (C) dorsal view, end of stage 16, (D) lateral view, stage 18, (E) ventral

view, stage 18, (F) dorsolateral view, stage 24, (G) lateral view, with details of (H), stage 24.

Abbreviations: an, angular; bs, basisphenoid; co, coronoid; col, columella; de, dentary; ex,

exoccipital; fr, frontal; hy, hyoid; ju, jugal; la, lacrimal; lat, laterosphenoid; ma, maxilla; na,

nasal; ops, opisthotic; par, parasphenoid; pfr, prefrontal; pl, palatine; po, postorbital; prm,

premaxilla; pt, pterygoid; qj, quadratojugal; qu, quadrate; sq, squamosal; su, surangular.

Diaphanized with KOH and stained with alizarin red S and alcian blue. Scale: 10 mm.

Figure 05. Skull of Melanosuchus niger. (A) stage 15, (B) stage 16, (C) stage 22, (D) end of

stage 24, (E) stage 19, (F) stage 20, (G) stage 22, (H) stage 26, (I) 42 days after hatching. Dorsal

view. Abbreviations: bo, basioccipital; bs, basisphenoid; ex, exoccipital; fr, frontal; ju, jugal; la,

Page 40 of 47



Draft

41

lacrimal; ma, maxilla; na, nasal; pa, parietal; pfr, prefrontal; po, postorbital; pp, palpebral; prm,

premaxilla; pt, pterygoid; so, supraoccipital; sq, squamosal. Diaphanized with KOH and stained

with alizarin red S and alcian blue. Scale: A, B, E, F, G and H 10 mm, C and D 5 mm.

Figure 06. Skull of Melanosuchus niger. (A) stage 15, (B) stage 16, (C) beginning of stage 19,

(D) end of stage 20, (E) beginning of stage 21, (F) beginning of stage 25, (G) end of stage 25 (H)

42 days after hatching. Ventral view. Abbreviations: an, angular; bo, basioccipital; bs,

basisphenoid; de, dentary; ec, ectopterygoid; ex, exoccipital; hy, hyoid; ju, jugal; ma, maxilla; pl,

palatine; prm, premaxilla; pr, prootic; pt, pterygoid; sp, splenial. Diaphanized with KOH and

stained with alizarin red S and alcian blue. Scale: 10 mm.

Page 41 of 47



Draft

Figure 01. Chondrocranium of Melanosuchus niger. (A) ventral view, stage 10, (B) the lateral view, stage 10, (C) the ventral view, stage 11, (D) dorsal view, stage 12, (E) lateral view, stage 12, (F) ventral view,

stage 12, (G) dorsal view, stage 14, (H) lateral view, stage 14. Abbreviations: ac, acrochordal cartilage; bp, basal plate; col, columella; ctr, common trabeculae; ef, epiotic fenestra; hf, hypoglossal foramen; hyf, hypophyseal fossa; ins, internasal septum; ios, interorbital septum; ip infrapolar process; lta, lamina transversalis anterior; mc, Meckel's cartilage; mef, metoptica fenestra; nc, nasal capsule; nac, nasal

concha; no, notochord; oa, occipital arch; oc, optic capsule; orf, orbitonasal fenestra; pia, pilae antotica; pm, pilae metoptica; pc, pterygoquadrate cartilage; pro, proatlas, ps, planum supraseptale; ptc,

parietotectal cartilage; qc, quadrate cartilage; sc, sphenethmoid commissure; tma, taeniae marginalis; tme, taeniae medialis; tn, tectum nasal; tr, trabeculae; ts, tectum synoticum; za, zona annularis. Diaphanized with KOH and stained with alizarin red S and alcian blue. Scale: A, B and C 2 mm; D, E, F, G and H 5 mm.

Page 42 of 47



Draft

199x267mm (300 x 300 DPI)

Page 43 of 47



Draft

Figure 02. Selected cross sections of the chondrocranium Melanosuchus niger. (A) stage 10, (B-C) stage 11, (D) stage 12, (E) stage 14, (F) stage 16. Abbreviations: an, angular; ar, articular; br, brain; bp, basal plate;

bs, basisphenoid; ctr, common trabeculae; de, dentary; hy, hyoid; ica, internal carotid artery; ios, interorbital septum; ju, jugal; ma, maxilla; mc, Meckel's cartilage; mn, mandibular nerve; man, maxillary nerve; nm, nictitating membrane; oc, optic capsule; pc, pterygoquadrate cartilage; pi, pituitary; pia, pilae antotica; po, postorbital; ps, planum supraseptale; pt, pterygoid; qc, quadrate cartilage; sp, splenial; su, surangular; to, tongue; tr, trabeculae; trg, trigeminal ganglia; II , optic nerve; III, oculomotor nerve; IV,

trochlear nerve. Stain: Haematoxylin and eosin. Scale: 3 mm.

168x199mm (300 x 300 DPI)

Page 44 of 47



Draft

Figure 03. Skull of Melanosuchus niger. (A) stage 15, (B) stage 16, (C) stage 21, (D) stage 22, (E) beginning of stage 24, (F) stage 28. Lateral view. Abbreviations: an, angular; co, coronoid; de, dentary; fr, frontal; ju, jugal; la, lacrimal; ma, maxilla; na, nasal; pfr, prefrontal; po, postorbital; pp, palpebral; prm,

premaxilla; pt, pterygoid; qj, quadratojugal; qu, quadrate; sq, squamosal; su, surangular. Diaphanized with KOH and stained with alizarin red S and alcian blue. Scale: 10 mm.

198x292mm (300 x 300 DPI)

Page 45 of 47



Draft

Figure 04. Skull of Melanosuchus niger. (A) lateral view, beginning of stage 16, (B) lateral view, end of stage 16, (C) dorsal view, end of stage 16, (D) lateral view, stage 18, (E) ventral view, stage 18, (F) dorsolateral view, stage 24, (G) lateral view, with details of (H), stage 24. Abbreviations: an, angular; bs, basisphenoid; co, coronoid; col, columella; de, dentary; ex, exoccipital; fr, frontal; hy, hyoid; ju, jugal; la, lacrimal; lat,

laterosphenoid; ma, maxilla; na, nasal; ops, opisthotic; par, parasphenoid; pfr, prefrontal; pl, palatine; po, postorbital; prm, premaxilla; pt, pterygoid; qj, quadratojugal; qu, quadrate; sq, squamosal; su, surangular.

Diaphanized with KOH and stained with alizarin red S and alcian blue. Scale: 10 mm.

202x194mm (300 x 300 DPI)

Page 46 of 47



Draft

Figure 05. Skull of Melanosuchus niger. (A) stage 15, (B) stage 16, (C) stage 22, (D) end of stage 24, (E) stage 19, (F) stage 20, (G) stage 22, (H) stage 26, (I) 42 days after hatching. Dorsal view. Abbreviations:

bo, basioccipital; bs, basisphenoid; ex, exoccipital; fr, frontal; ju, jugal; la, lacrimal; ma, maxilla; na, nasal; pa, parietal; pfr, prefrontal; po, postorbital; pp, palpebral; prm, premaxilla; pt, pterygoid; so,

supraoccipital; sq, squamosal. Diaphanized with KOH and stained with alizarin red S and alcian blue. Scale: A, B, E, F, G and H 10 mm, C and D 5 mm.

203x259mm (300 x 300 DPI)

Page 47 of 47



Draft

Figure 06. Skull of Melanosuchus niger. (A) stage 15, (B) stage 16, (C) beginning of stage 19, (D) end of stage 20, (E) beginning of stage 21, (F) beginning of stage 25, (G) end of stage 25 (H) 42 days after

hatching. Ventral view. Abbreviations: an, angular; bo, basioccipital; bs, basisphenoid; de, dentary; ec, ectopterygoid; ex, exoccipital; hy, hyoid; ju, jugal; ma, maxilla; pl, palatine; prm, premaxilla; pr, prootic; pt, pterygoid; sp, splenial. Diaphanized with KOH and stained with alizarin red S and alcian blue. Scale: 10

mm.

202x259mm (300 x 300 DPI)

Page 48 of 47



draft · 2019. 2. 7. · draft 2 ontogeny of the skull of melanosuchus niger (crocodylia:...

Documents