Comparative analysis of the integument of different tree frog species from Ololygon and Scinax genera ( Anura : Hylidae )

The integuments of ten treefrog species of two genera from Scinaxnae – O. angrensis (Lutz, 1973), O. flavoguttata (Lutz & Lutz, 1939), O. humilis (Lutz & Lutz, 1954), O. perpusilla (Lutz & Lutz, 1939), O. v-signata (Lutz, 1968), Scinax hayii (Barbour, 1909), S. similis (Cochran, 1952), O. trapicheroi (Lutz & Lutz, 1954) and S. x-signatus (Spix, 1824) – were investigated using conventional and histochemical techniques of light microscopy, and polarized light microscopy. All integuments showed the basic structure of the anuran integument. Moreover, the secretory portions of exocrine glands, such as serous merocrine and apocrine glands, were found to be restricted to the spongious dermis. Lipid content occurred together with the heterogeneous secretory material of the glands with an apocrine secretion mechanism. In addition, clusters of these apocrine glands were present in the ventrolateral integument of some species. Melanophores were also visualized in all examined hylids. However, the occurrence of iridophores, detected through polarized light microscopy, varied according to the species. The Eberth-Katschenko layer occurred in the dorsal integument from both genera, but it was only present in the ventral integument of O. albicans, O. angrensis, O. flavoguttata, O. perpusilla and O. v-signata. Although the integument of all treefrogs showed the same basic structure, some characteristics were genus-specific; however, these features alone may not be used to distinguish both genera.


INTRODUCTION
Many challenges confront biologists studying amphibians, since human activities have been prejudicial to natural biota.Hylidae is a large anuran family, and one of the most abundant and prominent groups of frogs in the Neotropics.Overall, 345 species of Hylidae are known to existin Brazil (SBH 2016).
In adult anurans, the integument consists of two firmly attached layers: the epidermis and the dermis, which is located just beneath the epidermis.The epidermis, formed by a stratified squamous epithelium, overlies the dermis of connective tissue, which is subdivided into two layers: the spongious dermis and the compact dermis.The spongious dermis is formed by loose connective tissue with pigment cells, such as melanophores (melanin producing cells) and iridophores (with reflective or iridescent structures) located just beneath the basal lamina (Bagnara et al. 1968).In addition to the pigment cells, blood vessels, alveolar mucous, serous, mixed and granular glands also occur.The compact dermis is composed of a series of alternating layers of collagenous fiber bundles arranged in a crisscross manner (Fox 1986a, b, Azevedo et al. 2005, de Brito-Gitirana and Azevedo 2005).Nevertheless, integument structure varies according to the body region (de Brito-Gitirana and Azevedo 2005, Felsemburgh et al. 2007).In addition, different types of exocrine glands occur in the anuran integument, withtheir secretory portions restricted to the spongious dermis (Duellmann andTrueb 1994, Brizzi et al. 2002).
In general, anurans show a wide array of colors related to specialized cells named chromatophores.Color and reflectivity are important mechanisms that allow anurans to change their integument color, enabling the maintenance of body temperature and avoiding detection by predators (Duellman and Trueb 1994).Three types of chromatophores are important for amphibian coloration: melanophores (with a black or brownish pigment named melanin), xantophores (with yellow colored pigments), and iridophores (with reflective or iridescent structures).These pigment cells are located in the spongious dermis just beneath the basal lamina, and their arrangement was called the Dermal Chromatophore Unit (DCU) (Bagnara et al. 1968).However, the exact mechanism of DCU organization is still unknown.Some anurans, like arboreal frogs, exhibit diverse morphology in their typical integument outline, showing cutaneous adaptations to avoid evaporative water loss (Blaylock et al. 1976, Warburg et al. 2000, Barbeau and Lillywhite 2005).Such adaptations include exocrine glands secreting lipids, which are spread over the body surface, aided by an elaborate series of behavioral movements to form a useful barrier against water loss.In addition, the histological aspects of the anuran integument can contribute to the differentiation of anuran species from Proceratophrys Miranda-Ribeiro, 1920 (Felsemburgh et al. 2007).
The abundant fauna of amphibians of Brazil (SBH 2016) contrast with the present loss and degradation of natural habitats and the scarce knowledge on the morphological, taxonomical, biological, and geographical distribution of most species.The aim of the present work was to investigate the morphological features of the integuments of some hylids in order to characterize this tissue and determine whetherthere are species-specific structures, contributing to the knowledge of the integument biology.

MATERIAL AND METHODS
Nine species of male adult tree frogs belonging to Scinaxinae from both Ololygon Fitzinger, 1843 and Scinax Wagler, 1830 genera were collected in Serra dos Órgãos National Park (PARNASO,22°29'31"S,42°59'11.48"W), in the municipality of Teresópolis, during two years (from March 2007to November 2008).Ololygon angrensis (Lutz, 1973)   Samplings were carried out at night from 7 to 10 pm and all individuals collected (Figs 1-10) were carefully placed in small humid plastic bags, transported from the collectionsite to the field laboratory with a walk of no longer than one hour.They were kept in captivity until the next morning, when the fixation procedures were carried out.Individuals were euthanized with 0.5% xylocaine according to regulations of the Federal Council of Veterinary Medicine (Law 5.517/68 article 16, "f").The animals were fixed in 10% formaldehyde and subsequently maintained in 70% (v/v) alcohol.At the end of field expedition, the treefrogs were taken to the laboratory, to undergo histological analysis.
For light microscopic (LM) analysis, 3-5 mm thick sections from the ventral, ventrolateraland dorsal regions of the integument were processed according to standard histological techniques for paraffin embedding before sectioning, i.e., the sections were quickly washed in water, dehydrated (70%, 90%, twice in 100% ethanol; 30 minutes each), clarified twice in xylene (30 minutes each), infiltrated and embedded in paraffin.The 5-µm thick serial histological sections were stained with hematoxylin-eosin (HE) (Lillie and Fulmer 1976), which is the standard stain for histological examination of animal tissues, staining thenuclei and cytoplasm in blue with the extracellular matrix in pale pink.Mallory's trichrome (Lillie and Fulmer 1976) was used since it is a good stain for distinguishing cellular from extracellular elements and is especially suitable for studying connective tissue, staining the collagenous fibers in blue, red blood cells in orange, and nuclei in red.Staining with 1% Alcian blue (AB) 8GX at pH 2.5 (Mowry 1963, Kiernan 1990) was utilized to detect sulfated and carboxylated glycoconjugates (in light blue).In addition, HE-stained slices were observed under a light microscopeusing polarized light in order to detect iridophores (an iridescent cell), since these cell types possess reflective structures (Bagnara et al. 1968) that can be easily recognized under polarized light.Sections were analyzed using a Leica DM750 microscope, and the images were captured using a Leica DFC452 digital camera.

RESULTS
In general, the integuments of all species showed the basic morphological structure of the anuran integument, essentially being formed by an epidermal and dermal layer .
The epidermis was relatively thin and consisted of a partially keratinized stratified squamous epithelium supported by a dermis.Although the keratinocyte was the predominant cell type, native flask cells were also visualized.In addition, epidermal cells were organized into a basal layer, an intermediate layer and an outermost layer.The epidermal cells of the outermost layer were partially keratinized, since their nuclear profiles could be easily visualized.
The dermis was subdivided into the spongious dermis, composed of loose connective tissue, and the compact dermis, which rested on the hypodermis.Moreover, the compact dermis was formed by collagenous fibers organized in a series of alternating layers, compactly arranged in a crisscross manner.Between the spongious dermis and the compact dermis, irregular basophilic deposits occurred scattered through this boundary region, corresponding to the Eberth-Katschenko (EK) layer.
Another typical feature of these treefrogs was the occurrence of different exocrine glands, whose secretory portions were housed in the spongious dermis .From the secretory portion, a single unbranched excretory duct passed over the epidermis and opened on the integument surface.These ducts were lined by two layers of cuboid cells.13) Ventral region (AB-method).In all integument regions, the epidermis (E) rests on the dermis, which is subdivided into the spongious dermis (SD) and the compact dermis (CD).Iridophores (→) occur in the dorsal region; however, they are absent in both ventrolateral and ventral regions.Melanophores (_) in the spongious dermis.Both serous (Ø) and apocrine glands (¬) occur in the spongious dermis.No glandular cell reacts to AB-method, suggesting that secretory units is made up of serous cells.The EK-layer (AE) exhibits its typical basophilic staining.Note clusters of apocrine glands (¬) with heterogeneous content in the ventrolateral integument.The EK-layer () exhibits typical alcianophilic reaction of its glycoconjugate content.Large blood vessels occur in the hypodermis.17) Ventral region (AB-method).The melanophores (_) are numerous and located just beneath of the epidermis as well as around de glandular secretory units.They occur also in the hypodermis, but absent in the ventral integument.Iridophores (→) occur in the spongious dermis of the dorsal region just beneath the epidermis.No iridophore is visualized in the ventral region of the integument.Clusters of apocrine glands (¬) with heterogeneous intake predominate at ventrolateral integument; inset of Fig. 15: Observe the granular content with dense stained core () intermingled with cytoplasm material (*).The EK-layer () is continuous in the ventral region, but discontinuous in the ventral integument.Serous glands (Ø) occur in all body regions.Note blood vessels (AE) in the spongious dermis.CD = compact dermis.
Depending on the dye affinity to their secretion, the merocrine glands of the serous and mixed types were visualized.Serous glands are formed by only serous-secreting cells, which have spherical nuclei and an acidophilic cytoplasm.Mixed secretory units are made up of mucous cells (nuclei generally flattened and displaced to the basal portion of the secretory cells and basophilic cytoplasm) and serous cells.It is noteworthy that mucous glands essentially formed by mucous cells did not occur in the integuments of all tree frogs (Table 3 and Figs 11-55).
Considering the apocrine glands, their secretory portion consisted of syncytial units, varying according to their content and dye affinity.Their secretory products were made up of small (20) Ventrolateral region (HE-staining) (21) Ventral region (HE-staining).The epidermis (E) is partially keratinized, and the outermost cell layer exhibits the nuclear profiles (→).Melanophores (_) occur in the spongious dermis of both dorsal and ventrolateral regions; they are more frequent in the dorsal integument.Small clusters of apocrine glands with heterogeneous content occur in the ventrolateral integument.In the ventral region, the lipid content (ë), mixed with basophilic material, is easily visualized in the apocrine glands (Fig. 21).In the dorsal region, the EK-layer () is continuous, but discontinuous in the ventrolateral integument, being less alcianophilic in the ventral region when compared to other integument regions.CD = compact dermis; H = hypodermis.acidophilic granules, seemingly due to their protein content.Nevertheless, some apocrine glands revealed that this secretion is a mixture of basophilic and lipid contents, intermingled with cytoplasmic material; their rounded nuclei were displaced to the cell basal domain.In some glands, the secretory product was constituted of acidophilic cytoplasmic material mixed with heterogeneous material, which revealed slight basophilic reaction usually associated with lipid material (Table 4).
In O. angrensis, the secretory product of the apocrine glands exhibited basophilic granules with an acidophil core after staining with Mallory´s trichrome (Figs 14-17).Furthermore, these secretory granules were also observed in S. similis  and contained lipids associated with basophilic material.We suggest that the basophilic staining was due to carbohydrates of glycolipids, since they showed no alcianophilic reaction typical of acid polysaccharides.In O. albicans, O. angrensis, O. flavoguttata and S. hayii, apocrine glands with heterogeneous content were more frequent in the ventrolateral integument, occurring as small clusters .
Pigment cells, such as melanophores and iridophores, were identified in the integument, occurring in the spongious dermis, just beneath the basal lamina.While melanophores were identified through the typical brownish color of their melanin granules under light microscopy, iridophores were visualized by polarized light microscopy through their reflective or iridescent pigments .Melanophores also occurred in the hypodermis of O. angrensis, O. v-signata and S. similis (36)(37)(38)(39)(40)(52)(53)(54)(55), while iridophores were only detected in the hypodermis of S. similis.The occurrence of both pigment cells varied according to the species and integument region (Table 5).
The Eberth-Katschenko (EK) layer occurred between the spongious and compact dermis and was recognized through its typical basophilic and alcianophilic reaction after using the HE-and AB-methods, respectively (  (34) Ventrolateral region (HE-staining) (35) Ventral region (AB-method).The spongious dermis houses both apocrine glands with heterogeneous content (¬) and serous glands (Ø).Melanophores (_) occur in the dorsal integument, but they are not identified in either the ventrolateral or ventral region.Iridophores did not occur in all integument regions.The EK-layer (AE) is a well defined continuous layer occurs as irregular deposits between the spongious and compact dermis of the ventral region.Cutaneous elevations (â) are separated by grooves (Ú) in the ventral region.CD = compact dermis.trapicheroi and S. x-signatus (Figs 14-21, 32-40, 52-55).These slight elevations were formed by the epidermis, followed by the dermis, being separated by grooves.
In this study, the integument showed the basic structure as already described for other anurans, i.e., the epidermis rests on a dermis, which is divided into a spongious and a compact dermis.The majority of anurans display this structural pattern of the integument (Elkan 1968, Goniakowska-Witalinska and Kubiczek 1998, Warburg et al. 2000, de Brito-Gitirana and Azevedo 2005, Delfino et al. 2006, Felsemburgh et al. 2007, Gonçalves and de Brito-Gitirana 2008, Felsemburgh et al. 2009).
In general, the epidermis varied from 4-5 cell layers.However, in O. humilis and S. x-signatus the ventral epidermis was thicker, and it may be related to species habitat.Nevertheless, Melanophores (_) and iridophores (→) organized as chromatophore units occur in both dorsal and ventrolateral integument.Iridophores exhibit alcianophilic reaction.Note serous glands (Ø) and apocrine glands with granular content (¬) in the spongious dermis.In the ventral region, cutaneous elevations (¬) are separated by prominent grooves (Ú).The EK-layer (AE) occur in the dorsal integument but not in the ventral integument.Moreover, the epidermis (E) of the ventral region is more developed than other integument regions.E = epidermis; CD = compact dermis; H = hypodermis.
we did not find detailed behavioral data in the literature to support this explanation.In mammals, in some body areas, friction and other forces dictate the thickness of the lining epithelium, since the number of epithelial cell layers is related to epithelial resistance (Ham 1977, de Brito-Gitirana 2015).
Anuran glands have received significant attention.They have been described as being of different types, like mucous, serous, lipid (or wax), and mixed (seromucous) glands.However, some authors have named the granular glands as poison or serous glands (Mills and Prum 1984, Duellmann and Trueb 1994, Brizzi et al. 2002).
In this study, well-established histological criteria to categorize the cutaneous gland of mammals (Ham 1977, Kierzenbaum 2004) were used in order to adopt a coherent histological classification, especially in reference to anuran glands, since they exhibit variable morphology.Thus, on the basis of how their secretory products are released to the external environment, the exocrine glands can be classified as holocrine, apocrine or merocrine.Holocrine glands release both secretions and entire cells, while apocrine glands release the secretory product and cytoplasmic matrix of the apical portion of the cell.In merocrine cells, no cytoplasm is lost and the gland uses exocytosis  45) Ventral region (AB-method).Melanophores (_) occur in the spongious dermis of the dorsal integument, and in the ventrolateral region as isolated groups.They are absent in the ventral integument.In the dorsal region, the EK-layer (AE) is continuous and well stained by the AB-method.Isolated serous glands (Ø) occur in all integument regions.The apocrine glands with granular content (¬) are visualized in the ventrolateral integument, where they are more developed.In the ventral region, cutaneous elevations (â) are also separated by grooves.The dermis contains several small blood vessels.E= epidermis; CD = compact dermis.to release its secretory product to the extracellular space (Ham 1977, Kierzenbaum 2004).
Given that the secretory cell remains intact, according to the type of secretionproduced, the merocrine gland of mammals is classified as serous, mucous or mixed.Serous glands are essentially composed of serous cells with large spherical nuclei and an acidophilic cytoplasm that is individualized by the cell membrane.Mucous glands are composed by the mucus-secreting cells, which exhibit an irregular shape; the nuclei are basally located, and their cytoplasm is basophilic (through HE-staining), alcianophilic (through AB-staining to detect acid glycoconjugates), and/or PAS positive (detect neutral glycoprotein).Mixed glands are made up of both serous and mucous cells constituting the same secretory portion (Ham 1977, Kierszenbaum 2004, de Brito-Gitirana 2013).
Actually, the granular gland described by Fox (1986a, b) and Duellman and Trueb (1994) is a kind of apocrine gland since its secretory product is a mixture of cytoplasm and secretory product.Furthermore, this glandular type is composed of a syncytium since the large mass of cytoplasm with many nuclei is not separated into individual cells by a cell membrane.In fact, the apocrine glands of anurans exhibit a high level of polymorphism.
According to Warburg and co-workers (2000), lipid content represents the main adaptation of xeric-inhabiting arboreal frogs, enabling them to remain exposed throughout the year, even during dry seasons.Cutaneous lipids in tree frogs -Phyl-  55) Ventral region (Mallory´s trichrome staining).The epidermis (E) is slightly ticker when compared to those of other hylids, as in the compact dermis (CD).Melanophores (_) are visulized in both dorsal and ventrolateral integument just beneath the epidermis.Serous glands are present in all regions; however, some of them show slightly alcianophilic content (→) in both ventrolateral and ventral regions.The apocrine glands (¬) with granular content occur in both dorsal and ventrolateral regions.The EK-layer (AE) is visualized in both dorsal and ventrolateral integument but is absent in the ventral integument.lomedusa sauvagei Boulenger, 1882, P. iherengii Boulenger, 1885, P. boliviana Boulenger, 1902, Ranoid gracilenta (Peters, 1869), R. caerulea (White, 1790), Polypedates maculatus (Gray, 1830) -are a specialized adaptation to reduce dehydration in arid environments (Amey and Grigg 1995, Lillywhite 2004, Barbeau and Lillywhite 2005, Gomez et al. 2006).These lipids are spread over the body by complex self-wiping behavior to form an effective barrier that reduces evaporative water loss (Barbeau and Lillywhite 2005).
In this study, clusters of glands were observed in the ventrolateral integument of O. albicans, O. angrensis, O. flavoguttata, S. hayii.These glandular accumulations are probably present in a specialized region of the integument that provides special functions.
In all hylids, examined in this study, melanophores occurred in the dorsal integument, but they did not always occur in the ventral integument.In contrast to melanophores, iridophores were visualized only in the dorsal region of O. albicans, O. angrensis, O humilis, and S. similis, while they occurred only in the ventral integument of S. albicans.
In all examined species, at least in the dorsal region of the integument, the Eberth-Katschenko (EK) layer was visualized as an acellular layer that was restricted to a region between the spongious and compact dermis.Moreover, the EK-layer was usually continuous in the dorsal integument, showing its typical basophilic and alcianophilic stainings, which were due to the glycoconjugate content.In R. icterica and L. catesbeianus, the EK-layer contained both dermatan sulfate and calcium, and occurred as scattered aggregates throughout the spongious dermis (Pelli et al. 2007(Pelli et al. , 2010)).These mineral consists of calcium phosphate deposits (Katchburian et al. 2001).Moreover, calcium of the EK-layer was more concentrated in the dorsal integument of male toads, but no significant difference was detected in the integument of females (Azevedo et al. 2005).Elkan (1968) suggested that the absence of the EK layer in some anuran species may be correlated with the fixative type and storage time.Nevertheless, in this study, all hylids were fixed in the same manner, using the same fixative, and the presence or absence of the EK-layer varied according to the specimen and integument region.In addition, the EK-layer was absent in the ventral region of the integument of the species of Scinax, probably being a genus-specific feature.For some authors (Katchburian et al. 2001, Mangione et al. 2011), the EK-layer might be a remnant of an ancestral dermal skeleton.Toledo and Jared (1993) suggested that the calcium located in the EK layer participates in hydric balance, affecting the hydric absorption and retention.On the other hand, Azevedo and et al. (2007) demonstrated that hyaluronic acid (HA) occurs in the spongious dermis, suggesting that the entire spongious dermis acts as a hydric reservoir since HA, an important component of connective tissue matrices, is involved in promoting matrix assembly, tissue hydration and viscosity of some fluids (Laurent et al. 1996).However, the functional significance of the presence of calcium in the EK-layer remains unclear.
Although the usual patterns observed in the Ololygon and Scinax species, their integuments revealed histological characteristics.Thus, histological methods can be efficient to help characterize and differentiate of anuran integuments, thereby improving their taxonomy.
Figures 11-13.Light micrograph of the integument of O. albicans: (11) Dorsal region (HE-staining); (12) Ventrolateral region (HE-staining); (13) Ventral region (AB-method).In all integument regions, the epidermis (E) rests on the dermis, which is subdivided into the spongious dermis (SD) and the compact dermis (CD).Iridophores (→) occur in the dorsal region; however, they are absent in both ventrolateral and ventral regions.Melanophores (_) in the spongious dermis.Both serous (Ø) and apocrine glands (¬) occur in the spongious dermis.No glandular cell reacts to AB-method, suggesting that secretory units is made up of serous cells.The EK-layer (AE) exhibits its typical basophilic staining.Note clusters of apocrine glands (¬) with heterogeneous content in the ventrolateral integument.The EK-layer () exhibits typical alcianophilic reaction of its glycoconjugate content.Large blood vessels occur in the hypodermis.

Figures
Figures 14-17.Light micrograph of the integument of O. angrensis: (14) Dorsal region (AB-staining); (15) Ventrolateral region (HE-staining), inset (Mallory´s trichrome staining); (16) Ventral region (HE-staining); (17) Ventral region (AB-method).The melanophores (_) are numerous and located just beneath of the epidermis as well as around de glandular secretory units.They occur also in the hypodermis, but absent in the ventral integument.Iridophores (→) occur in the spongious dermis of the dorsal region just beneath the epidermis.No iridophore is visualized in the ventral region of the integument.Clusters of apocrine glands (¬) with heterogeneous intake predominate at ventrolateral integument; inset of Fig.15: Observe the granular content with dense stained core () intermingled with cytoplasm material (*).The EK-layer () is continuous in the ventral region, but discontinuous in the ventral integument.Serous glands (Ø) occur in all body regions.Note blood vessels (AE) in the spongious dermis.CD = compact dermis.

Figures
Figures 18-21.Light micrograph of the integument of O. flavoguttata: (18) Dorsal region (HE-staining); (19) Dorsal region (AB-method);(20) Ventrolateral region (HE-staining) (21) Ventral region (HE-staining).The epidermis (E) is partially keratinized, and the outermost cell layer exhibits the nuclear profiles (→).Melanophores (_) occur in the spongious dermis of both dorsal and ventrolateral regions; they are more frequent in the dorsal integument.Small clusters of apocrine glands with heterogeneous content occur in the ventrolateral integument.In the ventral region, the lipid content (ë), mixed with basophilic material, is easily visualized in the apocrine glands (Fig.21).In the dorsal region, the EK-layer () is continuous, but discontinuous in the ventrolateral integument, being less alcianophilic in the ventral region when compared to other integument regions.CD = compact dermis; H = hypodermis.

Figures
Figures 27-31.Light micrograph of the integument of O. humilis: (27) Dorsal region (HE-staining); (28) Dorsal region (AB-method);(29) Ventrolateral region (HE-staining) (30) Ventral region (Mallory´s trichrome staining); (31) Ventral region (AB-method).In the dorsal region, the spongious dermis is poorly developed.Melanophores (_) are visualized in all integument regions; however, iridophores (→) are visualized only in both dorsal and ventrolateral integument.Both pigment cells are located just beneath the epidermis.Alcianophilic reaction is observed in cytoplasm of iridophores as well as in the EK-layer (AE) of the dorsal integument.The EK-layer is absent in the ventral integument.Apocrine glands with heterogeneous content (¬) occur in both ventrolateral and ventral integument.In S. humilis, mixed glands (Ú) are visualized in the ventral region, being formed by serous and mucous cells.Mucous cells exhibit alcianophilic reaction.E = epidermis; CD = compact dermis.

Figures
Figures 32-35.Light micrograph of the integument of O. perpusilla: (32) Dorsal region (HE-staining); (33) Dorsal region (AB-method);(34) Ventrolateral region (HE-staining) (35) Ventral region (AB-method).The spongious dermis houses both apocrine glands with heterogeneous content (¬) and serous glands (Ø).Melanophores (_) occur in the dorsal integument, but they are not identified in either the ventrolateral or ventral region.Iridophores did not occur in all integument regions.The EK-layer (AE) is a well defined continuous layer occurs as irregular deposits between the spongious and compact dermis of the ventral region.Cutaneous elevations (â) are separated by grooves (Ú) in the ventral region.CD = compact dermis.
Figures 41-45.Light micrograph of the integument of O. trapicheroi: (41) Dorsal region (Mallory´s trichrome staining); (42) Dorsal region (AB-method); (43) Ventrolateral region (AB-method); (44) Ventral region (HE-staining); (45) Ventral region (AB-method).Melanophores (_) occur in the spongious dermis of the dorsal integument, and in the ventrolateral region as isolated groups.They are absent in the ventral integument.In the dorsal region, the EK-layer (AE) is continuous and well stained by the AB-method.Isolated serous glands (Ø) occur in all integument regions.The apocrine glands with granular content (¬) are visualized in the ventrolateral integument, where they are more developed.In the ventral region, cutaneous elevations (â) are also separated by grooves.The dermis contains several small blood vessels.E= epidermis; CD = compact dermis.
Figures 52-55.Light micrograph of the integument of S. x-signatus: (52) Dorsal region (HE-staining); (53) Dorsal region (AB-method); (54) Ventrolateral region (Mallory´s trichrome staining); (55) Ventral region (Mallory´s trichrome staining).The epidermis (E) is slightly ticker when compared to those of other hylids, as in the compact dermis (CD).Melanophores (_) are visulized in both dorsal and ventrolateral integument just beneath the epidermis.Serous glands are present in all regions; however, some of them show slightly alcianophilic content (→) in both ventrolateral and ventral regions.The apocrine glands (¬) with granular content occur in both dorsal and ventrolateral regions.The EK-layer (AE) is visualized in both dorsal and ventrolateral integument but is absent in the ventral integument.

Table 1 .
List of individuals used in this study.

Table 4 .
Occurence of apocrine glands in the integument of Oloygon spp.and Scinax spp.

Table 3 .
Occurence of exocrine merocrine glands in the integument of Oloygon spp.and Scinax spp.

Table 5 .
Occurence of pigment cells in the integument of Oloygon spp.and Scinax spp.

Table 6 ;
. The EK-layer was visualized in the dorsal region of the integument of all Oloygon species, but absent in all species of Scinax.

Table 6 .
Occurrence of the EK-layer in the integument of Oloygon spp.and Scinax spp.The EK-layer can occur as a continuous or discontinuous layer.