IntroductionMany authors have described the architecture of the

reproductive system of Amphibia in order to explain several different reproductive strategies that occur in that taxon (e.g., de Oliveira, Zanetoni and Zieri, 2002; de Oliveira and de Souza Santos, 2004; Sever et al., 2004). These strategies seem to be suggested by morphological and functional variations in the reproductive organs (Duellman and Trueb, 1994; Kardong, 2002). Various techniques have been employed, from the staining procedure for histological studies (de Oliveira and Zeiri, 2005) to Transmission Electron Microscopy for ultrastructural analysis (Palmer et al., 1997; Amaral et al., 2000; Sretarugsa et al., 2001). These techniques recently have become very important due to their potential taxonomic value. However, most of the papers

do not include both sexes, therefore, descriptions tend to be incomplete within the species level (e.g., Hermosilla, Urbina and Cabrera, 1983; Hoque and Saidapur, 1994; de Oliveira and de Souza Santos, 2004).

Cuba is inhabited by 52 species of frogs belonging to the genus Eleutherodactylus, 98% of which are endemic. Most studies had been focused on systematics, natural history, bioacustics (Díaz and Cádiz, 2008) and recently on biogeographical origin and irradiation of some species (Rodríguez et al., 2010). The ground-dwelling frog, Eleutherodactylus planirostris, is one of the most widespread anurans in Cuba. It has the typical strong sexual dimorphism, a tuberculate and brown dorsum with two major pattern polymorphisms: mottled or pale dorsolateral stripes (Díaz and Cádiz, 2008).

Studies dealing with the morphological characterization of the reproductive system of Cuban amphibians are scarce. The few works that have been published do not describe the morphology of the gonads in both sexes (Fontaine, 2005; Rodríguez, 2008), neither included the use of Transmission Electronic Microscopy (Fontaine, 2005). Therefore, this paper aims at describing the structure and ultrastructure of the gonads of Eleutherodactylus planirostris in both sexes.

Herpetology Notes, volume 5: 281-290 (2012) (published online on 22 August 2012)

1 División de Colecciones Zoológicas. Instituto de Ecología y Sistemática. Carretera de Varona, Km 3 ½, Capdevila, Boye-ros, AP 8029, CP 10800, La Habana, Cuba. e-mail: manueliturriaga83@gmail.com

2 Departamento de Biología Animal y Humana. Facultad de Biología. Universidad de la Habana. Calle 25 no 455 e/ I y J, Vedado, Plaza de la Revolución, La Habana, Cuba. e-mail: yami@fbio.uh.cu, anita@fbio.uh.cu

Abstract. A description of the gonads of Eleutherodactylus planirostris was performed by using light microscopy and Transmission Electron Microscopy. The general histological architecture of its gonads is very similar to other anurans. Testes of E. planirostris are paired ovoid organs constituted by a mass of seminiferous tubules surrounded by a layer of fibrous connective tissue. Germinal tissue is situated within seminiferous tubules in an arrangement of spermatocyst. Germinal cells were classified in six types during spermatogenesis: spermatogonia, spermatocyte I, spermatocyte II, early and late spermatids and spermatozoon. The spermatogenesis is like in other anuran species. Morphology of the spermatozoon head and tail exhibits interespecific difference and could be a useful tool in Taxonomy. During the germinal development junctional complexes of the type gap junction between spermatocytes and Sertoli cells were identified. Moreover, mitochondria, smooth endoplasmic reticula and a Golgi apparatus which play an important role in spermatogenesis were found. Ovaries are lobulated organs, with a central cavity, covered by a transparent ovarian capsule. They are composed of germinatives and folliculars cells. Germinatives cells were classified in five types: oogonias, previtellogenic oocyte, oocyte at the beginning vitellogenesis stage, vitellogenic and postvitellogenic oocytes. A junctional complex between follicular cells and oocytes was observed. A Golgi apparatus and mitochondrias were found in the previtellogenic oocyte cytoplasm; however, many ribosomes and yolk platelels were observed. All these organelles provide the synthesis of RNAr and proteins used during embryonic development. The role of Sertoli cells in spermatogenesis is discussed.

Keywords. Eleutherodactylus, gonads, spermatogenesis, vitellogenesis, junctional complexes, Sertoli cells.

Manuel Iturriaga1, Yamilka Rodríguez2, Ana Sanz2

Structural and ultrastructural description of the gonads of Eleutherodactylus planirostris (Anura: Eleutherodactylidae)

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Materials and MethodsFive females and five males of Eleutherodactylus planiros-tris were collected in the Parque Metropolitano, Havana, Cuba (23006´31´´N, 82024´55´´W), between May and August 2006. The individuals were anesthetized with ether and were dissected through a longitudinal incision from the groin to the thoracic re-gion. The reproductive organs were removed and fixed in 10% neutral formalin solution. The material was processed by histo-logical routine for dehydration, clarification and embedded in paraffin. The samples were cut with a microtome to 5 μm and the sections obtained were stained with haematoxylin/eosin or Go-mory trichrome. The histological analysis was made with a light

microscope Carl Zeiss and micrographs were taken with a digital camera Motic Digital Microscope (DMB Series) coupled to the microscope. Another three animals of both sexes were dissected as described above and their gonads were fixed in 2.5% gluta-raldehyde (Applichem). The gonads were processed for Trans-mission Electron Microscopy (TEM) by inclusion in epoxy resin (Merck). The blocks were cut to 40-60 nm by an ultra-microtome Leica Ultracut, using diamond knives. The sections were contras-ted using 3% uranyl acetate and 0.3% lead citrate (PolySciences) and were observed with a transmission electron microscope JEOL JEM 1010-80 kV.

Figure 1. Micrographs under light microscopy of testis and seminiferous tubules of Eleutherodactylus planirostris stained with haematoxylin/eosin: (A) Longitudinal section of testis; (B) Cross-section of a seminiferous tubule; (C) Seminiferous tubule showing a small cyst of spermatogonias and a cluster of spermatozoa; (D) Cross-section of seminiferous tubules showing cells in different stages of development and the interstitial tissue located between them. Abbreviations are: SPC, spermatocytes SPZ, spermatozoa; SC, Sertoli´s cell; SPG, spermatogonias; SPT; spermatids; IT: interstitial tissue.

Structural and ultrastructural description of the gonads of Eleutherodactylus planirostris 283

Results

Male

The testes in Eleutherodactylus planirostris are paired organs located in the abdominal cavity and linked to kidneys. Covering each testis there is a thin layer of connective tissue, called albuginea tunic, which sends trabecula to inner testi. It is not pigmented and therefore seminiferous tubules can be observed inside testi (fig. 1A). A connective tissue delimits these tubules and both form the morphological unit of testis. This connective

tissue has blood vessels, collagen fibers, fibroblasts and Leydig interstitial cells.

Each seminiferous tubule has a germ tissue with a lot of spermatogenetic cysts or spermatocysts (fig. 1B). These cysts are clusters of germ cells in different stages of differentiation that occur due to a close morphofunctional relation with the Sertoli cells. The development of the cells is synchronical in the cysts.

The different cell types of spermatogenesis were identified by both light microscopy and TEM.

Figure 2. Micrographs under Transmission Electron Microscopy of masculine cells in different stages of development and somatic cell of Eleutherodactylus planirostris: (A) Spermatogonia; (B) Spermatocyte; (C) Cyst of spermatocytes (SPC) showing a Sertoli cell (SC); (D) Spermatids; (E) Leydig cell (LC) in interstitial tissue.

Spermatogonia are cells that begin the spermatogenic process. These cells are the largest in the spermatogenetic lineage. Under light microscopy, their nuclei are irregular, little pigmented and the chromatin is granulated. Under TEM, a prominent Golgi apparatus, many mitochondria and lipid inclusions in their cytoplasm can be observed (fig. 2A). Usually, they are very close to the wall of the seminiferous tubules and are associated with Sertoli cells.

Spermatocytes I are spherical cells, slightly smaller than the spermatogonia. In both microscopy techniques, the chromatin is slightly condensed in their nuclei (figs. 1B, 2B). Spermatocytes II are difficult to observe by both microscopy techniques since they quickly pass to their second meiotic division. They are haploid cells, quite smaller than spermatocytes I and have more compact nuclei. Generally, spermatocytes II are observed in different phases of the first meiotic division, with the nuclear material showing different degrees of coiling (fig. 2C).

The spermatids are the product of the second meotic division. They can be seen under light microscopy in two forms. The early spermatids which are round cells having a smaller size than spermatocyte II and with more compacted chromatin (figs. 1D, 2D). The late spermatids are easy of to identify because their bodies are very elongated and their nuclei even more compact. The cystic arrangement is altered and organized in bundles by Sertoli cells. The presence of late spermatids and cysts in bundles are signs of the beginning of the spermiogenesis process.

Spermatozoa occur in clusters inside the cysts (figs. 1B, 1C, 1D). They are characterized by an extraordinary nuclear coiling and a cytoplasmic reduction. The spermatozoon head is slender, mainly represented by the acrosome and nucleus. Under TEM, the acrosome is located at the top of the head. It is elongated and filled with a substance of moderate electronic density. Between the acrosome and the nucleus appears a subacrosomal space (fig. 3A). The part that connects head and neck with the tail of the spermatozoon can be distinguished

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Figure 3. Micrographs under Transmission Electron Microscopy of head and tail of sperm in Eleutherodactylus planirostris: (A) Cross-sectional area of a sperm head, showing the acrosome (Ac), sub-acrosomal space (SS) and nucleus (Nu); (B) Cross-section of a sperm tail showing the undulating membrane, axoneme (Ax), yuxtaxonemal fiber (Yf), axial sheath (As) and axial fiber (Af); (C) Details of the above, showing the axoneme (Ax), yuxtaxonemal fiber (Yf) and the axial sheath (As).

only at the ultrastructural level under TEM. It consists of a small portion of cytoplasm and proximal and distal centrioles, which are oriented perpendicularly. The tail is filiform, slightly stained and generally oriented towards the luminar area of the seminiferous tubules. It is constituted of two parts: the axoneme and an

undulating membrane as accessory structure (fig. 3B). The axoneme is located below the distal centriole and consists of nine pairs of peripheral microtubule doublets that surround one central pair. The dynein arms are very short or absent (Fig. 3C). The undulating membrane consists of a fiber yuxtaxonemal part associated with

Structural and ultrastructural description of the gonads of Eleutherodactylus planirostris 285

Figure 4. Micrographs under light microscopy of the ovary and some oocytes of Eleutherodactylus planirostris stained with haematoxylin-eosin (A, B, C) and Gomory trichomic (D, E): (A) Longitudinal section of ovary, the arrow shows central cavity; (B) Oogonias (OG) and vitellogenic oocyte (OC); (C) Pre-vitellogenic oocyte showing follicular cells (FC), nucleus (Nu) and cytoplasm (Cyt); (D) Perinucleolar stage showing nucleoli (Nul) and cytoplasm with yolk inclusions (Yk); (E) Cross-section of a vitellogenic oocyte where vitteline platelets are visible.

the axoneme, a sheath or wrapping axial and axial fiber (figs. 3B, 3C).

During their development, the spermatozoa are arranged in very organized bundles due to their association with the Sertoli cells. When they reach maturity, they are released from the bundle and reach the locular lumen and the ductuli efferentes (fig. 1C).

FemaleThe ovary of Eleutherodactylus planirostris is a

lobulated organ with a central cavity covered by a tenuous and transparent ovarian capsule (fig. 4A). It is located in the upper region of the abdominal cavity, linked to the kidneys and occupying an ample zone during the reproductive stage.

In addition, female gonads are composed of primordial germinative cells, oogonia and follicular cells. The primordial germinative cells are situated in the periphery of the ovary. Each oocyte is covered by a single layer of follicular cells made of smooth or square epithelial tissue. Externally, there is a theca, that is, a connective tissue that irrigates follicular cells.

In this study oocytes were classified in four stages based upon their size, diameter, presence and amount of nucleoli, characteristics of the cytoplasm and amount of vitellus: 1) pre-vitellogenic oocyte, 2) oocyte at the beginning of the vitellogenesis, 3) vitellogenic oocyte and 4) post-vitellogenic oocyte.

The oogonia are arranged in peripherical nests in the ovary (fig. 4B). These cells contain a scarce cytoplasm

and a large and oval nucleus (50-150 μm of diameter) with uniformly distributed chromatin.

The pre-vitellogenic oocyte (diameter between 150 y 500 μm) has a large nucleus in central position, there are a few nucleoli located on the periphery of the nuclear envelope (fig. 4C). The cytoplasm is homogeneous although it exhibits slight granulations and is more stained than oogonia. Under TEM, in the cytoplasm of pre-vitelogenic oocytes, a Golgi apparatus and mitochondria are dispersed and not forming a Balbiani body. The plasma membrane projects microvilli into the follicular cells and the follicular cells send cytoplasm projections to the microvilli of the oocyte. Both states are true interconnected and thus form junctional complexes. Externally, the pre-vitellogenic oocyte has a theca composed of fibroblasts, blood vessels and collagen fibers. After the theca and closer to the oocyte, there is one single layer of smooth epithelial cells (fig. 5A). A vitelline envelope has not been observed.

The oocyte at the beginning of the vitellogenesis presents a diameter between 500 and 950 μm. The nucleus keeps its central position and has few nucleoli and many chromosomes in the periphery (fig. 4D) which are beginning the perinucleolar stage. As development advances, the cytoplasm acquires an acidophilic character and distinct cytoplasmatic zones can be recognized, the peripherical and the internal cytoplasm. The peripherical region is filled with vitellus which is lacking in the internal cytoplasm (centripetal deposition). The nucleus outline is more irregular and

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Figure 5. Micrographs under Transmission Electron Microscopy of oocytes in various development stages of Eleutherodactylus planirostris: (A) Periphery of a previtellogenic oocyte showing a follicular cell and the junctional complexes; (B) Periphery of a vitellogenic oocyte showing a follicular cell, a theca fibroblast and collagen fibers that comprise the yolk and cytoplasmic projections retracted. Abbreviations are: follicular cell (FC); junctional complexes (JC); fibroblast (Fb); collagen (Co); yolk (Yk), cytoplasmic projections (CP).

nucleoli increase in number. The vitelline envelope and square epithelial tissue are already present.

The vitellogenic oocyte exhibits a diameter between 950 and 1950 μm and differentiation between the animal and the vegetative pole is visible. The nucleus is displaced towards the animal pole, the vitellogenesis is intense and the vitellus progressively occupies all the external cytoplasm, mainly in the vegetative pole. Nucleoli are absent, cytoplasm is completely granular, vitelline platelets and a vitelline envelope is clearly identifiable (fig. 4E). Moreover, under TEM, external follicular cells present retracted cytoplasmic projections as it occurs in the pre-vitellogenic oocyte (fig. 5B).

The post-vitellogenic oocyte shows a diameter greater than 2000 μm. In this stage the oocyte presents the same histological characteristics as the vitellogenic oocyte. The vitelline envelope is easily identifiable and the epithelium is smooth. It is an oocyte that will be ovulated. However, under TEM, this oocyte can not be observed since its size does not allow a proper observation.

Companion cellsSertoli cells could be observed under TEM (fig. 2E).

They have a few lipid droplets in their cytoplasm and lysosomes can not be distinguished. The nucleus is composed of irregular patches of heterochromatin and contains a nucleolus inside. No evidence was found that these cells degenerate and could be reabsorbed at the end of this process. In the interstitial tissue, Leydig cells are large, also with a large nucleus and elongated cytoplasm. They have a large numbers of mitochondria and smooth endoplasmic reticula.

.

Discussion

Male

Testes of E. planirostris keep the structural pattern found in most anurans (Duellman and Trueb, 1994; Kardong, 2002). The albuginea tunic is colorless, however, in some species of this genus it can be pigmented as occurs in E. glamyrus, E. atkinsi and E. cuneatus (Fontaine, 2005). The pigment cells can be found in different organs, constituting an extracutaneous pigmentary system of unknown function (de Oliveira and Zieri, 2005). However, according to Palmer et al. (1997) and Sanz and Uribe (1999), this pigmentation in reptiles protects testes against solar radiation.

Concerning the general histological architecture

observed in E. planirostris, a great similarity with other species such as Calyptocephalella gayi (previously Caudiverbera caudiverbera) (Hermosilla, Urbina and Cabrera, 1983), Hyla pulchella andina (Montero and Pisanó, 1992), Pleurodema thaul (Díaz-Páez and Ortiz, 2001) and Physalaemus cuvieri (de Oliveira, Zanetoni and Zieri, 2002) was verified. All cells are in the same developmental degree in each cyst. Spermatocysts or spermatogenic cysts are a diagnostic feature of anurans within vertebrates (Kardong, 2002).

Concerning the spermatogenetic cell lineage, the spermatogonia are the largest cells and they rest on adjacent zones of the basal lamina of the seminiferous tubules, associated with the follicular and Sertoli cells (Kardong, 2002; Gilbert, 2006). They are very similar in various groups of anurans (e.g., Cavicchia and Moviglia, 1983; Montero and Pisanó, 1992; de Oliveira, Zanetoni and Zieri, 2002).

The spermatocytes are generally observed in the prophase of the first meiotic division, with different stages of chromatinic and chromosomal condensation (Gilbert, 2006). Under TEM, in spermatocytes I, a large number of mitochondrias were found in their cytoplasm, which indicates that the spermiogenesis process is beginning. This could be related with the largest size found in this cell type under light microscopy. The spermatocytes I at this stage of growth do not greatly increase in size (Gilbert, 2006). At this stage, the relationship of spermatocytes with Sertoli cells becomes visible in the form of junctional complexes of the type gap juntion. This type of relationship in mammals ensures the passage of molecules and ions that are necessary for proper completion of spermatogenesis and, apparently, also it could have the same role in amphibians (Browder, Erickson and Jeffrey, 1991; Gilbert, 2006). The spermatocytes II are smaller than the preceding and very difficult to find because they quickly enter in the second meotic division (Sanz and Uribe, 1999; Paniagua, 2002).

In this work, two stages for spermatids are established, early spermatids and late spermatids, similarly to what some authors have found (e.g., Hermosilla, Urbina and Cabrera, 1983; Díaz-Páez and Ortiz 2001; de Oliveira, Zanetoni and Zieri, 2002). The most significant features are cellular and nuclear elongation, which occur simultaneously with the chromatin condensation during late spermatid formation.

The spermatozoon of E. planirostris presents a large, thin and undulating tail, similar to E. bressleare; and a narrow and acuminate head like E. turquinensis

Structural and ultrastructural description of the gonads of Eleutherodactylus planirostris 287

(Fontaine, 2005). The presence of organelles that ensure the differentiation of sperm, as the Golgi apparatus does during the formation of the acrosome and the subsequent location of this structure in front of the nucleus, coincides with reports by Shalan et al. (2004) for amphibians and other vertebrates. The sperm nucleus, located behind the acrosome, is tapered by it, and has a small nuclear fossa. In the cross sections at the level of the head, the presence of a subacrosomal space between the acrosome and the nucleus is observed. This is in accordance with findings in other species of leptodactylids by Aguiar-Jr., Giaretta and Recco-Pimentel (2006) that describe a nuclear fossa at the base of the nucleus and a nuclear space in its front end separating the acrosome, although these authors did not distinguish a perforatorium.

The tail is filiform and consists of the axoneme entirely occupied and an undulating membrane and an accessory structure, as in other anurans of the families Leptodactylidae and Bufonidae (Kwon and Lee, 1995). The structures that conform the undulating membrane, like the yuxtaxonemal fiber, sheath or wrapper axial and axial fiber have been also reported in other anurans (e.g., Costa et al., 2004; Aguiar-Jr., Giaretta and Recco-Pimentel, 2006).

FemaleThe ovary of E. planirostris exhibits the same

structural pattern as in the rest of the anurans (Duellman and Trueb, 1994; Kardong, 2002). It consists of paired organs which have a central cavity and are covered by a colorless thin layer according to de Oliveira and de Souza Santos (2004). However, Fontaine (2005) found in E. glamyrus, a pigmented ovary and Fontaine (2005) considered this as a homology with the albuginea tunic of males.

Oogenesis was divided in two fundamental stages: previtellogenic and vitellogenic. Oogonias are the elements that form the nests germinative cells and are located in the periphery of the ovary (de Oliveira and de Souza Santos, 2004). The nests of germinative cells are a typical feature of anurans and also occur in teleost fishes and reptiles (Uribe, 2003b). These nests guarantee the production of a germinal lineage along the life cycle (Kardong, 2002; Gilbert, 2006).

In this study, oocytes were classified in four categories on the basis of histological and morphological changes. Nevertheless, classifications vary depending on the author and the technique employed. Some researchers rely only on size and coloration changes (Montero and Pisanó, 1991; Díaz-Páez and Ortiz, 2001). Others attend

to diameter only (Hoque and Saidapur, 1994) and some use powerful techniques which enable the study of ultrastructural changes (Sretarugsa et al., 2001).

The previtellogenic oocyte of E. planirostris shows the same characteristics observed by de Oliveira and de Souza Santos (2004) and Fontaine (2005). However, the number of nucleoli was fewer than that of Scinax fuscovarius (de Oliveira and de Souza Santos, 2004). These authors explain that this feature could have a differentiation at interespecific level. An external theca is found which is conformed by fibroblasts, that is, connective cells that play an important role in the synthesis of the extracellular matrix (Estrada and Uribe, 2002; Paniagua, 2002), blood vessels and collagen fibers. The entry of vitellus causes a dicrease in the acidophilic character of the cytoplasm (Gilbert, 2006).

The vitellogenic and post-vitellogenic oocytes exhibit similar histological characteristics. The nucleus moves towards the animal pole and lacks nucleoli. However, the presence of nucleoli has been found in E. varleyi and E. turquinensis in these stages (Fontaine, 2005). Uribe (2003b) recorded an increase in number of nucleoli during the vitellogenic stage in urodels. The presence of nucleoli guarantees the synthesis of RNAr and pre-assembles the synthesis proteic machine (Gilbert, 2006). Perhaps their presence in the vitellogenic oocyte is due to a not sufficient amount of RNAr.

The vitelline envelope, outside the plasma membrane of the vitellogenic oocyte, appears to be very thick. This envelope undergoes molecular changes during the transit of the oocyte along the oviduct, which are essential in order to be fertilized (Browder, Erickson and Jeffrey, 1991).

Concerning the complexes laying between the follicle cells and the oocyte plasma membrane, there is evidence in mammals showing that these complexes have a leading role in the passage of nutrients and molecules regulating the oocyte development (Browder, Erickson and Jeffrey, 1991; Gilbert, 2006). In the case of amphibians, these complexes could be the path for reaching certain components needed for the formation of vitellus, previously formed in the liver or other organs (Uribe, 2003a, b).

Companion cellsSertoli cells described above have an irregular nucleus

and absence of lysosomes in the cytoplasm. This is consistent with Smita et al. (2004) in their study of the Sertoli cells of two species of salamanders. They found that the absence of lysosomes in the cytoplasm of Sertoli

Manuel Iturriaga et al. 288

cells is indicative of a process of active spermatogenesis, coinciding with the reproductive peak. On the other hand, the cytoplasm filled with lysosomes is a feature of a testicular regression.

However, the absence of lysosomes during active spermatogenesis is interesting due to the role of the Sertoli cells in the process of the phagocytosis of cellular debris, including mitochondrias during spermiogenesis in that sense (Gilbert, 2006). Further observations should be made.

The presence of Leydig cells in the interstitial tissue highlights is very important, due to their endocrine activity present at least in mammals (Pudney, 1999; Gilbert, 2006). When these cells were observed under TEM, there were abundant mitochondrias and smooth endoplasmic reticula, which are characteristic organelles of steroid-producing cells (Jiménez and Merchant, 2003). This suggests a possible endocrine function in the spermatogenesis process of E. planirostris, which may be similar to reports in mammals.

Acknowledgements. We are very grateful to Rafael Oliva, from the Departamento de Anatomía Patológica, Hospital “Ramón González Coro”, La Habana and Nilda Almaguer and Emir Pérez from the Laboratorio de Biología Celular, Facultad de Biología, Universidad de La Habana for their help during the laboratory work. We thank María Lourdes Segura and Luis Felipe Jiménez from the Facultad de Ciencias, Universidad Nacional Autónoma de México and Helen Díaz-Páez from the Universidad de Concepción, Chile, for all the bibliography offered so kindly. Special thank to Mabel Alfonso for printing many papers needed in this study. Jans Morffe, Luis de Armas and Pedro Pablo Herrera from the Instituto de Ecología y Sistemática, La Habana and Elier Fonseca from the Facultad de Biología, Universidad de La Habana for their critical revision of previous drafts of the manuscript.

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Accepted by Mirco Solé