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- MATERIALS AND METHODS
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Ceratophryidae represent a monophyletic group of terrestrial and aquatic frogs inhabiting lowlands of South America where they are more diverse in semiarid environments of the Chaco region. Adult morphology of ceratophryids presents some features associated to terrestrial and fossorial life such as hyper-ossified skulls, spade feet for digging, among others. For anurans, different mineralized structures have been described in the integument as calcium reservoirs and related to the terrestrial life and water balance (e.g., the calcified layer and dermal ossifications). We describe the ontogeny of the integument in the three genera of ceratophryids (Chacophrys, Ceratophrys, and Lepidobatrachus) that inhabit in semiarid environments. Data obtained demonstrated the early acquisition of metamorphic transformations in the integument layers in larvae of Ceratophrys cranwelli and Lepidobatrachus spp. and a continuous increment in the thickness of them up to old postmetamorphic stages. The integument of ceratophryids develops calcium deposits as the calcified layer during postmetamorphic stages. Furthermore, dorsal shields are also present in adult stages independently of terrestrial versus aquatic lifestyles. While the calcified layer seems to be a feature of a fully developed integument, in which their layers have acquired the adult thickness, dorsal shields develop at premetamorphic stages in L. llanensis and postmetamorphic individuals of C. cranwelli. In ceratophryids, similar to other studied taxa (e.g., Brachycephalus spp.) dorsal shields develop via an intramembranous ossification in which the calcified layer does not precede its differentiation. Within anurans, the occurrence of dorsal shields in the monophyletic ceratophryids suggested a distinctive evolutionary history in the lineage. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.
The integument of vertebrates is a continuous and heterogeneous cover formed by two layers; the epidermis and the dermis that are different in origin and structural organization (Dhouailly, 2009). This double-layered organization has particular features involving different functions for vertebrate lineages that provide important information for understanding their history, lifestyles, and relationships between organisms and the environment (Felsemburgh et al., 2006). In amphibians, the integument is unique among vertebrates due to its higher permeability with functional consequences such as cutaneous respiration and osmoregulation (Bentley and Yorio, 1976). Furthermore, the presence of mineralization and ossifications associated with the integument has been described as a possible reservoir of calcium associated to water balance (Elkan, 1968; Bentley and Yorio, 1976; Sampson et al., 1987; Toledo and Jared, 1993; Stiffler, 1995; Azevedo et al., 2005; Witzmann et al., 2010).
The presence of calcium in the amphibian integument may be recognized in four types of morphological structures: dermal scales (Zylberberg and Wake, 1990; Castanet et al., 2003); the calcified layer (Elkan, 1968; Toledo and Jared, 1993; Azevedo et al., 2005); osteoderms (Ruibal and Shoemaker, 1984; Vickaryous and Sire, 2009); and dorsal shields (Trueb, 1973; Fabrezi, 2006; Campos et al., 2010).
Scales are only present in caecilians and are formed by mineralized collagen fibers hosted in the stratum spongiosum of the dermis. Bone is absent (Zylberberg and Wake, 1990; Castanet et al., 2003).
The calcified layer has been described in the skin of some frogs (Elkan, 1968). This layer, also known as Eberth–Kastschenko layer or fundamental layer is composed of glycosaminoglycans associated with calcium accumulations forming a thin sheet (continuous or distributed in patches) below the dermis (Elkan, 1968). Spatial and temporal variation has been described within and among species (Elkan, 1968; Toledo and Jared, 1993). The calcified layer has been recorded in species of Alytidae, Bufonidae, Ceratophryidae, Dendrobatidae, Hylidae, Leptodactylidae, Microhylidae, Pelobatidae, Rhacophoridae, and Ranidae (Elkan, 1968; Toledo and Jared, 1993; Azevedo et al., 2005; Fabrezi et al., 2010; Pelli et al., 2010). It was assumed that the calcified layer acts in water balance and protection against desiccation in terrestrial frogs as it has hydrophilic properties by the presence of glycosaminoglycans (Elkan, 1968; Toledo and Jared, 1993; Azevedo et al., 2005); further it is frequent in terrestrial taxa, absent in the aquatic pipids but some aquatic frogs such as Pseudis spp. and Barbourula busuangensis have calcified layer (Elkan, 1968; Fabrezi et al., 2010).
The term osteoderm names, in a broad sense, any ossification hosted in the dermis (Romer, 1956; Strahm and Schwartz, 1977; Zylberberg and Wake, 1990; O'Leary et al., 2004; Scheyer and Sander, 2004). Osteoderms vary greatly in size, shape, structure, ornamentation, and joints (Vickaryous and Hall, 2006). The osteoderms represent hard elements in which bone cells are immersed in an organic matrix which may combine premineralized bone, lamellar bone, or fibrolamellar bone associated with Sharpey's fibers (Vickaryous and Sire, 2009). The anuran osteoderms were described as small flattened elements (up to 3 mm) immersed in the stratum spongiosum of the dorsal dermis of the head and trunk (Ruibal and Shoemaker, 1984). Osteoderms were recorded in Phyllomedusa bicolor, P. vaillanti, Gastrotheca weinlandii, Megophrys nasuta, Hylophractine augusti (Ruibal and Schoemaker, 1984), and Brachycephalus spp. (Campos et al., 2010). Although developmental data are not available for the osteoderms in amphibians, it was assumed that they are the result of metaplastic ossification that occurs by direct transformation of dermal cells in skeletal tissue in the absence of osteoblasts (Ruibal and Shoemaker, 1984; Zylberberg and Castanet, 1985; Zylberberg and Wake 1990; Scheyer and Sander, 2004; Vickaryous and Sire, 2009).
Dorsal shields are unusual among anurans (some dendrobatids, ceratophryids, and in Brachycephalus spp.) and they could be considered larger osteoderms (Trueb, 1973; Ruibal and Shoemacker, 1984; Vickaryous and Sire, 2009). Dorsal shields have been considered as special features of the integument (Moss, 1972; Ruibal and Shoemacker, 1984; Vickaryous and Sire, 2009) and related to terrestrial lifestyles (DeMar, 1966; Fabrezi, 2006; Clemente-Carvalho et al., 2009; Dilkes, 2009; Vickaryous and Sire, 2009; Campos et al., 2010). They are large bony plates (more than 5 mm in diameter) placed on, or fused to, the presacral vertebrae (Lynch, 1971; Trueb, 1973; Ruibal and Shoemaker, 1984; Fabrezi, 2006; Clemente-Carvalho et al., 2009; Campos et al., 2010).
The co-occurrence of the calcified layer and osteoderms in some frogs led to propose that this layer might represent the early stage of differentiation from which dermal skeletal structures develop (Ruibal and Shoemaker, 1984). Differently, Guardabassi (1963) interpreted the calcified layer as the vestiges of the dermal armor of primitive amphibians. Paleontological data recorded dorsal bony armors in different lineages of dissorophid temnospondyl of the Carboniferous and the Triassic (DeMar, 1966; Dilkes and Brown, 2007; Dilkes, 2009; Buchwitz and Voigt, 2010; Witzmann et al., 2010) which, together with other features (e.g., strong and hyperossified skeletons), suggested a scenario of terrestrial and semiarid conditions in which the thickening of the integument involved a minor loss of moisture through the skin (DeMar, 1966).
From these data, some generalizations emerge: (1) the integument of frogs may present a distinct calcified layer in the dermis; (2) the integument of frogs may present dorsal dermal ossifications immersed in the dermis; (3) the co-occurrence of both types of structures, in some taxa, could suggest that the calcified layer would be the initial stage of osteorderm development; and (4) both types of structures could represent vestiges of the extensive ossification (dermal armors) of the integument of primitive amphibians.
The Ceratophryidae Tschudi, 1838 are considered a distinctive group of neotropical frogs. Much has been discussed about the relationships of these genera but there is a strong consensus for the monophyly of the clade (see Reig and Limeses, 1963; Lynch, 1971; Laurent, 1986; Maxson and Ruibal, 1988; Haas, 2003; Fabrezi, 2006; Frost et al., 2006; Grant et al., 2006, Pyron and Wiens, 2011; among others). The clade is composed of the following taxa: Chacophrys Reig and Limeses, 1963 (one species), Lepidobatrachus Budgett, 1899 (three species), and Ceratophrys Wied-Neuwied, 1824 (eight species). Adults of Chacophrys and Ceratophrys are terrestrial, and adults of Lepidobatrachus are aquatic. Ceratophrys species are distributed in tropical areas of South America, and Ceratophrys cranwelli Barrio, 1980 and Ceratophrys ornata (Bell, 1843) are endemic to the Chaco region and sympatric with Lepidobatrachus species and Chacophrys pierottii (Vellard, 1948). As the Chaco region comprises semiarid environments with wet summers, these frogs are active during this season. In the Chaco, ceratophryids produce an epidermal cocoon during the dry season that reduces water loss and protects them until the next rainy season (Ruibal and Shoemaker, 1984; McClanahan et al., 1994).
Among the Ceratophryidae, dorsal shields are formed by a variable number of bony plates resting on the flattened neural spines of the presacral vertebrae; these shields are attached to the vertebral skeleton by ligaments (Fabrezi, 2006). Within ceratophryids, dorsal shields show a remarkable morphological and ontogenetic variation. In Ceratophrys, dorsal shields have been described in C. cranwelli, C. aurita, and C. ornata, and become differentiated at postmetamorphic stages (Lynch, 1971; 1982; Wild, 1997; Fabrezi, 2006). In these species, dorsal shields form a dorsal armor in which medial and lateral shields cover vertebrae II–VII and their transverse processes (Fabrezi, 2006). The number of shields is variable with three to five medial shields and three or more non-symmetric pairs of lateral shields. In Lepidobatrachus the shields are present in L. asper and L. llanensis with a smaller size and formed only by one or two shields of medial position which develop at larval stages in L. llanensis (Reig and Cei, 1963; Lynch, 1971; 1982; Fabrezi, 2006).
Consequently, the Ceratophryidae—being monophyletic, sharing similar habitats, and differing in lifestyles—represent an excellent model to explore the variation of the integument and test the generalizations mentioned above. For that, we present the study of morphological and histological variation in the integument during larval and adults stages and focus on mineralization and ossifications. We selected species of the three Ceratophryid genera living along the semiarid environments of the South American Chaco (C. pierottii, C. cranwelli, Lepidobatrachus asper, L. laevis and L. llanensis). Data about the presence/absence and timing of differentiation of the calcified layer and dorsal shields allow us to provide new information about the morphological variation to interpret the anuran evolution.
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- MATERIALS AND METHODS
- LITERATURE CITED
Metamorphic transformations from the larval to the adult integument in anurans imply changes associated from the aquatic larval life toward the terrestrial life of the adults. These transformations are generalized and described as events limited to the metamorphic stages (Duellman and Trueb, 1986; Yoshizato, 1992; Tamakoshi et al., 1998; Brown and Cai, 2007).
In Ceratophryids, the changes that take place in the transformation from larval to adult integument (incremental number of epidermal layers, development of glands, and differentiation of two dermal strata) present variation in the timing of their occurrence. While in C. pierottii these transformations occur at metamorphic stages, like most anurans; in C. cranwelli, L. laevis, and L. llanensis they are pre-displaced to prometamorphic stages, like other larval features (Fabrezi and Quinzio, 2008) (Fig. 1). With differences in the temporal sequence of these transformations, ceratophryids share a dorsal to ventral sequence of changes which is evidenced by the earlier appearance of advanced characteristics in the dorsal skin (Fig. 1). In spite of the early acquisition of the complete structural configuration in the skin, the thickness of the different strata of the integument in Ceratophryids presents a continuous increment up to advanced postmetamorphic stages (Table 1).
The mineralized structures related to the adult integument in anurans (calcified layer and dorsal shields) also present variation in the timing of differentiation among ceratophryids. The calcified layer is present only in the adult integument, and while dorsal shields develop at prometamorphic stages in L. llanensis its differentiation takes place in adult stages of C. cranwelli.
The calcified layer of the dermis has been described as a structure of frog skin whose absence in some aquatic taxa (Crinia signifera, in Myobatrachidae, Telmatobius, in Telmatobiidae; and Pipidae) led to propose that this layer is a feature of terrestrial anurans (Elkan, 1968; Toledo and Jared, 1993; Azevedo et al., 2005; Pelli et al., 2010). However, the calcified layer has been described in Barbourula busuangensis and in Pseudis spp. (Elkan, 1968) in which it is already completely differentiated at the end of the metamorphosis (Fabrezi et al., 2010). Furthermore, the aquatic Lepidobatrachus spp. also have calcified layer. Then, the calcified layer is present in certain aquatic species as well as it is absent in terrestrial taxa [e.g. Hyperolius spp., Rhinophrynus dorsalis, Limnodynastes dorsalis (Elkan, 1968)].
Different to those descriptions in Pseudis paradoxa (as P. platensis) presented by Fabrezi et al., (2010) and Quinzio (2011) in which the skin of the recently metamorphosed individual is identical to that of the adult, including the calcified layer, the calcified layer in ceratophryids differentiates during postmetamophic stages when the integument acquires the typical histological organization of the adult (Fig. 2; Table 1). Therefore, at least for the ceratophryids and P. paradoxa, the appearance of the calcified layer occurs when the integument has reached its final configuration.
Elkan (1968) suggested that this layer may be involved in the regulation of water balance due to the physiological properties of it components. This proposal is consistent with the presence of calcified layer for species that live in arid regions with high temperatures where the evaporation rate is high, such as the Chacoan terrestrial species of ceratophryids. However, it is also present in aquatic taxa (e.g. Lepidobatrachus spp. and P. paradoxa). Although other functions as mineral homeostasis, mechanical protection, or against desiccation (Elkan, 1968; Baldwin and Bentley, 1980; Toledo and Jared 1993; Azevedo et al. 2005; Lillywhite 2006) have been proposed for this layer, our findings do not provide arguments to discuss the function of the calcified layer.
Some authors proposed the co-occurrence of the calcified layer and ossifications in the dermis could represent that the calcified layer is the precursor site from which diverse ossifications develop in the skin (Ruibal and Shoemaker, 1984). From this generalization, the presence of the calcified layer should precede the differentiation of the shields but in L. llanensis dorsal shields appear before the calcified layer (Figs. 7 and 8). In C. cranwelli the calcified layer precedes the differentiation of the shields but it is absent in the area of the skin where the dorsal shield will develop. This fact could suggest that the calcified layer is replaced by the shields although the absence of the layer in the integument on the shields is also a feature of L. llanensis.
Development of dorsal shields by intramembranous ossification centers in an antero-posterior direction occurs during prometamorphic larval stages in Lepidobatrachus spp. without rigid link with presacral vertabrae (synchondrosis or synostosis) (Figs. 7 and 8). In C. cranwelli we could infer a sequential addition of ossification centers (beginning by the medial plates) that grow continuously in lateral and caudal directions. This suggestion is supported by the fact that specimens with higher SVL have more plates. Fabrezi and Quinzio (2008) staged individuals of C. cranwelli and found those larger than 75 mm reached 11 years by skeletochronology methods; this fact would suggested the differentiation of dorsal shields in C. cranwelli could occur when minimum size and age are achieved. Furthermore, specimens of C. cranwelli, C. ornata, and C. aurita smaller than 70 mm lack dorsal shields (Lynch, 1982; Ruibal and Shoemaker, 1984).
In Brachycephalus spp., dorsal shields are present and the calcified layer is absent on the skin of the shields (Ruibal and Shoemaker, 1984; Clemente-Carvalho et al., 2009; Campos et al., 2010). In Brachycephalus ephippium the development of dorsal shields occurs in a latero-medial sequence, from paravertebral shields (lateral shields in this study) to spinal shields (medial shields in this study) (Campos et al., 2010). In this species, the shields differentiate independently of the dermis as an intramembranous addition of osseous tissue derived from an osteo-chondrogenic membrane of the perichondry of the presacral vertebrae (Campos et al., 2010). Although ceratophryids and B. ephippium share the intramembranous development of dorsal shields they differ in the origin of the osteoblasts.
The amphibian osteoderms were defined as skeletal elements that develop by metaplasia (Ruibal and Shoemacker, 1984; Zylberberg and Wake, 1990); this definition was based on the histological organization of the adult osteoderm and differs from our findings in which the intramembranous ossification involves the normal differentiation of osteoblasts from mesenchymal cells of the hypodermis. The presence of osteoblasts during osteoderm development has also been described for turtles (Scheyer et al., 2008). Although the term osteoderm is used in tetrapods to identify any ossification related to the integument (Ruibal and Shoemacker, 1984; Zylberberg and Castanet, 1985; Zylberberg and Wake, 1990; Scheyer and Sander, 2004; Vickaryous and Hall, 2006; Vickaryous and Sire, 2009), developmental data are still scarce to provide a useful concept based on homology since dermal ossifications may involve diverse differentiation pathways (intramembranous, metaplastic ossification) and origins (hypodermis, perichondrial outgrowth).
The histological organization in diploë structure might indicate that osseous tissue grow faster in the early stages of development and gradually slow down (Witzmann and Soler-Gijón, 2010). This organization in Ceratophryids could also explain the presence of growth marks in the cortex of the shields.
The integration between the collagen fibers of the stratum compactum with the shield bone matrix (fibers seem to be engulfed by the apical growing of the external cortex of the shield) has similar features to those described for Sharpey's fibers whose function is related to give a strength but flexible attachment between dermal collagen fibers and skull co-ossifications in B. ephippium (Felisbino and Carvalho, 2000; Clemente-Carvalho et al., 2009; Witzmann and Soler-Gijón, 2010); and whose presence was observed in the inner surface of osteoderms in some amphibian temnospondyli [e.g., Gerrothorax and Peltobatrachus (Witzmann and Soler-Gijón, 2010)].
The temporal gap between the fossil amphibians and the origin of the anurans is too long to speculate about the homology of dorsal shields, as they were described in ceratophryids and Brachycephalus spp., with the osteoderms and dorsal armors of paleozoic amphibians. However, some structural resemblance may suggest an ancient and constrained capability of the tetrapod integument to generate mineralizations and ossifications.
In addition, the integument of ceratophryids presents calcium deposits as the calcified layer which develops at adult stages. Independently of this mineralized layer, some Ceratophryids bear dermal ossifications on their presacral vertebrae. These dorsal shields may appear before (Lepidobatrachus spp.) or after (Ceratophrys spp.) the metamorphosis, and vary in number, size, and shape to form a dorsal armor in Ceratophrys. Similar to other studied taxa (e.g., Brachycephalus spp.) calcium deposits and dermal ossifications in the adult skin have evolved in fossil amphibians but are rare among extant anurans. The presence of these calcified structures in the monophyletic lineage of ceratophryids suggested a distinctive evolutionary history.