Zoologischer Anzeiger 257 (2015) 29–38 Contents lists available at ScienceDirect Zoologischer Anzeiger journal homepage: www.elsevier.com/locate/jcz Comparative testis morphology of Neotropical anurans Gabriela Baroni Leite a,∗, Lilian Franco-Belussi b, Diogo B. Provete c,1, Classius de Oliveira b a Graduate Program in Animal Biology, Department of Biology, São Paulo State University (UNESP), 15054-000, São José do Rio Preto, São Paulo, Brazil b Department of Biology, São Paulo State University (UNESP), 15054-000, São José do Rio Preto, São Paulo, Brazil c Graduate Program in Ecology and Evolution, Department of Ecology, Institute of Biological Sciences, Federal University of Goiás, Goiânia 74001-970, Goiás, Brazil article info abstract Article history: Anuran testes have generally similar germinative and somatic anatomical components, but their Received 19 December 2014 morphology varies among species groups. However, little is known about how environmental and evo- Received in revised form 16 April 2015 lutionary factors influence testis shape and sperm quantity. Here, we tested the hypothesis that the Accepted 24 April 2015 arrangement of both germinal compartment (seminiferous locules containing germ cells) and somati- Available online 25 April 2015 cal compartment (interstitial fibers and somatic cells) differ between species with distinct reproductive modes and breeding activities. We compared the testes of eight species from two families of Neobatra- Keywords: chian anurans: Physalaemus centralis, Physalaemus cuvieri, Physalaemus marmoratus, Physalaemus olfersii, Reproductive biology Evolutionary morphology Pseudopaludicola sp. (aff. murundu) (Leptodactylidae: Leiuperinae), Crossodactylus caramaschii, Hylodes Hylodidae cardosoi, and Hylodes sazimai (Hylodidae). We built a 3D model of the testis to visualize the morphology Leptodactylidae and arrangement of seminiferous locules. We measured locular diameter and area, thickness of reticular Leiuperinae fiber bundles, and area of both collagen fibers and sperm bundles. Hylodids had the highest values for Urogenital morphology most characters, except for sperm and reticular fiber bundles. For example, their locular diameter was 50%, and locular area 20% larger than leiuperines, which usually had thicker reticular fibers bundles. These fibers provide greater stability to the large quantity of small, spherical seminiferous locules found in leiuperines. Conversely, hylodids had larger area of collagen fibers, which provides greater flexibility and resistance to a few large and flattened seminiferous loci. Our results can shed light on how tes- ticular morphology is influenced by evolutionary processes and ecological strategies, such as breeding seasonality and reproductive modes in anurans. © 2015 Elsevier GmbH. All rights reserved. 1. Introduction number, morphology, and maturation time of sperm (reviewed by Ramm and Scharer, 2014). As a result, testes are subjected to nat- Amphibians have the greatest diversity of reproductive modes ural selection pressures, which may influence the development of among tetrapods (Duellman and Trueb, 1994; Wells, 2007). Mor- testicular characteristics (Ramm and Scharer, 2014). phological characteristics of the urogenital system in amphibians Anuran testes are paired, smooth, rounded, and compact struc- are related to these reproductive modes, including internal fertil- tures, usually yellowish, and smaller than the ovaries of the same ization and viviparity. These characteristics make them excellent species. However, they are usually asymmetric in size, indepen- models for comparative studies on the evolution of vertebrate dently of age and health condition (Duellman and Trueb, 1994; Liu reproduction (Kühnel et al., 2010). Testes architecture is one of et al., 2011; Zhou et al., 2011; Mi et al., 2012) and position in abdom- the most important features in the evolution of male reproduc- inal cavity (Oliveira and Vicentini, 1998). Additionally, melanocytes tive characters, since their reproductive success is correlated to the have been reported on the tunica albuginea of testes in some species (reviewed in Oliveira and Franco-Belussi, 2012), and their presence seems to exhibit phylogenetic signal in Neotropical anuran lineages ∗ (Provete et al., 2012). In contrast to anurans, the testes of caudates Corresponding author at: Departamento de Biologia, Instituto de Biociências, Letras, e Ciências Exatas, Universidade Estadual Paulista, Rua Cristóvão Colombo, and caecilians have lobular segmentation, whose dimensions vary 2265, São José do Rio Preto, 15054-000, São Paulo, Brazil. Tel.: +55 1732212387. strongly among families (Wake, 1968; Ingersol et al., 1991). E-mail addresses: [email protected] (G.B. Leite), The anuran testis is covered by a thin tunica albuginea that [email protected] (L. Franco-Belussi), [email protected] (D.B. Provete), is rich in collagen fibers, but lack septa dividing the parenchyma [email protected] (C. de Oliveira). (Oliveira and Vicentini, 1998). They include seminiferous locules 1 Present address: CNPq post-doctoral fellow, Department of Biological and Envi- ronmental Sciences, University of Gothenburg, SE 405 30, Göteborg, Sweden. surrounded by an elastic fibrous layer that delimits the interlocular http://dx.doi.org/10.1016/j.jcz.2015.04.005 0044-5231/© 2015 Elsevier GmbH. All rights reserved. 30 G.B. Leite et al. / Zoologischer Anzeiger 257 (2015) 29–38 Fig. 1. Testicular morphology of Crossodactylus caramaschii, Hylodes sazimai, and H. cardosoi, showing the organ’s surface pigmented (A, B, C) and location of seminiferous locules (D, E, F). The photomicrograph is showing differences in the thickness of the tunica albuginea (arrowhead) and in the locular diameters (double arrow) between species (G, H, I). Staining: Hematoxylin/Eosin (G, H, I). area (interstitial region), which is formed by connective tissue how testis morphology evolved, but also the mechanisms responsi- (Lofts, 1976; Oliveira and Vicentini, 1998). This interstitial region ble for changes in some of its structures. Some aspects of testicular contains fibroblasts, blood vessels, efferent ductules, and testicular pigmentation have been analysed from the perspective of phyloge- melanocytes, giving a dark coloration to the testis of some species netic systematics (e.g., Grant et al., 2006) and phenotypic evolution (Oliveira et al., 2002, 2003; Oliveira and Franco-Belussi, 2012). (e.g., Provete et al., 2012). Thus, comparative data on locule and Germ cells in the seminiferous locules are supported by Ser- fiber arrangement may also provide new insights into how testis toli cells, forming spermatocysts. Each cyst contains cells at the anatomy has evolved in anurans. same spermatogenetic stage, establishing a synchrony in their Leptodactylidae (sensu Frost, 2014) is an exclusive Neotropical development, a common characteristic of amphibians (Cavicchia family of anurans. Most leptodactylids build foam nests into which and Moviglia, 1983; Rastogi et al., 1988; Báo et al., 1991; Santos they deposit eggs and where the initial tadpole development takes and Oliveira, 2008). However, internal characteristics of testicular place (Haddad and Prado, 2005). Conversely, hylodids usually lay components greatly vary interspecifically, e.g., sperm produc- eggs inside constructed subaquatic chambers in montane streams tion increases with testis size, improving reproductive success in the Brazilian Atlantic Forest (Haddad and Prado, 2005; Wells, (Emerson, 1997). Also, there is a strong relationship between sperm 2007). It seems that foam nests is a plesiomorphic character, while morphology and testis size, suggesting that sperm competition eggs in subaquatic chambers are more derived (Gomez-Maestre has influenced not only sperm (Zeng et al., 2014), but also testis et al., 2012). It is thought that placing eggs in foam nests is a strategy morphology. Previous studies have focused on the causes and con- to decrease water dependency, while species that lay eggs directly sequences of variation in testis size in amphibians (reviewed in in the water depend on high humidity (Gomez-Mestre et al., 2012). Ram and Scharer, 2014). However, little is known about how dif- Also, hylodids usually have continuous reproduction, whereas ferences in the histology of germinative (e.g., seminiferous locules) most of leptodactylids are explosive breeders. Therefore, these and somatic components (e.g., arrangement of fibers that support groups have contrasting traits that make them useful to understand the organ) are influenced by evolutionary and environmental fac- how testicular morphology is correlated with reproductive modes, tors. Thus, a comparative analysis could help to understand not only breeding activity, and environmental characteristics. Specifically, G.B. Leite et al. / Zoologischer Anzeiger 257 (2015) 29–38 31 Fig. 2. Histological characterization of the testes of Crossodactylus caramaschii, Hylodes sazimai, and H. cardosoi, showing the locular area (lines A, B, C), arrangement of collagen fibers (arrow), testicular melonocytes (*) (D, E, F), and reticular fibers beam (arrowhead) in the interstitium (G, H, I). L: seminiferous locule. Staining: Hematoxylin/Eosin (A, B, C), Picrossirius (D, E, F), and Gömöri’s reticulin (G, H, I). Bars = 25 ␮m. we expect that the arrangement of both testicular compartments on Santos et al. (2011), who showed that the size of seminifer- will differ between species of these families. ous locules of a hylid did not vary throughout the annual cycle.