Morphology of the Parotoid Macroglands in Phyllomedusa Leaf Frogs M

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Journal of Zoology. Print ISSN 0952-8369 Morphology of the parotoid macroglands in Phyllomedusa leaf frogs M. M. Antoniazzi1, P. R. Neves1, P. L. Mailho-Fontana1, M. T. Rodrigues2 & C. Jared1

1 Laboratório de Biologia Celular, Instituto Butantan, São Paulo, Brazil 2 Depto. Zoologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil

Keywords Abstract Amphibia; Anura; Phyllomedusa; parotoid; poison glands; skin. The parotoid macroglands of toads (bufonids) and leaf frogs (hylids) are used in passive defence against predators. The parotoids release poison when the amphib- Correspondence ian is bitten by a predator. Despite the apparent similarity, the anatomical and Carlos Jared, Laboratório de Biologia histological structure of these macroglands in hylids is poorly studied when com- Celular, Instituto Butantan, Av. Vital Brazil, pared with those of bufonids. In this paper, we focused on the morphology of the 1500, CEP: 05503-900 São Paulo, SP, Brazil. macroglands of P. distincta, a leaf frog endemic to the Brazilian Atlantic rainfor- Tel/Fax: 55-11-26279772 est, comparing their structure with those of bufonids. In addition, we compared Email: [email protected] the macrogland morphology of P. distincta with those from major clades of Phyl- lomedusa. All results revealed a macrogland morphology in leaf frogs distinct from Editor: Andrew Kitchener that of toads, suggesting that the term parotoid should be used only for those of bufonids. Received 19 July 2012; revised 25 March 2013; accepted 4 April 2013 doi:10.1111/jzo.12044

Introduction accordingly we restrict the use of the term ‘gland’ for skin mucous, granular and lipid glands, which consist of single Amphibian skin is characterized by the presence of mucous glandular types. glands, mainly associated with respiration and protection Parotoid macroglands are commonly found in anurans, against desiccation, and granular (or poison) glands, which particularly bufonids (Lutz, 1971; Toledo & Jared, 1995; provide an arsenal of chemical compounds used in defence Almeida et al., 2007; Jared et al., 2009, 2011) and hylids (Phyl- against microorganisms and predators (Duellman & Trueb, lomedusinae) (Lutz, 1966), but they are also present in other 1986; Fox, 1986; Toledo & Jared, 1993, 1995; Zug, 1993; amphibians, such as salamanders (Brodie Jr & Gibson, 1969; Stebbins & Cohen, 1995; Clarke, 1997). In addition to these Luther, 1971; Brodie Jr, 1983). They are, in general, poorly single microscopic glands spread over the whole integument, studied, possibly because they are believed to be similar to the poison glands of certain areas of the skin are greatly parotoids of bufonids, for which morphological descriptions enlarged and form accumulations that were named macrog- are more detailed (Toledo et al., 1992; Almeida et al., 2007; lands by Toledo & Jared (1995) in order to differentiate Jared et al., 2009, 2011). them from regular, isolated skin granular glands. The paro- The parotoids of the leaf frogs (Hylidae, Phyllomedusinae) toids, located in the postorbital and supratympanic region, have an apparently similar structure to those of toads, being are the best-known macroglands (Brazil & Vellard, 1925, longer and less conspicuous, but they have never been studied 1926; Wilber & Carroll, 1940; Tronchet, 1952; Lutz, 1971; morphologically. In both toads and leaf frogs, their position Hostetler & Cannon, 1974; Duellman & Trueb, 1986; Toledo suggests association with passive defence, an adaptation to & Villa, 1987; Toledo, Jared & Brunner, 1992; Hutchinson & avoid predation in frontal attacks by predators (Toledo & Savitzky, 2004; Almeida et al., 2007; Jared et al., 2009, Jared, 1995; Jared et al., 2009). In both cases the triggering 2011). Cannon & Palkuti (1976) and Tyler, Burton & Bauer mechanism for poison release seems to be activated when the (2001), based on the topographical anatomy of these glands, predator bites the amphibian (Jared et al., 2009, 2011). This gave etymological reasons for using the term parotoid paper focuses on the morphology of the parotoid macroglands instead of paratoid or parotid and we follow them. In addi- of Phyllomedusa distincta, a leaf frog endemic to the Brazilian tion, following Toledo & Jared (1995), we prefer to use Atlantic rainforest (Frost, 2013). The structural similarity ‘parotoid macrogland’ instead of ‘parotoid gland’ to avoid between the macroglands of P. distincta and those of repre- ambiguity in the identiﬁcation of the cutaneous glands, sentatives of major clades of Phyllomedusa reveals a distinc- because parotoids are clearly multiglandular structures; tive and different organization from those of toads, and leads

42 Journal of Zoology 291 (2013) 42–50 © 2013 The Zoological Society of London M. M. Antoniazzi et al. Parotoid morphology in leaf frogs us to suggest that the use of the term ‘parotoid macrogland’ ments in 4% formaldehyde (made from paraformaldehyde) should be restricted to the bufonids. buffered in 0.1 M phosphate buffer, pH 7.2 (Junqueira, 1995) for 48 h. The skin samples and the macroglands were embed- Materials and methods ded in glycol metachrylate (Leica historesin, Leica TM, Wetzlar, Germany), sectioned 2–4-mm thick and stained with Five specimens of P. distincta Lutz (1950; Fig. 1), collected at toluidine blue-fuchsin. A pair of parotoids was also embedded Estação Biológica de Boracéia, São Paulo State, Brazil, had in paraffin both in transverse and longitudinal orientations, their left parotoid macroglands manually compressed for and the sections were stained with picrosirius and examined by poison release. Subsequently, the animals were sacrificed with polarized microscopy for collagen fibres (Junqueira, Bignolas an overdose of thionembutal, and fragments of the dorsal & Brentani, 1979). Glycol metachrylate sections were also and ventral skin and both entire macroglands were removed, submitted to bromophenol blue, periodic acid-Schiff (PAS) fixed and prepared for light microscopy. Specimens of Phyl- combined with alcian blue pH 2.5 and the von Kossa method lomedusa, P. distincta (MZUSP 35048), P. vaillant (MZUSP (Bancroft & Steven, 1990), for detection of proteins, neutral 86303, MZUSP 86309), P. hypochondrialis (MZUSP 38452), and acid mucosubstances, and calcium, respectively. Some P. burmeisteri (MZUSP 81254), P. tomopterna (MZUSP fresh fragments of the dorsal skin were fixed in 10% formalde- 80910), P. bicolor (MZUSP 66178, MZUSP 66180), P. mega- hyde with calcium chloride 1.3% and sucrose 7.5%, sectioned in cephala (MTR 21354) were used for a comparison of macro- a cryostat after embedding in Jung-Tissue Tec (Leica), and gland microanatomy. stained with Sudan black B, for identification of lipids. Four pairs of parotoids were cut transversely to the longitu- Light micrographs were obtained in a Leica DMLB dinal axis in four pieces and fixed together with the skin frag- microscope, equipped with a MPS 60 photographic system.

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Figure 1 Phyllomedusa distincta (a) Lateral view of an adult specimen showing the whole length of one of the parotoids along the body; (b) Cephalic portion of a parotoid (arrow) forming a postorbital and supratympanic protuberance; (c) Parotoid opened in horizontal plane showing the honeycomb-like arrangement of the alveoli (*).

Polarized microscopy was done in an Olympus BX60 light skin. In the cephalic region, they occur in a postorbital and microscope equipped with PM-C 35 DX photographic system. supratympanic location (Fig. 1a,b), and are quite wide and The anatomy of the parotoids in Phyllomedusa hypochon- prominent. As they extend backwards on both sides of the drialis, P. tomopterna, P. bicolor, P. burmeisteri, P. vaillanti dorsum, they become thinner and eventually end in the pos- and P. megacephala was investigated under a stereomicro- terior dorsal region (Fig. 1a). When longitudinally sectioned, scope after making an incision in the skin. the macroglands show a honeycomb form composed of glan- For comparison, the parotoids of the toad Rhinella marina dular alveoli (Fig. 1c). were studied as a representative species of genus Rhinella. Following similar methods used for P. distincta, two pairs of parotoids of two Rhinella marina collected in Belterra, Pará Skin histology of P. distincta state, Brazil, were dissected, fixed in 4% buffered formalde- The skin of P. distincta is characterized by the presence of hyde and embedded in paraffin. After microtomy, the sections many glands penetrating the dermis; glands are more numer- were stained with haematoxylin-eosin and photographed in an ous dorsally (Fig. 2a). The dorsal skin also possesses a large Olympus BX51 light microscope equipped with a digital number of pigment cells, arranged in characteristic dermal camera and with the software Image-Pro Express (Media chromatophore units (Duellman & Trueb, 1986) (Fig. 2a). Cybernetics, Rockville, MD, USA). The epidermis is thin and composed of four to five cell layers, with a discrete stratum corneum. The dermis is com- Results posed of two layers, the stratum spongiosum, which is more superficial, and the stratum compactum, below. The stratum Parotoid macrogland anatomy of spongiosum is characterized by a layer of pigment cells (mainly in the dorsum), underlying the epidermis, blood vessels, P. distincta and mucous, granular and lipid glands, which are distributed The parotoids form a pair of well-defined and elongated dor- both in the dorsal and ventral skin (Figs 2a–g and 3a–d). The solateral skin structures, which stand out from the rest of the stratum compactum is poorly organized and is mainly

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Figure 2 Phyllomedusa distincta (a) Histo- logical general view of the dorsal skin showing a type 1 mucous gland (M1) and a lipid gland (Li) inserted in the dermis (D). m m 20 m 20 m Below the epidermis (E) the pigmentary cells (P) show a characteristic arrangement. Blood (c) (d) vessel (BV). Toluidine blue-fuchsin staining. (b) Type 1 mucous glands (M1) with secretion in the lumen (*). Toluidine blue-fuchsin staining. (c) Type 2 mucous glands (M2) composed of different cells, some of them with metachromatic granules (arrows). Toluidine blue-fuchsin staining. (d) Type 1 mucous gland (M1) with all secretory cells positive to protein. Bromophenol blue method. (e) Type 2 mucous gland (M2) with a few cells positive 20 mm 20 mm to bromophenol blue (arrows). Bromophenol blue method. (f) Type 2 mucous gland (M2) with all the secretory cells positive to neutral (e) (f) (g) mucopolysaccharides, but only a few cells positive to acid mucopolysaccharides (arrows). PAS, + Alcian Blue pH 2.5 method. (g) Type 1 mucous gland (M1) with all the secretory cells exclusively positive to PAS. 20 mm 20 mm 20 mm PAS + Alcian blue, pH 2.5 method. PAS, periodic acid-Schiff.

All glandular types are enveloped by a monolayer of (a) (b) myoepithelial cells (Fig. 3c) and communicate with the skin surface through epithelial ducts (Figs 2a,g and 3b,d). The mucous glands are spherical and acinar, and are formed by a single layer of secretory cells with basal nuclei and an evident lumen; two distinct types are recognized according to the lumen and secretory cells they possess. Type 1 glands Ϯ 100 mm 20 mm measure about 37.0 2.4 mm in diameter, have a wide lumen and contain only one type of secretory cell (Fig. 2b), which is positive to PAS (Fig. 2g) and bromophenol blue (Fig. 2d). (c) Type 2 glands measure about 34.5 Ϯ 3.5 mm in diameter, have a narrower lumen and are formed by two different cell types (Fig. 2c), both positive to bromophenol blue (Fig. 2e) and PAS, but distinguishable by the secretory granules preferen- tially accumulated in an apical position, which are also positive to alcian blue, pH 2.5 (Fig. 2f). The lipid glands are larger than the mucous glands, with an acinar arrangement and a narrow lumen. The secretory epithelium is formed by a monolayer of columnar cells of only one type, with basal nuclei and granules of irregular size, and showing a pale content when stained with toluidine blue- fuchsin (Fig. 2a). The histochemistry reveals that these granules are highly positive to Sudan black B (Fig. 3a), and slightly 20 mm positive to PAS and alcian blue pH 2.5 (Fig. 3b). The granular glands are larger than the mucous and lipid (d) glands and are flattened, with the larger axis measuring about 166.6 Ϯ 26.3 mm and 54.3 Ϯ 13.2 mm in height. They are syncytial, with peripheral nuclei, and are completely filled with a large number of spherical granules. These granules show low affinity to toluidine blue (Fig. 3c) and are highly positive to bromophenol blue (Fig. 3d).

Parotoid macrogland histology of P. distincta In transverse histological sections, the macroglands are characterized by the accumulation of large, elongated, syncytial granular glands, measuring about 371.4 Ϯ 25.0 mm in diam- m 20 m eter and 1277.8 Ϯ 327.8 mm in height. These are arranged side by side deep in the dermis, below the layer formed by the much Figure 3 Phyllomedusa distincta (a) Lipid glands (Li) are located just smaller regular skin glands (Fig. 4a). In longitudinal sections, below the epidermis (E) and are positive to Sudan Black B method; (b) the macroglands show the circular profiles of the elongated Lipid gland (Li) shows a few cells near to the glandular duct (*) that are granular glands immersed in a homogeneous matrix of con- positive to acid mucopolisaccharides (arrows). PAS + Alcian Blue pH 2.5 nective tissue rich in blood vessels (Fig. 4b). Polarized micro- method; (c) Granular gland (G) with nuclei of the secretory syncytium scopy of transverse and longitudinal sections, stained with (large arrows) and myoepithelial cell layer (thin arrow). Epidermis (E); picrosirius, show that the matrix of connective tissue forms a dermis (D). Toluidine blue-fuchsin staining; (d) Granular gland (G) with framework of collagen fibres mainly of type I, which are rec- the secretory granules positive to bromophenol blue indicating protein ognized by their reddish colour (Fig. 4c). The characteristic content. Dermal stratum spongiosum (Ss) and stratum compactum granular glands that comprise the macroglands, similarly to (Sc); secretory duct (*); myoepithelial layer (arrow). Bromophenol blue the regular skin glands, are enveloped by a myoepithelial method. PAS, periodic acid-Schiff. layer, which seems to be more developed when compared with the other cutaneous glands (Fig. 4d) and with elements in the cytoplasm that are quite positive to PAS (Fig. 4e). The large comprised of collagen fibres. Finally, the innermost skin layer, syncytia are composed of a peripheral region, where the nuclei the subcutaneous tissue, is rich in blood vessels and nerves. are arranged, and of an internal region, full of spherical gran- There is no evidence of the presence of a calcified dermal layer ules morphologically distinct from those observed in the between the stratum spongiosum and the stratum compactum, granular glands present in the rest of the skin; they are loosely even when the von Kossa method is applied. arranged, more spherical and heterogeneous in size, and with

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Figure 4 Phyllomedusa distincta (a) Trans- verse histological section of the macrogland showing the accumulation of large, elongated granular glands (G), making this region much thicker than the rest of the skin. Epidermis 150 mm (E); dermis (D); pigmentary cell layer (P); mucous gland (M); lipid glands (Li); subcutaneous tissue with blood vessels (arrow). (b) (c) Toluidine-blue-fuchsin staining; (b) transverse section of the macrogland with some large granular glands (G) immersed in the connective tissue (Ct) of the dermis. Toluidine-blue- fuchsin staining; (c) polarized microscopy of the macrogland showing the dermal connective tissue mainly composed of collagen type I (Col). Large granular glands (G). Picrosirius staining; (d) detail of a large granular gland of 100 mm 200 mm the macrogland showing the syncytium full of granules (g) and with peripheral nuclei (Sy). (d) (f) Myoepithelial layer (My). Toluidine blue- fuchsin staining; (e) detail of the myoepithelial layer of the large granular gland of the macrogland. The myoepithelial cells are full of granules highly positive to neutral mucopolysaccharides. PAS method. (f) Same region of Fig. 4e, showing the secretory granules (g) highly positive to protein. Periphery of the (e) syncytium (Sy). Myoepithelial layer (arrow). 10 µm1010 mm mm Bromophenol blue method. PAS, periodic acid-Schiff.

a high affinity to toluidine blue-fuchsin (Fig. 4d). Although show that, after compression, some granular syncytia remain positive to bromophenol blue, they are not so intensely stained untouched, while others are observed in different stages of as the granules of the granular glands in the rest of the skin filling (Fig. 5c). Poison release leads to the collapse of glandu- (Fig. 4f). lar myoepithelia and the nucleated peripheral region of the Each secretory unit of the macroglands is connected to the syncytia. Sections of the compressed parotoids observed exterior through an epithelial duct, forming a canal through under polarized microscopy after Picrosirius staining indicate which the secretion is liberated (Fig. 5a,b). Transverse and that no significant changes are seen in the homogeneous longitudinal serial sections of the ducts reveal the flattened arrangement of the collagen fibres that form the glandular shape of the ductal canal and of the ductal wall that is com- framework (Fig. 5d). posed of three layers of epithelial cells (Fig. 5b). The whole length of the ductal canal is open to the passage of the gland’s Parotoid macroglands of other secretion (Fig. 5a); in deeper sections passing through the Phyllomedusa pigment layer some secretory granules were observed inside the ducts (Fig. 5b). All of the analysed species of Phyllomedusa have honeycomb- When a phyllomedusine macrogland is manually com- like alveoli in the dorsolateral area. In Phyllomedusa hypo- pressed, its poisonous secretion is liberated through pores in chondrialis and P. tomopterna the macroglands are not the form of drops on the skin surface. The histological sections prominent and were only detected after an incision of the skin

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20 mm 20 mm Figure 5 Phyllomedusa distincta (a) Longitu- (c) dinal section of the ductal region (*) of a large granular gland (G) of the macrogland. Note the open lumen along the whole length of the duct. Epidermis (E); dermis (D); pigmentary layer (P).Toluidine blue-fuchsin staining. (b) Same region of Fig. 5a in a transverse section through the duct at the level of the pigmentary cells (P). Note the secretory granules (g) inside the lumen. Toluidine blue-fuchsin staining. (c) Manually compressed macrogland. Many large glands are collapsed (G*) while (d) others are full of secretion (G). Dermis (D). Toluidine blue-fuchsin staining. (d) Polarized microscopy of the macrogland after manual compression. The arrangement of the collagen ﬁbers (Col) in the dermis is similar of that observed in the intact macrogland. Large 100 mm 150 mm granular glands of the macrogland (G). Picro- sirius staining.

adjacent to cephalic region. In the other examined species differentiated glands are arranged around the duct (Fig. 6d,e) (Phyllomedusa bicolor, P. burmeisteri, P. vaillanti and similar to those observed in other bufonids (see Jared et al., P. megacephala), macrogland structure is very similar to that 2009, 2011). observed in P. distincta. In contrast to P. distincta, when the parotoids of R. marina and other Rhinella are submitted to manual compression, their poisonous secretion is liberated in the form of jets. His- Parotoid macroglands of Rhinella marina tological sections show that, after compression, poison release The parotoids of R. marina are large and irregular, with a empties many of the large granular glands, causing the total polygonal proﬁle, and restricted to a more anterior position, collapse of several syncytial alveoli (for details, see Jared et al., just behind the tympani (Fig. 6a). The external pores are very 2009 and Mailho-Fontana, 2012). prominent. When longitudinally sectioned, they show a honeycomb-like internal organization very similar to that of Discussion P. distincta (Fig. 6b). As in P. distincta, transverse histological sections through Knowledge of the morphology of anuran parotoids is based the parotoids of R. marina show large, elongated, syncytial mainly on histological and ultrastructural studies in bufonids, granular glands arranged side by side in the dermis, below a which describe the secretory syncytium, the secretory granules layer of smaller regular skin glands juxtaposed to the epider- and the myoepithelial layer (Hostetler & Cannon, 1974; mis (Fig. 6c). They are also enveloped by a myoepithelial layer Cannon & Hostetler, 1976; Toledo et al., 1992; Almeida et al., and their secretion granules, in contrast to those from the rest 2007; Jared et al., 2009, 2011). Despite the great differences in of the skin, are highly positive to alcian blue pH 2.5, weakly size and in morphology of the secretion granules of skin positive to PAS and negative to bromophenol blue. granular glands and the parotoid glandular units (alveoli), it is Each secretory unit of R. marina parotoids is connected to believed that both structures have the same epithelial origin the exterior through an epithelial duct, which is lined by a very (Toledo et al., 1992; Toledo & Jared, 1995). In P. distincta, the thick epithelium, which obstructs the ductal canal and forms different grades of positive reaction to bromophenol blue indi- an epithelial plug (Fig. 6d). The plug is better observed in cate qualitative and/or quantitative differences in the proteins transverse sections through the duct (Fig. 6f). Superﬁcial present in both types of glands. In the Rhinella species studied

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Figure 6 Rhinella marina (a) Specimen showing one of the parotoids, just behind the tympanus. Note the polygonal shape of the macrogland. Parotoid pores (arrows). (b) Parotoid sectioned in horizontal plane showing the honeycomb-like arrangement of the alveoli(*). (c) Low magnification of a transverse histological section of the macrogland showing the arrangement of the large, elongated granular glands (G). Haematoxylin- eosin staining (d) longitudinal histological section of the ductal region of a large granular gland (G) of the parotoid. Note the ductal canal obstructed by an epithelial plug (pl). Cal- cified dermal layer (cc); pore (po); common mucous glands (arrows); differentiated (c) (d) (e) (f) mucous glands (*). Haematoxylin-eosin staining (e) transverse histological section of the duct near the skin surface showing the arrangement of the differentiated glands (*) around it. Calcified dermal layer (cc). pore (po). Haematoxylin-eosin staining (f) transverse histological section of the duct in a deeper plane, where it is completely obstructed by the epithelial plug (pl). 300 mm 100 mm 100 mm Haematoxylin-eosin staining.

so far, secretions from the skin and from the parotoids are after compression. In these toads, the pressurized poison totally distinct. While in the skin there is a predominance of breaks a plug and is squirted in toto in the form of jets (Jared proteins, in the parotoids the secretion is composed of sub- et al., 2009). Although a plug is absent in P. distincta and the stances that are reactive to PAS and alcian blue pH 2.5, but successive phases of the macrogland recovery after compres- not to bromophenol blue, indicating a non-protein secretion. sion have never been followed, it is expected that they regen- The conclusion is that even having a common origin with erate gradually and recover the poison inside the secretory the glands in the rest of the skin, the parotoids, especially syncytia, similar to what has already been observed in bufo- in bufonids, are well-differentiated structures strategically nids (Jared et al., unpubl. data). located for the rapid release of large quantities of poison in Additionally, bufonid parotoids have a type of differenti- case of a predator’s attack, and must not be considered as ated mucous gland that is arranged around the syncytial simple accumulations of granular glands. ducts, forming a complex structure resembling a rosette (Jared The connective tissue framework observed in the parotoids et al., 2009). These differentiated glands may play a role in the of bufonids is responsible for a macrogland honeycomb archi- physiology of the alveoli and/or in the ﬁnal composition of the tecture (Jared et al., 2009). The homogeneous collagen of the toad poison. Such structures were not observed in the macro- bufonid parotoid is very elastic and resistant, because, even glands of P. distincta, reinforcing the idea that they are func- after compression, the shape of the macrogland usually tionally distinct. remains unchanged, a phenomenon that was observed in the In contrast to the parotoids in bufonids (Toledo & Jared, histological sections examined under polarized microscopy 1995; Jared et al., 2009), in phyllomedusines the macroglands (Jared et al., 2009). In P. distincta, we detected a similar alveo- are not associated with any evident defensive behavioural dis- lar structure, but comparison of alveoli before and after com- plays such as lung inﬂation, head butting or crouching. pression shows that the secretory syncytia contain different However, many species of Phyllomedusa, including P. dis- volumes of secretion, probably depending on the amount of tincta, show a dead feigning (tanatosis) behaviour (Sazima, external pressure received. The differential content of post- 1974), in which the animal tightens the body and remains compression phyllomedusine gland differs from that of bufo- static, acquiring a foetal position. This behaviour could make nids (Jared et al., 2009), in which syncytia are totally emptied the macroglands more prominent and could contribute to

48 Journal of Zoology 291 (2013) 42–50 © 2013 The Zoological Society of London M. M. Antoniazzi et al. Parotoid morphology in leaf frogs poison release at the moment of a predator’s bite. The absence Our results suggest that bufonid parotoids are much more in P. distincta of the characteristic epithelial plug that specialized structures for poison release than their equivalent obstructs the ducts in bufonids (Jared et al., 2009) might facili- in phyllomedusines. Even considering the need for more tate poison release. The transverse serial sections through the studies covering a taxonomically more representative sample, whole length of the ducts in P. distincta showed that no plug is we suggest restricting the term parotoid to bufonid postorbital present. In contrast, the epithelial plug is quite evident in macroglands. Taking into consideration the characteristic bufonid parotoids and must be responsible for the permanent anatomy of the macroglands in most phyllomedusines, we turgidity of the parotoid secretory syncytia (Jared et al., 2009). suggest for them the term ‘dorsolateral macroglands’. Consid- In this case, pressure exerted by the bite of a potential preda- ering the diversity and the span of morphological variation in tor works as a trigger for the explosive release of poison in the size, habitat and body form in bufonids and phyllomedusines, form of powerful jets (Toledo et al., 1992; Toledo & Jared, it will be very interesting to know the variation of the struc- 1995; Jared et al., 2009). Conversely, when the parotoids of tural differences observed in their macroglands. P. distincta are compressed, no jets are observed. The poison is expelled from the pores in the form of droplets and accu- mulates on the macrogland skin surface. Acknowledgements In addition, phyllomedusines make use of an array of We thank Beatriz Maurício and Simone Jared for their tech- defensive morphological, behavioural and toxinological strat- nical assistance. CNPq, INCTTOX-CNPq and Capes pro- egies that are different from those of bufonids. The green vided financial support. IBAMA provided permission to colour of the body, which serves as efficient camouflage collect animals to M.T. Rodrigues (#193/2001) and to M.M. among leaves, is rapidly changed to aposematism when these Antoniazzi (#15964-1). anurans move among vegetation, exposing the yellow, orange and red colours of their flanks and limbs. Also, if a phyllomedusine is swallowed by a predator, many different bioac- References tive compounds (including opioids and emetic substances) are Almeida, P.G., Felsemburgh, F.A., Azevedo, R.A. & Brito- released, giving the swallowed anuran the chance to be regur- gitated (Sazima, 1974; Erspamer et al., 1993). Gitirana, L. (2007). Morphological re-evaluation of the Despite the tradition of designating all amphibian parotoid glands of Bufo ictericus (Amphibia, Anura, Bufo- postorbital/supratympanic macroglands as parotoids, we nidae). Contrib. Zool. 76, 145–152. have shown here that, at least between macroglands of bufo- Bancroft, J.B. & Steven, A. (1990). Theory and practice of his- nids and those of P. distincta, there are significant morpho- tological techniques. Edinburgh: Churchill Livingstone. logical differences that are supported by our preliminary Brodie, E.D. Jr (1983). Antipredator adaptations of salaman- observations in other phyllomedusines (P. burmeisteri, P. bi- ders: evolution and convergence among terrestrial species. color, P. rohdei, P. tetraploidea, P. bahiana, P. megacephala). In Plant, animal and microbial adaptations to terrestrial In these species, and also in P. vaillanti, P. burmeisteri, environment: 109–133. Margaris, N.S., Arianoutsou- P. tomopterna and P. hypochondrialis, our preliminary results Faraggitaki, M. & Reiter, R.J. (Eds). New York: Plenum. indicate the presence of a honeycomb-like macrogland, Brodie, E.D. Jr & Gibson, L.S. (1969). Defensive behavior structurally similar to that of P. distincta. Therefore, the and skin glands of the northwestern salamander, consistent morphological similarity in the macroglands of Ambystoma gracile. Herpetologica 25, 187–194. representatives of the major clades among Phyllomedusa Cannon, M.S. & Hostetler, J.R. (1976). The anatomy of the (Faivovich et al., 2010; Pyron & Wiens, 2011) suggests that, parotoid gland in Bufonidae with some histochemical find- despite variation in length and conspicuousness, this struc- ings, II Bufo alvarius. J. Morphol. 148, 137–160. ture seems to be widely distributed in the genus. On the Cannon, M.S. & Palkuti, G.A. (1976). The ‘parotoid’ gland of other hand, there is still no consensus about the presence of Bufonidae. Toxicon 14, 149–151. parotoid macroglands in all Phyllomedusinae (Lutz, 1950). Clarke, B.T. (1997). The natural history of amphibian skin They have been reported to be present in Phyllomedusa, but secretions, their normal functioning and potential medical absent in Phasmahyla guttata by Cochran (1955), who applications. Biol. Rev. 72, 365–379. described only a glandular dorsolateral ridge in this species. Cochran, D.M. (1955). Frogs of southeastern Brazil. Bull. U. As commented by Faivovich et al. (2010), additional work should be done to verify the homology between parotoids of S. N. Mus. 206, 1–423. Phyllomedusa and these dorsolateral glands of Phasmahyla, Duellman, W.E. & Trueb, L. (1986). Biology of amphibians. which is currently regarded as the sister group of the New York: McGraw-Hill Book Company. monophyletic Phyllomedusa. Also, additional anatomical, Erspamer, V., Falconieri Erspamer, G., Potenza, R.L., Barra, ultrastructural and histological data on these or similar D., Mignogna, G. & Bianchi, A. (1993). Pharmacological structures in Agalychnis, Cruziohyla and Phrynomedusa, the studies of ‘sapo’ from the frog Phyllomedusa bicolor skin – successive external groups for Phyllomedusa and Phas- a drug used by the peruvian matses indians in shamanic mahyla, would be an important contribution to understand- hunting practices. Toxicon 31, 1099–1111. ing further the phylogenetic history of phyllomedusines and Faivovich, J., Haddad, C.F.B., Baêta, D., Jungfer, K.H., of their glands. Álvares, F.R., Brandão, R.A., Sheil, C., Barrientos, L.S.,

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