Sequence of chondrocranial development in the oriental fire bellied toad Bombina orientalis

The vertebrate head as a major novelty is directly linked to the evolutionary success of the vertebrates. Sequential information on the embryonic pattern of cartilaginous head development are scarce, but important for the understanding of its evolution. In this study, we use the oriental fire bellied toad, Bombina orientalis, a basal anuran to investigate the sequence and timing of larval cartilaginous development of the head skeleton from the appearance of mesenchymal Anlagen in post‐neurulation stages until the premetamorphic larvae. We use different methodological approaches like classic histology, clearing and staining, and antibody staining to examine the larval skeletal morphology. Our results show that in contrast to other vertebrates, the ceratohyals are the first centers of chondrification. They are followed by the palatoquadrate and the basihyal. The latter later fuses to the ceratohyal and the branchial basket. Anterior elements like Meckel's cartilage and the rostralia are delayed in development and alter the ancestral anterior posterior pattern observed in other vertebrates. The ceratobranchials I–IV, components of the branchial basket, follow this strict anterior–posterior pattern of chondrification as reported in other amphibians. Chondrification of different skeletal elements follows a distinct pattern and the larval skeleton is nearly fully developed at Gosner Stage 28. We provide baseline data on the pattern and timing of early cartilage development in a basal anuran species, which may serve as guidance for further experimental studies in this species as well as an important basis for the understanding of the evolutionary changes in head development among amphibians and vertebrates.

The viscerocranium is derived from the neural crest and comprises the mandibular, hyoid and gill arches and their respective derivatives which form the lower jaw and its supporting structures (Cerny, Horáček, & Olsson, 2006;Kuratani, Matsuo, & Aizawa, 1997). The neurocranium is of mixed origin and encapsulates the brain and the sensory organs (Couly, Coltey, & le Douarin, 1992). Viscerocranium and neurocranium together form the chondrocranium whose elements undergo endochondral ossification in most species during further development (Morriss-Kay, 2001). Chondrocranial development begins, when mesenchymal cells form an aggregation (Hall & Miyake, 1995). These cells then interact with an epithelium, which is always at the site of future skeletogenesis, to initiate a condensation (Hall & Miyake, 2000). The condensed cells start to differentiate into chondroblasts and during further development into chondrocytes.
The chondrocytes undergo maturation which results in cartilage formation (Goldring, Tsuchimochi, & Ijiri, 2006). Predominantly, the cartilaginous chondrocranium is the earliest functional skeletal element to form (Rose, 2009). It enables food acquisition and additional capabilities. Therefore, the proper development of the chondrocranium is fundamental to successful further development.
Investigating the sequential development of cartilaginous elements in a variety of vertebrate species is essential for an understanding of evolutionary changes and for the identification of ontogenetic novelties as well as heterochronic events. The morphology of the chondrocranium of anuran tadpoles has been described in several species (e.g., Candioti, 2007;Candioti, Haas, Altig, & Peixoto, 2017;Haad, Candioti, & Baldo, 2014;Kolenc et al., 2013). Unfortunately, sequential descriptions of cartilage formation from the onset of chondrification until the premetamorphic stage in anurans are scarce (Lukas & Olsson, 2018a;Reiss, 1997) or outdated (Gaupp, 1906;Parker, 1876Parker, , 1879Stephenson, 1951;Stöhr, 1882;van der Westhuizen, 1961). Nevertheless, such investigations are important for questions regarding the origin of tetrapods and the evolution of morphological diversity of anurans. The foundation for this diversity may be laid during the ontogenetic process of cartilage formation.
The morphology of anuran tadpoles has several unique aspects such as the presence of the infrarostral and suprarostral cartilages as anterior parts of the lower and upper jaw, respectively (McDiarmid & Altig, 1999). Tadpoles of the anuran Bombina orientalis (Boulenger, 1890) display a generalized morphology close to the basal state in anurans (Cannatella & De Sá, 1993;McDiarmid & Altig, 1999) and are well suited for studies of the sequence of skeletal development.
B. orientalis belongs to the family Bombinatoridae which is closely related to the Alytidae. Both are part of the Discoglossidae, the second most basal branch of the Anuran phylogenetic tree (Feng et al., 2017). The cranial skeleton of the tadpole of B. orientalis has features typical for Discoglossidae such as the presence of two posterior processes at the pars alaris of the suprarostral cartilage and the reduced urobranchial process (Haas, 2003;Sokol, 1981). In B. orientalis, the suprarostral cartilage articulates with the cornua trabeculae via a synchondrosis (Svensson & Haas, 2005). The larval otic process, one of three processes which anchor the palatoquadrate onto the neurocranium, is flat in B. orientalis tadpoles (Sokol, 1981).
With the present work, we fill in a gap which exists in the scientific record between the description of early Gosner (1960) stages from Go1-20 (Prema, 1981;Sussman & Betz, 1978) and late larval development from Go 35 onward (Hanken & Hall, 1984;Maglia & Púgener, 1998)  The results of the present study form an important baseline for experimental studies of the development of the larval head skeleton in this species. We are for example using B. orientalis and other species to investigate the origin of the rostralia using loss-and gain-offunction methods. The interpretation of results from experimental manipulations requires a very good knowledge of normal development, as given by the present work. From November until February, they were kept at 8 C with minimum food supply to simulate natural behavior. After this cool down, they were kept at 24 C and fed ad libitum until the males started calling.
Mating and egg deposition took place in shallow water. Eggs were collected manually and cultured in 0.1X modified Barth's saline (Klein, 2001). Breeding temperature for different clutches ranged from 18 to 23 C. All embryos and larvae were staged according to the simplified staging table for anuran embryos and larvae (Gosner, 1960) and denominated as "Go stages." Developmental series from defined stages between Go 19 und Go 35 were taken from the clutch. Anesthesia was performed using 1% tricaine methane sulfonate  according to the animal welfare protocols at the Friedrich-Schiller-University Jena. Larvae were fixed in 4% phosphate-buffered formalin (PFA) or in Dent's fixative, depending on the specific further investigation. In total, 196 larvae were used in this study (Table 1). The specimens investigated are listed in Table 1. Slides, cleared-and-stained and whole mount antibody stained larvae are kept at the Institut für Zoologie und Evolutionsforschung, Friedrich-Schiller-University, Jena, Germany.

| Tissue staining
PFA-fixed specimens were used for serial sectioning. They were dehydrated and embedded in paraffin. Serial sectioning was performed using a rotary microtome (Microm, HM 355 S). Serial sections of 7 μm thickness were collected on microscope slides and stained according to Heidenhain's Azan technique (Heidenhain, 1915). Images were taken with an XC10 Olympus camera mounted on an Olympus BX51 microscope operated with dotSlide software. The clearing-andstaining procedure followed the protocol provided by Dingerkus and Uhler (1977) with the exception, that no alizarin red was used due to the absence of bones. Cleared-and-stained specimens were examined with a Zeiss Stemi 11 and images were taken by an attached camera (ColorView) operated by AnalySIS software. Specimens fixed with Dent's fixative were used for whole mount antibody staining. A monoclonal antibody against collagen II (116B3-collagen II, obtained from the Developmental Studies Hybridoma Bank) and Alexa 568 (Thermo Fischer Scientific) as fluorescent secondary antibody were used to specifically stain cartilages. Image stacks (10 μm z-plane, 1 AU) were produced using a confocal laser scanning microscope (LSM 510, Zeiss).

| 3D reconstruction
Images of serial sections were stacked with Fiji (Schindelin et al., 2012, RRID:SCR_002285). Stacks were aligned using the least squares (rigid) and the elastic non-linear block correspondence mode from the TrakEM2 plugin for Fiji . Aligned serial sectioning stacks and CLSM stacks were segmented using the Amira 6.0.1. 3D-analysis software (FEI Visualization Sciences Group, RRID: SCR_007353) for surface rendering. Surfaces were exported to the Wavefront OBJ file format and further processed using Maya 2020 (Autodesk, Inc.). Pictures were rendered using Autodesk Mudbox2017 (Autodesk, Inc.).

| RESULTS
At Go20 B. orientalis, tadpoles are laterally flattened and blood starts to circulate in the gills. The cornea and the tail fin become transparent until Go22 and the tail further elongates (Figure 1a

| Palatoquadrate
The palatoquadrate is part of the jaw apparatus and numerous muscles take their origin from this cartilage. It flanks the neurocranium

| DISCUSSION
We

| Developmental sequence of cartilage formation
As described in X. laevis, the development of the viscerocranium of B. orientalis also does not follow the strict anterior-posterior direction described in other vertebrate taxa (Gaupp, 1906;Gillis et al., 2012;Langille & Hall, 1987;Stöhr, 1882;Warth et al., 2017).  (Haas, 2003). At the anterior tip of the hypobranchial plate, a hypobranchial I is clearly distinguishable, but no syndesmotic connection occurs between hypobranchial I and ceratobranchial I other than described before (Haas, 1997). The presence of an urobranchial process has been reported in B. orientalis (Maglia & Púgener, 1998), but we cannot confirm this in any of the specimens investigated.
The parachordals are among the earliest cartilages to develop in many vertebrates (Langille & Hall, 1987;Stöhr, 1882;Warth et al., 2017). In X. laevis as well as in B. orientalis, they are the second cartilaginous structure of the neurocranium to chondrify. In both species, the parachordals proceed anteriorly to reach the anteriorposterior developing trabeculae cranii, which resembles a pattern seen in sturgeons (Warth et al., 2017). The three processi anchoring the palatoquadrate to the neurocranium develop in a strict anteriorposterior sequence: first the commissura quadratocranialis, second the processus ascendens, which is bombinatorid-typic with a high insertion, and third the flat larval otic process. This specific order of the developing processes of the palatoquadrate was already observed in X. laevis and in Ascaphus truei (Lukas & Olsson, 2018a;Reiss, 1997).

| The ancestral pattern of anuran cartilage formation
The present investigation confirms various discoglossid traits in B. orientalis. Tadpoles possess a suprarostral cartilage with two posterior processes, the suprarostral cartilage articulates with the cornua trabeculae via a synchondrosis which occurs during ontogenesis after both cartilages initially develop separated and a flat larval otic process.
Additionally, an urobranchial process and spiculae I-III are absent.
1 The mesenchymal Anlage of the ceratohyal is the first Anlage to appear during development and the ceratohyal is the first cartilage which chondrifies.
3 The neurocranium-anchoring processes of the palatoquadrate chondrify in anterior-posterior direction. First the commissura quadratocranialis, then the processus ascendens and last the larval otic processus.
4 Ceratobranchials develop in an anterior-posterior sequence.

| CONCLUSIONS
With developmental morphological studies, we gain insights in the

CONFLICT OF INTEREST
The authors declare no potential conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.