Ultrastructure of spermiogenesis and spermatozoa of Gymnotus cf. anguillaris and Brachyhypopomus cf. pinnicaudatus (Teleostei: Gymnotiformes)

Ultrastructure of spermiogenesis and spermatozoa of Gymnotus cf. anguillaris and Brachyhypopomus cf. pinnicaudatus (Teleostei: Gymnotiformes)

Tissue and Cell 39 (2007) 131–139 Ultrastructure of spermiogenesis and spermatozoa of Gymnotus cf. anguillaris and Brachyhypopomus cf. pinnicaudatus ...

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Tissue and Cell 39 (2007) 131–139

Ultrastructure of spermiogenesis and spermatozoa of Gymnotus cf. anguillaris and Brachyhypopomus cf. pinnicaudatus (Teleostei: Gymnotiformes) G.F. Franc¸a a,b , C. Oliveira b , I. Quagio-Grassiotto b,∗ a

Departamento de Biologia Celular, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, CP 6109, CEP 13.084-971, S˜ao Paulo, Brazil b Departamento de Morfologia, Instituto de Biociˆ encias, Universidade Estadual Paulista, CP 510, CEP 18.618-000, Botucatu, S˜ao Paulo, Brazil Received 24 October 2006; received in revised form 23 January 2007; accepted 20 February 2007 Available online 6 April 2007

Abstract The ultrastructure of spermiogenic stages and spermatozoa of representatives of two gymnotiform families, Gymnotus cf. anguillaris (Gymnotidae) and Brachyhypopomus cf. pinnicaudatus (Hypopomidae) were studied. Spermiogenesis of both species is characterized by lateral development of the flagellum and formation of a nuclear fossa. Some differences were found between these species, such as whether (B. cf. pinnicaudatus) or not (G. cf. anguillaris) nuclear rotation occurs, permanence of the cytoplasmic channel, and type and localization of the nuclear fossa. In the G. cf. anguillaris spermatozoon the nucleus is spherical with highly condensed chromatin. The nuclear fossa is shallow and lateral and is associated with the centriolar complex through stabilizing fibrils. The midpiece is short, with many vesicles, a cytoplasmic channel, and elongate mitochondria. In the B. cf. pinnicaudatus spermatozoon the ovoid nucleus is elongated lateral and posterior to the centriolar complex, and has highly condensed chromatin. The eccentric nuclear fossa is of the moderate type, and contains the entire centriolar complex. The midpiece is long, with numerous vesicles, elongate mitochondria, and no cytoplasmic channel. In both species the flagella are laterally disposed in relation to the nucleus and comprise of the classical 9 + 2 axoneme. Most of the characteristics found in the spermatozoa of these two species of Gymnotiformes are shared with species of Characiformes, whereas only a few are also found in Siluriformes. This suggests that Gymnotiformes and Characiformes may be more closely related than previously proposed. © 2007 Elsevier Ltd. All rights reserved. Keywords: Sperm; Morphology; Electron microscopy; Fish evolution; Knifefish

1. Introduction Gymnotiformes constitute an important component of the Neotropical freshwater fauna in the South and Central America occurring from Mexico to Argentina (Mago-Leccia, 1994). Currently, 135 species are known and distributed in 32 genera and 5 families (Albert and Crampton, 2005). ∗ Corresponding author at: Depto. Morfologia, Instituto de Biociˆ encias, Universidade Estadual Paulista, Campus de Botucatu, Distrito de Rubi˜ao Jr, s/n, CP 510, CEP 18.618-000, Botucatu, S˜ao Paulo, Brazil. Tel.: +55 14 3811 6264; fax: +55 14 3811 6264. E-mail address: [email protected] (I. Quagio-Grassiotto).

0040-8166/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tice.2007.02.003

The relationships among families of Gymnotiformes are still debated. Triques (1993) and Gayet et al. (1994), based on morphological data, recognized Apteronotidae as the most primitive family, due to the presence of a caudal fin. Also based on morphological data, Mago-Leccia (1994) divided the order into two suborders: Sternopygoidei, comprised of Sternopygidae and Apteronotidae; and Gymnotoidei, comprise of Rhamphichthyidae, Hypopomidae, Gymnotidae, and Electrophoridae. Alves-Gomes et al. (1995), combining molecular and morphological data, concluded that Sternopygidae did not represent a natural group and proposed the new family Eigenmanniidae. The cladogram presented by these authors shows that the order is divided into five

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groups: Sternopygidae; Electrophoridae and Gymnotidae; Hypopomidae and Rhamphichthyidae; Apteronotidae; and a new clade composed of their new family Eigenmanniidae. According to Albert and Crampton (2005), the genera Gymnotus and Electrophorus belong to a natural group (Gymnotidae), that is the sister group to all other Gymnotiformes. Rhamphichthyidae and Hypopomidae constitute the new superfamily Rhamphichthyoidea, while Sternopygidae and Apteronotidae belong to the new superfamily Sinusoidea. Ultrastructural studies of fish spermatozoa have shown that characters obtained from sperm ultrastructure and the process of spermiogenesis may be phylogenetically analyzed, thus providing important data for the elucidation of relationship patterns among several fish groups (Jamieson, 1991; Mattei, 1991). Although studies by Jamieson (1991) and Mattei (1991) present data related to structure of spermatozoa of nearly all major fish groups, the ultrastructure of the spermatozoa of only one gymnotiform species Apteronotus albifrons (cited as Sternarchus albifrons) (Jamieson, 1991) is provided. Therefore, information on sperm ultrastructure is sorely needed for this important clade of fishes. The main purpose of this study was to describe the ultrastructural characteristics of spermiogenesis and spermatozoa of Gymnotus cf. anguillaris (Gymnotidae) and Brachyhypopomus cf. pinnicaudatus (Hypopomidae). The data obtained were compared with those from other ostariophysian groups in order to get a better understanding of the possible relationships among Gymnotiformes, Siluriformes and Characiformes.

3. Results 3.1. Spermiogenesis in Gymnotus cf. anguillaris In G. cf. anguillaris, in the early spermatid, the nucleus, symmetrically encircled by the cytoplasm, has uncondensed chromatin, diffusely and homogeneously distributed (Fig. 1). The centriolar complex is lateral to the nucleus and anchored to the plasma membrane (Fig. 1). In the centriolar complex, the proximal centriole is anterior and perpendicular to the distal centriole. The latter is differentiated in to the basal body giving origin to the flagellum (Figs. 2 and 3). The centriolar complex moves towards the anterior direction, bringing along part of the plasma membrane and the initial segment of the flagellum. Due to this movement, the cytoplasmic channel, a space between the flagellum and the plasma membrane, is formed (Figs. 2–6). At the same time the nucleus undergoes a slight rotation, which causes it to be parallel to the initial segment of the flagellum (Figs. 2 and 6). In the nucleus, in the area in front of the centriolar complex, a small depression with the form of a double arc, the nuclear fossa, is formed (Fig. 6). The condensing chromatin forms thin filaments that progressively occupy the nucleus from the periphery to the central part (Figs. 4–6). The cytoplasm containing the mitochondria and other organelles moves in the direction of the initial segment of the flagellum, giving origin to a short midpiece (Fig. 6). Microtubules radiate from the centriolar complex (Fig. 4). Many vesicles originate near the border of the midpiece close to the plasma membrane (Figs. 4 and 5). In the late spermatid, the nucleus has highly condensed chromatin. The flagellar membrane does not form lateral projections or fins (Fig. 6).

2. Materials and methods

3.2. Spermatozoa of Gymnotus cf. anguillaris

The present study was conducted on adult males of Gymnotus cf. anguillaris (Gymnotidae) collected in the Tietˆe River, Botucatu, S˜ao Paulo, Brazil (22◦ 41 00.9 S 48◦ 19 58.1 W) and adult males of Brachyhypopomus cf. pinnicaudatus (Hypopomidae) collected in the Ba´ıa River, Bataypor˜a, Mato Grosso do Sul, Brazil (22◦ 43 19.4 S 53◦ 17 11.3 W). The fishes were identified and kept in the fish collection of Laborat´orio de Biologia e Gen´etica de Peixes (LBP), Departamento de Morfologia, Instituto de Biociˆencias, UNESP, Botucatu, S˜ao Paulo, Brazil, with the collection numbers LBP 2845 and LBP 2645, respectively. Gonad fragments were fixed in 2% glutaraldehyde and 4% paraformaldehyde in 0.1 M Sorensen phosphate buffer, pH 7.2. The material was postfixed for 2 h in the dark in 1% osmium tetroxide in the same buffer, stained in block with an aqueous solution of 5% uranyl acetate for 2 h, dehydrated in acetone, embedded in araldite, and sectioned and stained with a saturated solution of uranyl acetate in 50% ethanol, and with lead citrate (Reynolds, 1963). Electromicrographs were obtained using a Phillips – CM 100 transmission electron microscope.

The spermatozoon of G. cf. anguillaris has a head and a small midpiece, a single flagellum, and no acrosomal vesicle. In the head, the spherical nucleus is parallel to the centriolar complex and initial segment of the flagellum (Fig. 7). The nucleus has highly condensed chromatin in thin juxtaposed filaments. In the narrow cytoplasmic region apical to the nucleus, no organelles are seen (Figs. 7–11). The nuclear fossa facing to the centriolar complex is composed of a depression in the form of a double arc. The centriolar complex, although located close to the nucleus, remains outside of the nuclear fossa (Fig. 7). The centrioles are connected to each other by stabilizing structures that also connect each centriole to one arc in the nuclear fossa (Fig. 7). The proximal centriole is anterior and perpendicular to the distal centriole (Fig. 7). The midpiece is short and not well defined. It is concentrated around the medial distal region of the nucleus (Figs. 7–11). The midpiece has a cytoplasmic channel, elongate mitochondria and a large number of vesicles clustered in regions close to the plasma membrane (Figs. 7–12). Abundant microtubules radiate from the centriolar complex and are found throughout the entire midpiece (Figs. 8 and 9). Mito-

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Figs. 1–6. Spermiogenesis in Gymnotus cf. anguillaris. 1 and 2 – Early spermatids in longitudinal sections. 1 × 13800 and 2 × 13600; 3 – Longitudinal section of the spermatid midpiece, ×13600; 4 and 5 – Longitudinal and cross sections of spermatids, 4 × 23000 and 5 × 31500; 6 – Late spermatid in longitudinal section, ×22100. A: axoneme, D: distal centriole, F: flagellum, P: proximal centriole, M: mitochondria, N: nucleus, V: vesicles, asterisk: microtubules, black arrow: cytoplasmic channel, double white arrow: nuclear fossa, single white arrow: stabilization structures of the centriolar complex.

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Figs. 7–13. Spermatozoa of the Gymnotus cf. anguillaris. 13 and 14 – Spermatozoa in longitudinal sections, 13 × 22050 and 14 × 31500; 15 to 17 – Cross sections of spermatozoa head, 15 × 42000, 16 × 23500, 17 × 17000; 18 – Cross section of the spermatozoon midpiece, ×13800; 19 – Flagella in cross sections, ×63000. A: axoneme, C: centriole, D: distal centriole, F: flagellum, P: proximal centriole, M: mitochondria, N: nucleus, V: vesicles, asterisk: microtubules, black arrow: cytoplasmic channel, double white arrow: nuclear fossa, single white arrow: stabilization structures of the centriolar complex.

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chondria are separated from the flagellum by the cytoplasmic channel (Fig. 12). The flagellum is lateral and parallel to the nucleus and has the classical 9 + 2 microtubular pattern. The flagellar membrane does not have lateral projections or fins (Fig. 13). 3.3. Spermiogenesis in Brachyhypopomus cf. pinnicaudatus In B. cf. pinnicaudatus, in the early spermatid, the nucleus, symmetrically encircled by the cytoplasm, has uncondensed chromatin, diffusely and homogeneously distributed (Fig. 14). The centriolar complex is lateral to the nucleus and anchored in the plasma membrane (Fig. 14). In the centriolar complex, the proximal centriole is anterior and perpendicular to the distal centriole. The distal centriole, differentiated in to the basal body, gives origin to the flagellum (Fig. 14). As the centriolar complex migrates in the anterior direction it carries with it part of the plasma membrane and the initial segment of the flagellum (Fig. 15). Due to this movement a space between the flagellum and the plasma membrane, the cytoplasmic channel, is formed (Figs. 16–18). The process of chromatin condensation creates thin fibers that progressively and uniformly occupy the entire nucleus (Figs. 15 and 17). The cytoplasm containing the mitochondria and other organelles moves in the direction of the flagellum, giving arise to the midpiece (Figs. 16 and 17). The nuclear rotation occurs. At the end of nuclear rotation, the centriolar complex is contained within the nuclear fossa (Fig. 16). At this time the flagellum occupies a position medial to the nucleus. After the initial nuclear rotation, the nucleus undergoes a secondary lateral and posterior extension. It displaces the nuclear fossa and centriolar complex to a position more lateral and anterior in the spermatid (Fig. 17). At this time the midpiece elongates and the adjacent membranes of the cytoplasmic channel fuse with one another. Due to this process, the cytoplasmic channel closes (Fig. 19). In the final spermatid, the nucleus has highly condensed chromatin composed of thin juxtaposed filaments. The flagellar membrane does not form lateral projections or fins (Fig. 19). 3.4. Spermatozoa of Brachyhypopomus cf. pinnicaudatus The spermatozoon of B. cf. pinnicaudatus has a head, long midpiece, single flagellum, and no acrosomal vesicle. In the head, the ovoid nucleus, elongated lateral and posterior to the centriolar complex, has highly condensed chromatin in thin juxtaposed filaments (Figs. 20–22). An eccentric, of moderate type, highly irregular nuclear fossa is located at the anterior side of the nucleus (Figs. 20–23). The centrioles are completely contained within the nuclear fossa and connected to it by fibrillar stabilizing structures that also connect the centrioles to each other (Figs. 21 and 22). The proximal centriole is perpendicular, anterior and lateral to the distal centriole. The flagellum is eccentric in relation to

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the nucleus (Fig. 21). The long midpiece, thin, asymmetric, contains long ramified mitochondria and numerous vesicles (Figs. 20–22 and 25). The cytoplasmic channel found in the spermatid is not found in the spermatozoon. In the midpiece, microtubular arrays disposed in longitudinal position are found close to the axoneme (Figs. 21, 24, 25 and 27). A membranous compartment composed of numerous vesicles that encircle the axoneme is located posterior to the mitochondrial region (Fig. 26). The flagellum has the classical 9 + 2 microtubular pattern. The flagellar membrane does not have lateral projections or fins (Fig. 27).

4. Discussion In spermatozoa, the flagellum axis may be either perpendicular or parallel to the nucleus, depending on whether nuclear rotation occurs during spermiogenesis (type I spermiogenesis) or not (type II spermiogenesis) (Mattei, 1970). Usually, in the early spermatids the centriolar complex, anchored to the plasma membrane, is positioned laterally to the nucleus. Both types of spermiogenesis are found in the gymnotiforms here analyzed. In Gymnotus cf. anguillaris type II spermiogenesis occurs. Among the Ostariophysi descriptions of the occurrence of type II spermiogenesis are restricted to Characiformes of the family Acestrorhynchidae (Matos et al., 2000), and the inseminating species of the subfamily Glandulocaudinae (Burns et al., 1998; Pecio and Rafinski, 1999; Burns and Weitzman, 2005; Pecio et al., 2005). In Brachyhypopomus cf. pinnicaudatus type I spermiogenesis occurs. This type of spermiogenesis is the most common among Teleostei (Mattei, 1970). However, spermiogenesis in B. cf. pinnicaudatus is unusual because after the initial nuclear rotation, the nucleus undergoes a secondary lateral and posterior extension. This lateral and posterior extension of the nucleus is characteristic of spermiogenesis of the type II found in some Characiformes belonging to Characidae, specifically in the inseminating species of Glandulocaudinae (Burns et al., 1998; Pecio and Rafinski, 1999; Burns and Weitzman, 2005; Pecio et al., 2005). However, nuclear elongation in Glandulocaudinae is much more pronounced than in B. cf. pinnicaudatus. Another peculiarity of B. cf. pinnicaudatus spermiogenesis is the closure of the cytoplasmic channel. This also occurs in Characiformes of the family Erythrinidae (Quagio-Grassiotto et al., 2001a), as well as in the genus Triportheus of the family Characidae (Gusm˜ao-Pompiani, 2003), but does not occur in Glandulocaudinae (Burns et al., 1998; Pecio and Rafinski, 1999; Burns and Weitzman, 2005; Pecio et al., 2005). Among Siluriformes, closure of the cytoplasmic channel during the spermiogenesis was reported in the Diplomystidae (Quagio-Grassiotto et al., 2001b), the most primitive family of this order. The pattern of chromatin condensation in the nuclei of Teleostei depends on the type of protein associated with the DNA (Saperas et al., 1993). Condensation in thin juxtaposed

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Figs. 14–19. Spermiogenesis in Brachyhypopomus cf. pinnicaudatus. 7 – Early spermatid in longitudinal section, ×9750; 8 to 10 – Spermatids in longitudinal sections, 8 × 13250, 9 × 23000, 10 × 25200; 11 – Cross section of the spermatid midpiece, ×25200; 12 – Late spermatid in longitudinal section, ×22100. A: axoneme, D: distal centriole, F: flagellum, P: proximal centriole, M: mitochondria, N: nucleus, V: vesicles, black arrow: cytoplasmic channel, double arrow: nuclear fossa.

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Figs. 20–27. Spermatozoa of Brachyhypopomus cf. pinnicaudatus. 20 to 22 – Spermatozoa in longitudinal sections, 20 × 23000, 21 × 23000, and 22 × 23000; 23 – Centriolar complex in detail, ×46000; 24 – Cross section of posterior end of the nucleus, ×18900; 25 – Midpiece in cross section, ×42000; 26 – Longitudinal section of the anterior segment of flagellum, ×17000; 27 – Flagella in cross sections, ×31500. A: axoneme, D: distal centriole, F: flagellum, P: proximal centriole, M: mitochondria, N: nucleus, V: vesicles, asterisk: microtubules, double arrow: nuclear fossa, white arrow: stabilization structures of the centriolar complex.

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fibers, as found in G. cf. anguillaris and in B. cf. pinnicaudatus is also found in most Teleostei (Jamieson, 1991; Mattei, 1991). Among Ostariophysi this type of condensation is found in several Characiformes, such as those of the families Erythrinidae (Quagio-Grassiotto et al., 2001a), Curimatidae (Matos et al., 1998; Quagio-Grassiotto et al., 2003), and Acestrorhynchidae (Matos et al., 2000). It also occurs in several characid species, whether or not inseminated (Burns et al., 1998; Pecio and Rafinski, 1999; Gusm˜ao-Pompiani, 2003; Burns and Weitzman, 2005; Pecio et al., 2005), and in several Siluriformes (Emel’yanova and Makeyeva, 1991a, 1991b; Lee, 1998; Kim and Lee, 2000; Quagio-Grassiotto and Carvalho, 2000; Santos et al., 2001; Burns et al., 2002; Quagio-Grassiotto et al., 2005). Besides the different types of spermiogenesis, the spermatozoa of G. cf. anguillaris and B. cf. pinnicaudatus also differ from each other regarding to the form and position of the nucleus in relation to the flagellum, the form and position of the nuclear fossa, the length of the midpiece, the presence or absence of the cytoplasmic channel, and the presence of a membranous compartment following the initial segment of the flagellum. These two species have in common spermatozoa with similar patterns of chromatin condensation, asymmetric midpieces, similar form and number of mitochondria, and absence of flagellar fins. The spermatozoa of Apteronotus albifrons studied by Jamieson (1991) have some similarities with the species analyzed herein, i.e., an asymmetric midpiece, elongate mitochondria, and absence of flagellar fins. According to the most recent relationship pattern proposed for Gymnotiformes, Gymnotidae is the most basal family and the sister-group of all other families in the order (Albert and Crampton, 2005). Hypopomidae belongs to a second group, with Rhamphichthyidae, Sternopygidae, and Apteronotidae, being more related to Rhamphichthyidae (Albert and Crampton, 2005). The present data show that the spermatozoa of Hypopomidae are more similar to those of Gymnotidae than to the Apteronotidae (Jamieson, 1991). Therefore, the available data about spermatozoa ultrastructure of Gymnotiformes do not support the current hypothesis of relationship among the members of this order. Among Ostariophysi, the group whose spermiogenesis and spermatozoa are more similar to those found in G. cf. anguillaris is Acestrorhynchidae (Matos et al., 2000). G. cf. anguillaris and Acestrorhyncus falcatus (Matos et al., 2000) both have type II spermatozoa (sensu Mattei, 1970), similar patterns of chromatin condensation in thin juxtaposed fibers, short midpieces, the presence of a cytoplasmic channel, and the absence of flagellar fins. The main differences between these species are the nuclear fossa presence, conspicous centriolar stabilizing structures, elongate mitochondria, and clustereds vesicles in the midpiece of G. cf. anguillaris. The B. cf. pinnicaudatus spermatozoon shares with some inseminating Characidae of the subfamily Glandulocaudinae spermatozoon (Burns et al., 1998; Pecio and Rafinski, 1999; Burns and Weitzman, 2005; Pecio et al., 2005) a sim-

ilar form of the nucleus and the presence of microtubular arrays in the midpiece. Other characteristics are more similar to those found in Triportheus paranensis spermatozoa (Gusm˜ao-Pompiani, 2003). Among the characteristics shared between B. cf. pinnicaudatus and T. paranensis spermatozoon are the occurrence of long midpieces without cytoplasmic channels and the form and number of mitochondria. The absence of a cytoplasmic channel in the midpiece, as result of its closure during spermiogenesis, is also observed in the spermatozoa of species of Erythrinidae (Quagio-Grassiotto et al., 2001a), one of the most primitive Neotropical families of Characiformes (Calcagnotto et al., 2005). Moreover, this characteristic is also observed in Diplomystidae (QuagioGrassiotto et al., 2001b), the most primitive siluriform family (de Pinna, 1998). The relationships among the orders of Ostariophysi are still controversial. Rosen and Greenwood (1970) divided the superorder Ostariophysi into two clades, based either on the absence, Anotophysi (Gonorynchiformes), or presence, Otophysi (Cypriniformes, Siluriformes, Characiformes, and Gymnotiformes), of the Weberian apparatus. Roberts (1973), studying 22 morphological characters of Otophysi, suggested that Characiformes was the sister group of Gymnotiformes. Fink and Fink (1981, 1996), testing the previous hypothesis, found that Gymnotiformes was the sister group of Siluriformes. Dimmick and Larson (1996), combined morphological and molecular data, corroborated the hypothesis of relationship between Siluriformes and Gymnotiformes. However, when only molecular data was analyzed, Gymnotiformes appears as the sister group of Characiformes and both clades as the sister group of Siluriformes (Dimmick and Larson, 1996). A similar result was obtained by Ort´ı (1997), analyzing partial sequences of the ependymin gene. Saitoh et al. (2003), studied complete mitochondrial sequences of protein coding genes, found that Characiformes was the sister group of Gymnotiformes. More recently, Lavou´e et al. (2005), based on complete mitochondrial DNA sequences, suggested that Siluriformes and Characiformes might belong to a monophyletic clade that is the sister group of Gymnotiformes. The similarity observed between the spermatozoa of Gymnotiformes and those of Characiformes is in accordance with the hypothesis that suggests that Characiformes and Gymnotiformes are sister-groups.

Acknowledgements We would like to thank Hor´acio J´ulio Junior and Ricardo Teixeira for their help during collection expeditions, Ricardo Campos-da-Paz for the taxonomic identification of the species, and the E.M. Laboratory of IBB-UNESP, for allowing the use of their facilities. This research was supported by the Brazilian agencies FAPESP (Fundac¸a˜ o de Apoio a` Pesquisa do Estado de S˜ao Paulo), CNPq (Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico),

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and CAPES (Coordenac¸a˜ o de Aperfeic¸oamento de Pessoal de N´ıvel Superior).

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